Although the fetal liver is an active metabolic organ, its oxygen and glucose requirements have not previously been described. We measured hepatic blood flows and the oxygen and glucose differences across the liver in 12 late gestation fetal lambs in utero. Four animals were studied at least 1 wk postsurgically and again 2-5 d later to assess daily variations in hepatic blood flow and metabolism (group I). A second group of eight animals was studied 3-5 d postsurgically during a control period and during acute fetal hypoxia (group II). Under control conditions total hepatic blood flow averaged 400 ml/min per 100 g in both groups, and 75-80% was of umbilical origin. Liver blood flow and oxygen consumption were usually similar during repeated measurements, but in one animal varied considerably. During periods of normoxia, oxygen consumption for both the right and left lobes of liver was 4 ml/min per 100 g. Oxygen consumption of the whole liver accounted for 20% of total fetal oxygen consumption. This was achieved with oxygen extraction of 10-15%, so that hepatic venous blood was well oxygenated and provided an important source of oxygen for other fetal tissues. Under control conditions we could demonstrate no net hepatic uptake or release of glucose suggesting that the liver ultimately utilizes another carbon source to support its oxidative metabolism. During acute hypoxia total liver blood flow and its umbilical venous contribution both fell by 20%. Blood flow to the right lobe of the liver fell twice as much as that to the left lobe. Hepatic oxygen consumption was linearly related to oxygen delivery during the control and hypoxic periods. Consequently, right hepatic oxygen uptake fell by 45% whereas left hepatic oxygen uptake was unchanged, suggesting a functional difference between the lobes. During hypoxia glucose was released from both liver lobes; 6 mg/min per 100 g for the right lobe and 9 mg/min per 100 g for the left lobe. Total hepatic release of glucose was estimated to nearly equal umbilical uptake, so that 45% of the glucose available to fetal tissues was of hepatic origin. We conclude that the fetal liver responds to acute hypoxia by reducing its own oxygen consumption and releasing glucose to facilitate anaerobic metabolism.
J Bristow, A M Rudolph, J Itskovitz, R Barnes
Boronate affinity chromatography and ion exchange chromatography were used to measure the levels of glycosylated hemoglobins in normal and diabetic hemolysates, as well as the distribution of glucose adducts on alpha-NH2-valine and epsilon-NH2-lysine residues. When analyzed by ion exchange chromatography on BioRex 70 resin, the Hb Alc peak comprised 4.4 +/- 0.6% of 15 normal hemolysates and 9.1 +/- 2.1% of 15 diabetic hemolysates. The "Hb Alc" was rechromatographed on GlycoGel B boronate affinity resin that binds vicinal hydroxyl groups of covalently linked sugars. Only 70 +/- 5% of the hemoglobin adhered to the resin. Analysis by the thiobarbituric acid colorimetric test confirmed that the affinity resin effectively separated glycosylated from nonglycosylated hemoglobin. When corrected for nonglycosylated contaminants, the mean level of Hb Alc in normal hemolysates was 2.9 +/- 0.4%, a value considerably lower than those previously reported. In addition to Hb Alc, 5.2 +/- 0.5% of the remaining hemoglobin (Hb Ao) was glycosylated. In diabetics, glycosylated Ao was increased in parallel with Hb Alc. After reduction with [3H]borohydride and acid hydrolysis, glycosylated amino acids were first purified on Affi-Gel boronate affinity resin and then analyzed by ion exchange chromatography. The glucose adducts on Hb Ao were distributed as follows: alpha-chain N-terminal valine, 14%; alpha-chain lysines, 40%; beta-chain lysines, 46%. This study has revealed several pitfalls in the analysis of nonenzymatically glycosylated proteins. Peaks isolated by ion exchange chromatography or electrophoresis are likely to be contaminated by nonglycosylated proteins. Furthermore, both the thiobarbituric acid test and [3H]borohydride reduction show variable reactivity depending upon the site of the ketoamine-linked glucose.
R L Garlick, J S Mazer, P J Higgins, H F Bunn
Bradykinin (BK) increases short-circuit current (Isc) when added to the serosal side of rabbit or guinea pig ileum or rabbit colon. Significant effects on Isc are seen at concentrations as low as 10(-10) M. Anion substitution experiments and unidirectional 36Cl flux measurements indicate that this effect of BK on Isc is due to Cl secretion. The effect of BK on Isc can be partially blocked (60-70% inhibition) by cyclooxygenase inhibitors (indomethacin and/or naproxen) and completely blocked by the phospholipase inhibitor, mepacrine. The combined cyclooxygenase/lipoxygenase inhibitors BW 755 and eicosa-5,8,11,14-tetraynoic acid (ETYA) also completely block the effect of BK on Isc but the slow-reacting substance of anaphylaxis (SRS-A) antagonist FPL 55712 has no effect. None of the above inhibitors diminish the effect on Isc of other exogenously added secretory stimuli such as vasoactive intestinal peptide (VIP), theophylline, or prostaglandin E2 (PGE2). Prior desensitization of rabbit ileum to PGE2 blocks the effect on Isc of BK but not those of VIP or theophylline. Conversely, prior desensitization of rabbit ileum to BK greatly reduces the effect of PGE2 on Isc. BK also stimulates the synthesis of PGE2 in rabbit ileal and colonic mucosa and this effect can be blocked by prior addition of either indomethacin or mepacrine. These effects of BK are similar to those of exogenously added arachidonic acid (AA). AA also stimulates Cl secretion and increases PGE2 synthesis and its effect on Isc can be inhibited by prior desensitization to PGE2 or by prior addition of indomethacin. The above results indicate that BK stimulates active Cl secretion in both small and large intestine and suggest that this effect is due to the intracellular release of AA. Although the prostaglandins appear to be the major products of AA metabolism contributing to the secretory response, lipoxygenase products may also play a role.
M W Musch, J F Kachur, R J Miller, M Field, J S Stoff
This study examines the ontogenesis of somatomedin and insulin receptors in man. Particulate plasma membranes were prepared by ultracentrifugation from various tissues removed from fetuses after abortion and classified as less than 17, 17-25, and greater than 25 cm in length. The binding of iodinated insulinlike growth factors 1 (IGF-1) and 2 (IGF-2), somatomedin A (SMA), multiplication-stimulating activity (MSA), and insulin was examined at the different ages. In the liver, cross-reaction studies revealed separate insulin and IGF-2 receptors. The Scatchard plots of insulin binding to liver membranes were curvilinear and showed an increase in the concentration of insulin receptors with advancing age. A single IGF-2 receptor was found on liver and no alteration was observed during development. The brain contained a lower concentration of insulin receptors. A change in the brain receptors for somatomedins occurred during development. Early in gestation, a high concentration of a low-affinity IGF-1 receptor was found. After approximately the 17th wk of gestation a higher affinity IGF-1 receptor appeared, which then increased in concentration. Cross-reaction studies also revealed changes in the specificity of these receptors during development. In the youngest fetal group IGF-2 was preferentially bound. Around midgestation a separate IGF-1 receptor, indicated by the preferential displacement of iodinated IGF-1 by IGF-1, appeared. In contrast, iodinated IGF-2 bound to a receptor where IGF-1 and IGF-2 were equipotent.
V R Sara, K Hall, M Misaki, L Fryklund, N Christensen, L Wetterberg
Studies were performed in anesthetized opossums to investigate the influence of successive vagal stimuli on esophageal contractions. Mechanical activity was recorded manometrically 5 cm above the lower esophageal sphincter. Contractions in the esophagus were evoked by electrical stimuli of 2.5 mA, and 1-ms pulse duration applied to the cervical vagi, at various train durations and frequencies. Paired or multiple stimuli of 1-s train length were also tested at different interstimulus intervals (ISI). Paired stimuli at an ISI of less than or equal to 3 s and at a frequency of less than or equal to 10 Hz showed refractoriness, i.e., the contractions to the first stimulus inhibited the contraction to the second stimulus. A frequency of 50 Hz showed initial inhibition, i.e., the second stimulus inhibited the contraction to the first stimulus. Repetitive stimuli applied at a rate of 8/min (ISI 6.5 s) evoked contractions to each stimulus. At 15/min, every second or third contraction was inhibited. With stimuli applied at 30/min, contractions occurred only in response to the first and/or the last stimulus; depending upon the frequency of vagal stimulation. The intervening stimuli did not evoke any contractions. A long train stimulus produced an initial, a terminal, or both contractions depending on the stimulation value. These studies show that (a) vagal efferent stimulation causes initial inhibition and refractoriness in the esophageal smooth muscle; (b) the degree of initial inhibition increases with increasing frequency of stimulation; (c) the occurrence of contractions only at the onset and the end of a long train stimulus may be due to the influence of initial inhibition and refractoriness.
J S Gidda, R K Goyal
It has been postulated that host immune defects are responsible for the development and persistence of the hepatitis B surface antigen (HBsAg) carrier state. The nature of these defects is unknown, but the absence of a readily detectable antibody response to HBsAg (anti-HBs) may be important. The synthesis of both anti-HBs and antibody to hepatitis B core antigen (anti-HBc) in cultures containing peripheral blood mononuclear cells from chronic HBsAg carriers and from control (antibody-positive) patients was measured in the presence of pokeweed mitogen. Similar amounts of polyclonal IgG and IgM were synthesized by cultures containing lymphocytes from chronic carriers and controls. Anti-HBc was detectable in lymphocyte supernatants from 2 of 20 controls and from 21 of 29 carriers. The presence of anti-HBc synthesis in vitro correlated with high serum titers of anti-HBc. In contrast, anti-HBs was detected in lymphocyte supernatants from 6 of 20 controls (predominantly in those who had high serum titers of anti-HBs) but in none of the supernatants from 29 HBsAg carriers. In order to identify the mechanisms for the lack of detectable anti-HBs synthesis by chronic HBsAg carrier lymphocytes, co-culture experiments were performed using T and B lymphocyte fractions that had been purified by affinity chromatography. B lymphocytes from carriers co-cultured with allogeneic irradiated ("helper") T lymphocytes from controls synthesized normal amounts of IgG, IgM, and anti-HBc but still did not synthesize detectable amounts of anti-HBs. In the converse experiments, B lymphocytes from controls were co-cultured with irradiated T lymphocytes from carriers. The T lymphocytes from 16 of 24 carriers augmented anti-HBs production by control B cells normally, the remaining eight did not. Finally, mixtures of control B cells and control irradiated T lymphocytes were co-cultured with T lymphocytes from chronic HBsAg carriers. 5 of 12 carriers demonstrated active suppression of anti-HBs production, and in three this suppression was specific, as IgG and IgM production remained normal. We conclude that chronic HBsAg carriers have a specific B lymphocyte defect in anti-HBs production. In addition, defects in the function of regulatory T lymphocytes may contribute to the absence of anti-HBs synthesis in some HBsAg carriers.
G M Dusheiko, J H Hoofnagle, W G Cooksley, S P James, E A Jones
This paper is a cross-sectional study of spontaneous benign prostatic hyperplasia (BPH) in a single canine species. The effects of aging and hormonal changes on the growth, histology, and glandular secretory function of the canine prostate were studied in 42 male beagles ranging in age from 8 mo to 9 yr. The beagle prostate enlarges for at least 6 yr, whether normal or hyperplastic. In contrast, prostatic secretory function, determined by ejaculate volume and total ejaculate protein, declines markedly after 4 yr of age. These reciprocal growth and functional changes in the prostate are closely associated with a progressive increase in the incidence of BPH, which is already apparent in some dogs by age two. With age there is a modest decrease in serum androgen levels with no apparent change in serum 17 beta-estradiol levels. This suggests that the growth and functional changes that are associated with the development of BPH and are initiated very early in life reflect an altered sensitivity of the prostate to serum androgens or a response to the relative decrease in the serum androgen to estrogen ratio.
C B Brendler, S J Berry, L L Ewing, A R McCullough, R C Cochran, J D Strandberg, B R Zirkin, D S Coffey, L G Wheaton, M L Hiler, M J Bordy, G D Niswender, W W Scott, P C Walsh
Total and filaria-specific immunoglobulin E (IgE) levels were studied in cord blood from infants born in Madras, India, where filariasis and intestinal helminth infections are highly endemic. Increased total IgE levels were observed in 82% of 57 cord sera tested (geometric mean 12.6 ng/ml; range 1-1,900 ng/ml). 33 of these sera also contained IgE antibodies specific for filarial antigens as determined by solid-phase radioimmunoassay. Comparison of ratios of filaria-specific IgE to total IgE in paired maternal and cord sera suggested that cord blood IgE was derived from the fetus in most cases and not from transplacental antibody transfer. Our results suggest that prenatal allergic sensitization to helminth parasites occurs in the tropics. Such sensitization may contribute to the heterogeneity in host immune response and disease expression noted in filariasis and other helminth infections.
G J Weil, R Hussain, V Kumaraswami, S P Tripathy, K S Phillips, E A Ottesen
Vitamin K3 inhibits the conversion of benzo(a)pyrene to its more polar metabolites in an in vitro rat liver microsomal system. Vitamin K3 also inhibits benzo(a)pyrene metabolism in rat liver fragments and reduces its mutagenicity in the Ames test. Higher concentrations of vitamin K3 are required to comparably reduce benzo(a)pyrene metabolism when the microsomal system has been induced with 3-methylcholanthrene. High pressure liquid chromatography analysis of the products of benzo(a)pyrene metabolism shows a uniform reduction of all the metabolic products. When tumors were induced in ICR/Ha female mice by the intraperitoneal injection of benzo(a)pyrene, those mice given vitamin K3 before or both before and after benzo(a)pyrene had a slower rate of tumor appearance and tumor death rate as compared with those receiving benzo(a)pyrene alone. However, vitamin K1 increased the rate of tumor death while vitamin K deprivation and warfarin decreased the rate of tumor appearance and death in benzo(a)pyrene-injected mice. These studies indicate that vitamin K3 is an inhibitor of aryl hydrocarbon hydroxylase and reduces the carcinogenic and mutagenic metabolites in vitro, and inhibits benzo(a)pyrene tumorigenesis in vivo. That vitamin K1 enhances the benzo(a)pyrene effect while warfarin and vitamin K deficiency inhibit benzo(a)pyrene tumorigenesis indicates that vitamin K1, vitamin K deprivation, or possibly blockade of its metabolic cycle also modulates benzo(a)pyrene metabolism in vivo but by a mechanism or at a site different from the vitamin K3 effect. The vitamin K series should be considered as capable of serving a regulatory function in the metabolism of benzo(a)pyrene and possibly other compounds metabolized through the mixed function oxidase system.
L G Israels, G A Walls, D J Ollmann, E Friesen, E D Israels
Maintenance of chronic metabolic alkalosis might occur by a reduction in glomerular filtration rate (GFR) without increased bicarbonate reabsorption or, alternatively, by augmentation of bicarbonate reabsorption with a normal GFR. To differentiate these possibilities, free-flow micropuncture was performed in alkalotic Munich-Wistar rats with a glomerular ultrafiltrate total CO2 concentration of 46.5 +/- 0.9 mM (vs. 27.7 +/- 0.9 mM in controls). Alkalotic animals had a markedly reduced single nephron GFR compared with controls (27.4 +/- 1.5 vs. 51.6 +/- 1.6 nl/min) and consequently unchanged filtered load of bicarbonate. Absolute proximal bicarbonate reabsorption in alkalotic animals was similar to controls (981 +/- 49 vs. 1,081 +/- 57 pmol/min), despite a higher luminal bicarbonate concentration, contracted extracellular volume, and potassium depletion. When single nephron GFR during alkalosis was increased toward normal by isohydric volume expansion or in another group by isotonic bicarbonate loading, absolute proximal bicarbonate reabsorption was not substantially augmented and bicarbonaturia developed. To confirm that a fall in GFR occurs during metabolic alkalosis, additional clearance studies were performed. Awake rats were studied before and after induction of metabolic alkalosis associated with varying amounts of potassium and chloride depletion. In all cases, the rise in blood bicarbonate concentration was inversely proportional to a reduction in GFR; filtered bicarbonate load remained normal. In conclusion, a reduction in GFR is proposed as being critical for maintaining chronic metabolic alkalosis in the rat. Constancy of the filtered bicarbonate load allows normal rates of renal bicarbonate reabsorption to maintain the alkalotic state.
M G Cogan, F Y Liu
To study the effect of insulin on lipoprotein synthesis and secretion by the liver, apoprotein and lipid levels were measured in primary rat liver cell cultures grown on fibronectin-coated dishes. Triglycerides, phospholipids, apoprotein (apo) B, apo-E, and apo-C-III3 all accumulated in culture media linearly for periods up to 20 h. During incubations, cellular triglyceride contents increased slightly, while cellular apoprotein and phospholipid contents remained constant. In the absence of insulin, rates of accumulation in media of triglycerides, apo-B, apo-C-III3, and apo-E were 2.5 +/- 0.3 micrograms/mg and 33 +/- 5, 24 +/- 3, and 162 +/- 32 ng/mg cell protein per h, respectively. On gel permeation chromatography and density gradient ultracentrifugation, the majority of apoproteins in media were found to be associated with very low density lipoproteins (VLDL) and very little eluted or sedimented with albumin. Incubations in the presence of 50-800 microU/ml of insulin resulted in dose-dependent decreases of triglyceride, phospholipid, apo-B, and apo-E accumulation in the media, paralleled by increases in the cellular contents of these lipoprotein components. The inhibitory effects of insulin on secretion were reversible. Levels of apo-C-III3 and albumin were not affected by insulin. In addition to decreasing secretory rates, the proportion of apo-B, apo-E, and apo-C-III3 associated with VLDL also decreased after the addition of insulin. Concomitantly, the proportion of apo-B eluting with LDL and apo-C-III3, and apo-E eluting near albumin increased. Control experiments, in which exogenous 125I-VLDL or endogenously labeled [14C]VLDL were added to cultures, revealed that the insulin-induced differences in VLDL accumulation and the lipid association of media apoproteins were not due to differences in the processing of VLDL by cells cultured in the presence or absence of insulin. Therefore, it appears that insulin may inhibit the secretion of VLDL perhaps by reducing the intracellular association of lipids and apoproteins.
W Patsch, S Franz, G Schonfeld
Acute iron loading of rats, by intraperitoneal administration of iron-dextran (500 mg Fe/kg body wt 18-20 h before killing) decreased by 30% the rate of conversion of 5-amino-[14C]levulinate ([14C]ALA) into heme as measured with a recently described procedure for liver homogenates (1981. Biochem. J. 198: 595-604). The decrease in conversion of labeled ALA into heme caused by iron loading was shown to be due to a 70-80% decrease in activity of ALA dehydrase. The decrease in activity of ALA dehydrase caused by iron loading was not associated with a decrease in hepatic concentrations of GSH, nor could it be reversed by addition of dithiothreitol, Zn2+ or chelators of Fe2+ and Fe3+. Addition of FeSO4, ferric citrate, or ferritin to homogenates of control liver had no effect of activity of ALA dehydrase. The decrease in activity of ALA dehydrase, caused by iron-dextran, was mirrored by a reciprocal increase in ALA synthase. Iron-dextran potentiated the induction of ALA synthase by allylisopropylacetamide. However, this potentiation could be dissociated from the decrease in ALA dehydrase caused by iron loading.
H L Bonkowsky, J F Healey, P R Sinclair, J F Sinclair, S I Shedlofsky, G H Elder
Nitrous oxide, by inactivating cobalamin in vivo, produces a suitable animal model for cobalamin 'deficiency.' The synthesis of folate polyglutamate with tetrahydrofolate as substrate is severely impaired in the N2O-treated rat, but is normal with formyltetrahydrofolate as substrate. Methionine restores the capacity of the N2O-treated rat to utilize tetrahydrofolate the minimum effective dose being 16 mumol. S-Adenosylmethionine was somewhat less effective than methionine but 5'methylthioadenosine, a product of S-adenosylmethionine metabolism, was significantly more effective than methionine in correcting the defect in folate polyglutamate synthesis. 5'Methylthioadenosine is metabolised to yield formate. It is suggested that these compounds have their effect in correcting folate polyglutamate synthesis by supplying formate for the formylation of tetrahydrofolate. Formyltetrahydrofolate, at least in the cobalamin-inactivated animal, is the required substrate for folate polyglutamate synthesis. Cobalamin is concerned with the maintenance of normal levels of methionine and this in turn is a major source of formate through S-adenosylmethionine and 5'methylthioadenosine.
J Perry, I Chanarin, R Deacon, M Lumb
Metabolism of arachidonic acid (AA) via the cyclooxygenase pathway reduces glucose-stimulated insulin release. However, metabolism of AA by the lipoxygenase pathway and the consequent effects on insulin secretion have not been simultaneously assessed in the endocrine islet. Both dispersed endocrine cell-enriched pancreatic cells of the neonatal rat, as well as intact islets of the adult rat, metabolized [3H]AA not only to cyclooxygenase products (prostaglandins E2, F2α, and prostacyclin) but also to the lipoxygenase product 12-hydroxyeicosatetraenoic acid (12-HETE). 12-HETE was identified by coelution with authentic tritiated or unlabeled 12-HETE using four high performance liquid chromatographic systems under eight mobile-phase conditions and its identity was confirmed by gas chromatography/mass spectrometry using selected ion monitoring. The predominant effect of exogenous AA (5 μg/ml) was to stimulate insulin release from pancreatic cells grown in monolayer. This effect was concentration- and time-dependent, and reversible. The effect of AA upon insulin release was potentiated by a cyclooxygenase inhibitor (indomethacin) and was prevented by either of two lipoxygenase inhibitors (5,8,11,14-eicosatetraynoic acid [ETYA] and BW755c). In addition, glucose, as well as two structurally dissimilar agents (the calcium ionophore A23187 and bradykinin), which activate phospholipase(s) and thereby release endogenous AA in several cell systems, also stimulated insulin secretion. The effects of glucose, glucagon, bradykinin and high concentrations of A23187 (5 μg/ml) to augment insulin release were blocked or considerably reduced by lipoxygenase inhibitors. However, a lower concentration of the ionophore (0.25 μg/ml), which did not appear to activate phospholipase, was resistant to blockade. Exogenous 12-HETE (up to 2,000 ng/ml) did not alter glucose-induced insulin release. However, the labile intermediate 12-hydroperoxy-ETE increased insulin release. Furthermore, diethylmaleate (which binds intracellular glutathione and thereby impedes conversion of the lipoxygenase intermediates hydroperoxy-ETE and leukotriene A4 to HETE and leukotriene C4, respectively) potentiated the effect of glucose and of exogenous AA. Finally, 5,6-epoxy, 8,11,14-eicosatrienoic acid (a relatively stable epoxide analogue of leukotriene A4) as well as two other epoxy-analogues, potentiated glucose-induced insulin release. We conclude that dual pathways of AA metabolism exist in islet endocrine cells and have opposing regulatory effects on the beta cell—an inhibitory cyclooxygenase cascade and a stimulatory lipoxygenase cascade. Labile products of the latter pathway may play a pivotal role in stimulus-secretion coupling in the islet.
Stewart Metz, Michael VanRollins, Robert Strife, Wilfred Fujimoto, R. Paul Robertson
To investigate serum requirements for optimal erythropoiesis in vitro, we studied the response of erythroid progenitor cell proliferation in culture to platelet-derived growth factor (PDGF). Human bone marrow cells cultured with platelet-poor plasma-derived serum (PDS) form fewer erythroid colonies than do cells cultured with human whole blood serum or fetal calf serum (P less than 0.05). Treatment of washed platelets with thrombin releases a low molecular weight (less than 100,000) factor that enhances colony growth. This secreted factor appears to be PDGF, based upon the ability of partially purified and electrophoretically pure PDGF to restore colony-forming capacity of PDS-containing cultures to 70-96% of the level found in control cultures with whole blood serum or fetal calf serum. Enhancement of colony growth by PDGF was noted only in marrow cultures supplemented with erythropoietin and PDS. Presence of bioactive erythropoietin in PDGF preparations was excluded by assay in hypertransfused, polycythemic mice, and in fasted rats. Although PDGF stimulates erythroid burst formation in marrow cultures containing optimal concentrations of burst-promoting activity (BPA), it does not influence proliferation of circulating erythroid bursts, regardless of BPA concentration added to culture. We conclude that PDGF is a serum determinant of optimal erythroid progenitor cell proliferation in marrow culture. The activity of PDGF is distinct from that of the apparent erythroid specific growth factors erythropoietin and BPA.
N Dainiak, G Davies, M Kalmanti, J Lawler, V Kulkarni
Since unstable hemoglobins have been considered a source of reactive oxygen radicals, and oxidative membrane damage a prehemolytic event, we examined the erythrocyte membranes of six patients (three splenectomized) with hemoglobin Köln disease. In the hydrogen peroxide stress test, the patients' erythrocytes generated more than twice the malonyldialdehyde (a lipid peroxidative product) than control erythrocytes. Fluorescence spectra of lipid extracts of the patients' erythrocytes showed an excitation maximum at 400 nm and an emission maximum of 460 nm, characteristic of malonyldialdehyde lipid adducts. Two types of membrane polypeptide aggregates were found in the erythrocytes of the splenectomized patients. The first, which were dissociable by treatment with mercaptoethanol, contained disulfide-linked spectrin, band 3 and globin. The second, not dissociable by mercaptoethanol, had an amino acid composition similar to that of erythrocyte membranes and spectrin (unlike globin) and like that of aggregates produced by the action of malonyldialdehyde on normal erythrocyte membranes. Atomic absorption spectroscopy of hemoglobin Köln erythrocytes showed no increase in calcium content implying that these cross-links were not due to calcium-stimulated transglutaminase. Using a micropipette technique, we demonstrated that erythrocytes containing membrane aggregates from splenectomized patients were less deformable while aggregate-free erythrocytes from non-splenectomized patients had normal deformability. We conclude that the erythrocyte membranes in hemoglobin Köln disease show evidence of lipid peroxidation with production of malonyldialdehyde, and that the nondissociable membrane aggregates formed in this disease are likely cross-linked by malonyldialdehyde. Because the erythrocytes containing membrane aggregates from splenectomized patients with unstable hemoglobin disease show decreased membrane deformability, we hypothesize that this abnormality results in premature erythrocyte destruction in vivo.
T P Flynn, D W Allen, G J Johnson, J G White
Although pyridoxal phosphate is known to inhibit gelation of purified hemoglobin S, antisickling activity has never been demonstrated for intact erythrocytes. We incubated washed erythrocytes at 37 degrees C either in buffer alone, or with added pyridoxal phosphate or pyridoxal, washed these cells, suspended them in untreated buffer, and compared the percent modified hemoglobin, the oxygen affinity, and the extent of sickling under hypoxia. Pyridoxal phosphate modified intracellular hemoglobin more slowly than pyridoxal. Pyridoxal phosphate lowered the oxygen affinity of normal cells, but had no effect on oxygen binding by sickle cells. Pyridoxal increased the oxygen affinity of normal and sickle erythrocytes equally. Pyridoxal phosphate significantly inhibited sickling of sickle or sickle trait erythrocytes (P less than 0.001). Inhibition of sickling by pyridoxal phosphate was largely independent of oxygen binding; whereas inhibition of sickling by pyridoxal was almost entirely dependent on increased oxygen binding. Although pyridoxal phosphate and pyridoxal both inhibit sickling by modification of hemoglobin S, they differ in the kinetics of whole cell modification, the effect on oxygen affinity of intact cells, and the mechanism of action of the antisickling activity.
J A Kark, P G Tarassoff, R Bongiovanni
Spontaneous cytotoxicity mediated by natural killer (NK) cells is impaired in several human diseases including systemic lupus erythematosus (SLE). The precise mechanism(s) by which NK activity is suppressed in patients with SLE is generally unknown. The present study was designed to focus on cellular defects per se in NK cells from patients with SLE. It was observed that the usual enhancing effect of interferon (IF) and IF inducers was markedly impaired in SLE patients. Of 24 SLE patients studied, 17 had significantly decreased NK activity relative to controls. NK activity had a significant negative correlation with clinical activity score (r = -0.56, P less than 0.005) but was not correlated with corticosteroid dose, antinuclear antibody titers, total hemolytic complement (CH50), or sedimentation rate. Furthermore, significant depressions in NK activity correlated with variations in disease activity in six patients followed serially. Depressed NK function could not be reversed by prolonged in vitro incubation at 37 degrees C or with protease treatment. Furthermore, depressed NK activity was not altered by removal of glass adherent cells nor was a suppression of NK activity in normal controls seen by the addition of SLE peripheral mononuclear cells. No reversal of depressed activity to normal levels was seen by the addition of indomethacin nor did the supernatants from SLE cell cultures cause a suppression of normal NK function. NK activity in SLE patients did not respond normally to IF inducers (poly-I:C and concanavalin A) even if the SLE patients had normal NK function. The response of SLE cells to exogenous IF was also impaired. The number of effector-target conjugates was quantitated with several target cells (K562, Yac-1, Fravel) in SLE patients and controls. A significant correlation between the proportion of glass nonadherent mononuclear cells that formed effector-target conjugates with these various targets and the magnitude of NK lysis was observed. However, SLE and normal subjects had equal numbers of effector-target conjugates independent of NK function. Release of a soluble cytotoxic factor was induced with concanavalin A, and was markedly impaired in SLE patients relative to normal controls. Thus, impaired NK cell function in SLE does not appear to be related to cell-mediated suppressive mechanisms or to the deletion of effector cells; rather, the decreased NK activity may be related to an impaired release of a soluble cytotoxic factor.
W L Sibbitt Jr, P M Mathews, A D Bankhurst
Acquired abnormalities of connective tissue metabolism in inflammatory diseases often persist when lesional tissue is maintained in in vitro culture. Although connective tissue cells are exposed to inflammatory cell-derived mediators in vivo and such mediators have been shown to alter connective tissue cell behavior, it is unclear whether the persistence of metabolic defects in vitro could result from remote in vivo exposure to these mediators. An in vitro model was used to test whether transient exposure of normal fibroblasts to inflammatory mediators could lead to metabolic alterations that persist during in vitro culture. Short-term exposure of human foreskin fibroblasts in vitro to supernates of mitogen-activated peripheral blood mononuclear cells led to persistent abnormalities of prostaglandin E2 (PGE2) metabolism. Fibroblasts previously exposed to mononuclear cell products synthesized more than twice as much PGE2 when stimulated compared with similarly stimulated but previously unexposed control fibroblasts of the same strain. The enhanced PGE2 synthesis persisted for as long as 20 wk and 19 cell generations after the original exposure to mononuclear cell products. Exposure of fibroblast populations to mononuclear cell products may, thus, lead to metabolite alterations that are still evident after multiple cell generations.
J H Korn
The antiarrhythmic action of lidocaine has been attributed solely to its direct electrophysiological effects on the heart. However, lidocaine is particularly effective in treating ventricular arrhythmias associated with increased sympathetic activity, e.g., in myocardial infarction and digitalis toxicity. We tested the hypothesis that lidocaine administered intravenously depressed cardiac sympathetic nerve activity (CSNA). We measured CSNA in six dogs in control state and after lidocaine in doses of 0.625, 1.25, and 2.5 mg/kg i.v. over 2 min. These doses of lidocaine produced graded decreases of CSNA of -8 +/- 2, -18 +/- 1, and -41 +/- 5%, respectively (P less than 0.05, mean +/- SE). In six additional experiments the bolus of lidocaine was followed by an infusion for 20 min (1.25 mg/kg followed by 100 micrograms/kg per min and 2.5 mg/kg followed by 200 micrograms/kg per min). Infusion of lidocaine maintained depression of CSNA at a level that was 23 +/- 3 and 35 +/- 5% less than control (P less than 0.05), respectively, at plasma lidocaine levels of 5.2 +/- 0.6 and 7.5 +/- 1.4 micrograms/ml, respectively. CSNA returned to control during recovery periods. CSNA did not decrease with the passage of time or administration of vehicle. In five dogs with vagi intact, carotid sinuses isolated and held at a pressure of 100 mmHg, and aortic baroreceptors denervated, administration of lidocaine (2.5 mg/kg followed by 200 micrograms/kg per min) decreased renal nerve activity to 71 +/- 8% of control. Increases in left ventricular systolic pressure and maximum derivative of pressure with respect to time (dP/dtmax) resulting from electrical stimulation of preganglionic sympathetic nerves were not significantly altered by lidocaine, but were markedly attenuated by hexamethonium, a ganglionic blocker. In conclusion, lidocaine administered intravenously produces dose-dependent and sustained decreases in cardiac sympathetic nerve activity. These decreases can occur with therapeutic plasma levels. We speculate that this effect is due to central nervous system effects of the drug and that this effect may contribute to the antiarrhythmic actions of lidocaine.
B D Miller, M D Thames, A L Mark
To determine whether intravenous infusion of individual amino acids stimulated gastric acid secretion in man, graded doses of phenylalanine, tryptophan, glycine, alanine, histidine, and NaCl control were infused on separate days in nine healthy subjects. Intravenous infusion of phenylalanine and tryptophan significantly stimulated gastric acid secretion to 50 and 52%, respectively, of the acid secretory response to intragastric peptone. Intravenous alanine and histidine were without effect, whereas glycine produced a slight response. Serum gastrin concentrations did not significantly change during intravenous amino acid infusion, except in response to 0.1 M phenylalanine. However, the increase in serum gastrin occurred 2 h after acid secretion had significantly increased in response to the 0.025 M phenylalanine infusion. Plasma amino acid concentrations were measured during intravenous amino acid infusion and in response to a steak meal in five of the subjects. At a time when acid secretion was significantly increased during intravenous infusion of phenylalanine and tryptophan, plasma amino acids were similar to, or less than, that observed after the steak meal, suggesting that circulating levels of these three amino acids have a physiologic effect on gastric secretion in man. Intravenous infusion of a combination of graded doses of phenylalanine plus a continuous infusion of 0.01 M tryptophan shifted the dose-response curve to the left and resulted in a significantly greater response than to either amino acid alone. In five subjects with parietal cell vagotomy, intravenous phenylalanine and tryptophan stimulated acid secretion, whereas histidine was without effect, similar to normal subjects. These studies indicate that intravenous infusion of small amounts of phenylalanine (0.025 M, 3.1 mmol/h) and tryptophan (0.01 M, 1.25 mmol/h) stimulated gastric acid secretion at plasma concentrations similar to those observed after a steak meal, suggesting a physiologic role for circulating levels of these amino acids on gastric acid secretion. Because acid secretion increased at a time when serum gastrin was unchanged and since there was no correlation between changes in serum gastrin and acid secretion, the responses to phenylalanine and tryptophan are probably mediated by a nongastrin-related mechanism(s). Since both phenylalanine and tryptophan stimulated secretion in vagotomized subjects, the response is vagally independent. These observations suggest that circulating levels of these two amino acids have either a direct or indirect effect on or near the human parietal cell.
K E McArthur, J I Isenberg, D L Hogan, S J Dreier
The sympathoadrenal responses to acute and chronic hypoxic exposure at 10.5 and 7.5% oxygen were determined in the rat. Cardiac norepinephrine (NE) turnover was used to assess sympathetic nervous system (SNS) activity, and urinary excretion of epinephrine (E) was measured as an index of adrenal medullary activity. The responses of the adrenal medulla and SNS were distinct and dependent upon the degree and duration of hypoxic exposure. Chronic hypoxia at 10.5% oxygen increased cardiac NE turnover by 130% after 3, 7, and 14 d of hypoxic exposure. Urinary excretion of NE was similarly increased over this time interval, while urinary E excretion was marginally elevated. In contrast, acute exposure to moderate hypoxia at 10.5% oxygen was not associated with an increase in SNS activity; in fact, decreased SNS activity was suggested by diminished cardiac NE turnover and urinary NE excretion over the first 12 h of hypoxic exposure, and by a rebound increase in NE turnover after reexposure to normal oxygen tension. Adrenal medullary activity, on the other hand, increased substantially during acute exposure to moderate hypoxia (2-fold increase in urinary E excretion) and severe hypoxia (greater than 10-fold). In distinction to the lack of effect of acute hypoxic exposure (10.5% oxygen), the SNS was markedly stimulated during the first day of hypoxia exposure at 7.5% oxygen, an increase that was sustained throughout at least 7 d at 7.5% oxygen. These results demonstrate that chronic exposure to moderate and severe hypoxia increases the activity of the SNS and adrenal medulla, the effect being greater in severe hypoxic exposure. The response to acute hypoxic exposure is more complicated; during the first 12 h of exposure at 10.5% oxygen, the SNS is not stimulated and appears to be restrained, while adrenal medullary activity is enhanced. Acute exposure to a more severe degree of hypoxia (7.5% oxygen), however, is associated with stimulation of both the SNS and adrenal medulla.
T S Johnson, J B Young, L Landsberg
A specific stimulation of tubulin tyrosinolation in human polymorphonuclear leukocytes (PMN) is induced by the synthetic peptide chemoattractant, N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe), and this stimulation of tyrosinolation in PMN is completely inhibited in the presence of various reducing agents. Further studies to characterize the mechanism of stimulation of tyrosinolation in PMN have revealed that conditions that inhibited the respiratory burst in stimulated PMN, e.g., an anaerobic atmosphere, or addition of antioxidants such as cysteamine, azide, or 2,3-dihydroxybenzoic acid, also inhibited the peptide-induced stimulation of tyrosinolation in these cells. Moreover, the sulfhydryl reagent, N-ethylmaleimide, depressed tyrosinolation in resting PMN and completely inhibited the fMet-Leu-Phe-induced stimulation. In contrast, addition of diamide, which preferentially oxidizes cellular glutathione, significantly stimulated tyrosinolation both in resting and fMet-Leu-Phe-stimulated PMN. Furthermore, resting levels of tyrosinolation in seven patients with chronic granulomatous disease (CGD), whose oxidative metabolism is severely depressed, were 35-45% lower (P less than 0.01). Most strikingly, PMN from CGD patients failed to respond to fMet-Leu-Phe or the Ca2+-ionophore A23187, which also induced stimulation of tyrosinolation in normal resting PMN. Methylene blue normalized the depressed tyrosinolation in resting CGD PMN, although it did not increase tyrosinolation in stimulated PMN. These results are consistent with the idea that the characteristic activation of the oxidative metabolism and the associated changes in the redox state in stimulated PMN are coupled to the induction of stimulation of tubulin tyrosinolation in these cells.
J Nath, J I Gallin
Administration of the antioxidant vitamin E to rats, prior to administration of either streptozotocin or alloxan, provided protection against the diabetogenic effect of both these agents. This was demonstrated by their response to a glucose load, their pancreatic insulin content and light microscopy findings. In addition, rats whose antioxidant state was depleted, by being maintained on a vitamin E and selenium-deficient diet, demonstrated increased diabetogenic susceptibility to normally nondiabetogenic doses of streptozotocin. These findings provide indirect support for the suggestion that the chemical agents streptozotocin and alloxan may exert their diabetogenic effect by acting as oxidants or free radical producers.
A E Slonim, M L Surber, D L Page, R A Sharp, I M Burr
An organic compound that inhibits drug binding in uremia has been isolated from the sera of chronic renal failure patients, and its chemical structure has been determined. Addition of the compound to normal human sera in vitro resulted in drug binding defects similar to those seen in uremia. The purification of this substance was accomplished by n-butyl chloride extraction of acidified (pH 3.0) uremic sera followed by column chromatography, thin-layer chromatography, and paper electrophoresis. From analytical studies including ultraviolet and fluorescence spectroscopy, gas chromatography, chemical ionization and electron impact mass spectrometry, and proton nuclear magnetic resonance spectroscopy, the chemical structure of the uremic binding inhibitor was deduced to be 2-hydroxybenzoylglycine. This confirms the hypothesis that the drug binding defect in uremia is due to the accumulation of endogenous metabolic products rather than an intrinsic structural defect in albumin.
D M Lichtenwalner, B Suh, M R Lichtenwalner
Myeloperoxidase (MPO), a heme enzyme present in the azurophilic granules of human polymorphonuclear neutrophils (PMN), is important in the oxygen-dependent microbicidal activity of PMN. MPO deficiency, defined as the lack of PMN peroxidative activity, is a common genetic defect of human PMN. The purpose of our study was to characterize the structural basis for this loss of enzymatic activity, using protein biochemical and immunochemical techniques to examine PMN from three subjects with partial MPO deficiency and from five subjects with complete MPO deficiency.
William M. Nauseef, Richard K. Root, Harry L. Malech
Diamine oxidase (DAO; EC 220.127.116.11) is an enzyme found in high activity in the mature cells of the upper villus of rat small intestinal mucosa and in very much lower activity in all other tissues in the nonpregnant rat. This study was designed to determine whether a provocative test for increasing the level of plasma DAO activity by heparin administration could be used to monitor the extent and severity of acute, severe, small intestinal mucosal injury. In adult rats, small intestinal loops of varying lengths were perfused with 2,100 mosM sodium sulfate solution for 60 min to produce selective damage to villus epithelium. Plasma postheparin DAO (PHD) activity (180 min after 400 U/kg i.p. heparin) was measured 7 h after initiation of perfusion. With increasing length of intestinal mucosal injury, there was a progressive decrease in both basal and plasma PHD activity. The decrease in plasma PHD activity closely reflected the length of intestinal mucosa injured (n = 128, r = 0.86, P less than 0.001), and it was much more sensitive (threshold limit of detection = 13% of total length, range = 67 U/ml for 100% length of injury) than unstimulated basal levels of plasma DAO (threshold = 40%, range = 2.1 U/ml). Our previous data have suggested that DAO is unique among intestinal mucosal enzymes in that circulating levels can serve as a marker of mucosal injury; this study illustrates that the addition of a low-dose heparin administration enhances the use of DAO even further as a sensitive, quantitative, circulating marker for monitoring the extent of small intestinal mucosal injury in the rat.
G D Luk, T M Bayless, S B Baylin
Because it is unclear whether age-related bone loss results from increased bone resorption, decreased bone formation or both, we measured the serum level of bone Gla-protein (BGP), a specific marker for bone turnover, in 174 women, ages 30 to 94 yr. Serum BGP increased linearly with aging (r = 0.44, P less than 0.001) from 4.4 +/- 0.4 (mean +/- SE) in the 4th decade to 8.9 +/- 0.9 ng/ml in the 10th decade. This increase correlated inversely (P less than 0.001) with concomitant decreases in bone mineral density at the lumbar spine, midradius, and distal radius. Using partial correlation coefficients, serum BGP still correlated positively with age (r = 0.31, P less than 0.001) after creatinine clearance was fixed but not with creatinine clearance (r = -0.04, NS) when age was fixed. Urinary hydroxyproline (r = 0.29, P less than 0.001), an index of bone resorption, and serum alkaline phosphatase (r = 0.31, P less than 0.001), an index of bone formation, also increased with age and these increases correlated with increases in serum BGP (r = 0.39, P less than 0.001 and r = 0.43, P less than 0.001, respectively). Serum immunoreactive parathyroid hormone concentrations (r = 0.39, P less than 0.001) and urinary cyclic AMP excretion (r = 0.38, P less than 0.001) increased, suggesting that PTH secretion increased with age; these increases correlated significantly with increases in serum BGP. A subgroup of 32 women who were found to have vertebral fractures, hip fractures, or both had significantly higher values for serum BGP than the remainder. These data suggest that overall bone turnover increases in women with aging and, especially considering the concomitant decrease in skeletal mass, do not support the view that age-related bone loss results primarily from decreased bone formation.
P D Delmas, D Stenner, H W Wahner, K G Mann, B L Riggs
In long-term well adapted kidney transplant recipients we have found a close correlation between the T helper (TH):T suppressor/cytotoxic (TS/C) subset ratios and the presence of T cells that respond in the autologous mixed lymphocyte reaction (AMLR). In 21 recipients with T cell E rosette levels ranging between 53 and 86% and TH:TS/C ratios between 0.15 to 2.10, ratios of greater than 0.8 correlated with AMLR responses (13/13), and ratios of less than 0.8 with AMLR nonreactivity (7/7). By contrast, the allogeneic MLR showed no apparent correlation with the TH:TS/C ratios or with the AMLR pre- or postoperatively. It was found that the AMLR in 22 of 23 normal individuals was markedly inhibited by autologous T cells obtained from peripheral blood lymphocytes, exposed to 3,000 rad (Tx) and added as a third component to the cultures. In contrast, 13 of 13 kidney transplant recipients failed to exhibit this Tx AMLR inhibitory cell population. The "naturally occurring" T inhibitory cells, fractionated by an affinity column chromatography procedure into x-irradiated TH and TS/C subsets, inhibited the AMLR to the same extent as unseparated Tx cells. In cell interchange studies performed in four of five HLA identical donor-recipient pairs the Tx cells of the (normal) donor inhibited the recipient AMLR (immunosuppressed), but recipient Tx cells failed to inhibit the donor AMLR. Finally T cells, primed in AMLR and allogeneic MLR for 10 d were tested for AMLR or allogeneic MLR inhibitory activity. Allogeneic MLR primed x-irradiated cells, inhibited both the AMLR and allogeneic MLR while AMLR x-irradiated primed cells inhibited neither reaction. The Tx AMLR inhibitor found in normal peripheral blood, appears to be a cell that is highly sensitive to the effects of biologic or pharmacologic immunosuppressive agents.
L Fuller, C Flaa, D Jaffe, J Strauss, G K Kyriakides, J Miller
We have investigated the molecular basis of hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency in a patient who presented with the Lesch-Nyhan syndrome. A catalytically incompetent form of HPRT has been isolated from this patient's erythrocytes and lymphoblasts. This enzyme variant, which we have termed HPRTKinston, is indistinguishable from the normal enzyme in terms of its intracellular concentration and maximal velocity, but differs with respect to its isoelectric point (more basic) and Michaelis constants for both substrates (markedly elevated). The tryptic peptides of HPRTKinston were mapped by reverse-phase high pressure liquid chromatography in an attempt to define the precise abnormality in its primary structure. Sequence analysis of the single aberrant tryptic peptide in HPRTKinston revealed an aspartic acid to asparagine amino acid substitution at position 193. Electrophoretic analysis of the CNBr peptides of HPRTKinston confirmed the location of the proposed mutation. This amino acid substitution can be explained by a single nucleotide change in the codon for aspartic acid 193 (GAC leads to AAC). This is the first specific mutation described at the molecular level in a patient with the Lesch-Nyhan syndrome.
J M Wilson, W N Kelley
The contribution of fibrin stabilization to clot strength, measured as the static elastic modulus, was evaluated in human plasma by two independent procedures. In the first approach, amine inhibitors of fibrin stabilization were examined for their effects on the rigidity of normal plasma clots. It is a unique property of these inhibitors that they do not interfere with the reversible aggregation of fibrin molecules, i.e., do not delay clotting time, but selectively prevent only the formation of gamma-glutamyl-epsilon-lysine protein-to-protein linkages. Though the compounds tested were of different chemical structures and potencies, a fivefold reduction in clot strength was obtained in each instance. This value of 20% of normal seems to correspond to the rigidity of the Factor XIII-deficient plasma clot because, as demonstrated by the second approach, when a plasma specimen that genetically lacked the fibrin stabilizing factor was supplemented by the addition of measured amounts of the purified zymogen, a fivefold increase in clot strength could be achieved. The described procedure of evaluating Factor XIII in terms of correcting the elastic modulus of a deficient plasma clot is considered an important assay for the functional competence of purified preparations of the zymogen for the purpose of therapeutic application.
L Shen, L Lorand
Primary IgA nephropathy (Berger's disease) is characterized by renal deposits of IgA, the origin of which is still unknown. However, several clinical and biological findings suggest that these immunoglobulins might have a mucosal origin, and that such patients should present mucosal abnormalities. This paper reports the results of the immunohistomorphometrical analysis of tonsillar plasma cells from seven patients suffering from Berger's disease and seven controls also with recurrent tonsillitis. IgG, IgA, and IgM-secreting cells were enumerated after immunofluorescent staining of serial frozen-cut sections from 20 tonsils. In controls, a predominance of the IgG-secreting population, similar to this reported in the literature was observed (65% of IgG secreting cells and 29% of IgA plasma cells), while an inversion in the patients' plasma cells percentages was evidenced (IgG:37%, IgA:56%). This increment in the IgA population was paralleled by an augmentation of the number of dimeric IgA-secreting cells (75% of IgA plasma cells), stained both for cytoplasmic IgA and J chain. In controls, the latter cells were in similar proportions as previously reported by others (45% of IgA plasma cells). These results demonstrate an imbalance in the IgA-producing system of patients with Berger's disease, which is in keeping with the hypothesis favoring a mucosal origin for the mesangial IgA present in their kidneys.
M C Bene, G Faure, B Hurault de Ligny, M Kessler, J Duheille
Canine cyclic hematopoiesis is an autosomal recessive disease characterized by regular 11-13-d cycles of the neutrophil, reticulocyte, and platelet counts caused by a defect in regulation of marrow stem cell proliferation. Treatment with lithium abrogates cycling of the cell counts in these grey collie dogs. Aware of the defective lymphopoiesis associated with adenosine deaminase and purine nucleoside phosphorylase deficiencies, we hypothesized that abnormal purine or pyrimidine metabolism might be present in these dogs. Using high pressure liquid chromatography, we measured erythrocyte purine and pyrimidine nucleotide levels and plasma purine and pyrimidine nucleosides and bases in normal and grey collie dogs before and during lithium treatment. During neutropenic periods in the grey collies, erythrocyte ATP, GTP, and UTP levels were significantly elevated. Normal dogs made neutropenic with cyclophosphamide did not show such elevations. Lithium treatment normalized the levels of erythrocyte ATP, GTP, and UTP in the grey collies and eliminated the differences between normal and grey collie nucleotide levels. Plasma thymine levels were markedly increased during neutropenia in the grey collie but were not increased in cyclophosphamide-treated normal dogs. The finding of abnormal concentrations of purine and pyrimidine metabolites in these dogs suggest that a metabolic derangement in purine or pyrimidine metabolism may be the cause of the defective stem cell proliferation in this disease.
W R Osborne, W P Hammond, D C Dale
In clonal cultures of erythroid burst-forming units (BFU-E) obtained from blood, the accumulation of fetal and adult hemoglobins (Hb F and Hb A) was measured by radioligand immunoassay. Inclusion of adherent mononuclear cells in the culture promoted a striking increase in the relative amount of Hb F in each of 44 experiments with 14 donors. In two-thirds of the instances, this was accounted for by a selective increase in the absolute amount of Hb F. The differential effect on Hb F and Hb A accumulation was achieved without altering the maturity of the erythroid cells, their mean hemoglobin content, or the asynchrony of the production of the two hemoglobins. Virtually all bursts produced Hb F, and the population of BFU-E as a whole, rather than a selected subset, appeared to be the target of adherent cell action. When the adherent cells were excluded from the culture input, the base-line value of Hb F was reproducible for each donor over a period of several months, and correlated with the number of in vivo circulating F cells.
J Javid, P K Pettis
To determine whether the pulmonary vasodilation produced by isoproterenol is mediated solely by its beta adrenergic effects, we studied the hemodynamic responses to isoproterenol in three groups of dogs with pulmonary vasoconstriction produced by continuous ventilation with 10% oxygen: (a) hypoxia alone, (b) hypoxia and propranolol 0.3 mg/kg i.v. bolus followed by an infusion of 5 micrograms/kg per min, and (c) hypoxia after pretreatment with an inhibitor of cyclooxygenase, either indomethacin or meclofenamate 5 mg/kg s.c. twice daily for 2 d prior to study. All groups had similar values for mean pulmonary artery pressure (PAPm) and pulmonary vascular resistance (PVR) during room air and hypoxic ventilation. Isoproterenol in doses of 0.0025, 0.005, and 0.05 micrograms/kg per min produced a dose-related decline in PAPm and PVR during hypoxia in group 1. Despite beta-blockade with propranolol (group 2), isoproterenol at all three doses significantly reduced PAPm and PVR. The responses to isoproterenol were comparable in the presence or absence of propranolol; at 0.05 micrograms/kg per min the effects of isoproterenol were blunted, but not abolished, by propranolol. Similar results were observed even when five times the dose of propranolol was given. Isoproterenol at all three doses had no effect, however, on PAPm and PVR in the cyclooxygenase inhibitor-pretreated group. These data suggest that the pulmonary vasodilator effects of isoproterenol are not mediated solely by pulmonary vascular beta adrenergic receptors, and that vasodilator prostaglandins may play a role in the responses to this drug.
L J Rubin, J D Lazar
The immunoglobulin-synthesizing activities of peripheral blood mononuclear cells from 57 untreated patients with Hodgkin's disease and 47 normal subjects were compared. Cumulative amounts of IgM and IgG synthesized and secreted by unstimulated and pokeweed mitogen-stimulated cells over a 7-d period were determined in a solid-phase radioimmunoassay. Synthesis of IgM in unstimulated cultures and of both IgM and IgG in cultures stimulated with pokeweed mitogen was markedly reduced in patients with Hodgkin's disease, whereas the mean level of the spontaneous IgG synthesis was enhanced. The degree and frequency of in vitro abnormalities were not influenced by disease stage or histology. Depression of pokeweed mitogen-induced immunoglobulin synthesis did not correlate with excessive number of monocytes and it was unaffected by removal of phagocytic cells or addition to the cultures of monocytes from normal individuals. On the other hand, monocytes isolated from blood of patients with Hodgkin's disease were even more effective than normal monocytes in supporting pokeweed mitogen-induced immunoglobulin synthesis by normal phagocyte-depleted mononuclear cells. Synthesis of both IgM and IgG induced by pokeweed mitogen remained subnormal after addition to patient B cell cultures of autologous irradiated T cells or allogeneic normal T lymphocytes. T cells from patients with Hodgkin's disease appeared at least as effective as normal T cells in helping pokeweed mitogen-induced immunoglobulin production by normal B cells. However, when normal T cells were co-cultured with B cells from patients with Hodgkin's disease, spontaneous IgG synthesis declined, whereas the addition of patient T cells to normal B cells resulted in an increase of spontaneous IgG synthesis. In patients showing depression of pokeweed mitogen-induced immunoglobulin synthesis the lymphoproliferative response and immunoglobulin synthesis stimulated by Staphylococcus aureus bacteria of the Cowan first strain, a T cell independent B cell mitogen, were also markedly reduced. These studies demonstrate impairment of immunoglobulin synthesis by cultured lymphocytes from untreated patients with Hodgkin's disease after stimulation with polyclonal B cell activators and suggest that the in vitro abnormalities may be, at least in part, the result of a preexisting in vivo activation of lymphocytes in Hodgkin's disease patients.
S Romagnani, G F Del Prete, E Maggi, G Bellesi, G Biti, P L Rossi Ferrini, M Ricci
We have studied the accessibility of Factor Xa to neutralization by the heparin-antithrombin complex within plasma and whole blood. This serine protease was detected by measuring the concentrations of activation fragments (F2/F1+2) cleaved from prothrombin. The levels of F2/F1+2) were quantitated by means of a sensitive and specific radioimmunoassay. Our findings indicate that the binding of Factor Xa to "activated" platelets but not to phospholipid micelles results in the protection of the above enzyme from inactivation by the heparin-antithrombin complex. This sequestration of Factor Xa is not affected by the liberation of platelet release proteins or the molecular heterogeneity of the mucopolysaccharide preparations used. The magnitude of enzyme protection is strongly correlated with the extent of prothrombin activation at the time of heparin addition. On this basis, we suggest that high in vivo rates of thrombin generation may lead to the sequestration of Factor Xa on the platelet surface and hence allow this serine protease to resist the action of heparin until the complex is cleared from the circulation.
J M Teitel, R D Rosenberg
Canine tracheal epithelium secretes Cl via an electrogenic transport process that appears to apply to a wide variety of secretory epithelia. To examine the mechanisms involved, intracellular chloride activity, acCl, was measured with Cl-selective intracellular microelectrodes. The results indicate that when the rate of secretion was minimal acCl was 37 mM; with stimulation of secretion the intracellular voltage depolarized, but acCl was not significantly altered, at 39 mM. These findings indicate that: (a) Cl is accumulated across the basolateral membrane under nonsecreting and secreting conditions at an activity 3.8 and 2.4 times, respectively, that predicted for an equilibrium distribution; (b) Cl exit across the apical membrane may be passive with an electrochemical driving force of 22 mV; and (c) stimulation of secretion enhanced the rate of Cl entry across the basolateral membrane, since Cl transport increased without a change in acCl. In the absence of Na in the extracellular fluid, acCl approached the value expected for an equilibrium distribution. This finding suggests that "uphill" entry of Cl into the cell against its electrochemical gradient is dependent upon, and energized by, the entry of Na down its gradient. Submucosal bumetanide, a loop diuretic, also decreased the rate of Cl secretion and decreased acCl, indicating an inhibition of Cl entry. These findings indicate that Cl entry into the cell is directed against its electrochemical gradient and is mediated by a Na-coupled, bumetanide-inhibitable, transport process at the basolateral membrane and that Cl may exit passively down a favorable electrochemical gradient across the apical membrane.
M J Welsh
The in vitro immune response of systemic lupus erythematosus (SLE) lymphocytes to nucleosides conjugated to keyhole limpet hemocyanin (KLH) (A,G,C,T-KLH) was investigated. The nucleosides were chosen not only because they are a part of nucleic acid antigen and involved in autoimmunity, but also because nucleoside covalently bound to either soluble IgG or cells had been shown to induce unresponsiveness in mice. A significant proliferation index was induced in SLE lymphocytes, as compared with normal or rheumatoid arthritis (RA) lymphocytes in vitro [in (A,G,C,T)-KLH, 1 microgram/ml; stimulation index = M +/- SE, SLE 2.10 +/- 0.26, RA 1.06 +/- 0.14, normal 1.12 +/- 0.12 P less than 0.05]. Lymphocytes from SLE patients responded specifically to low doses of (A,G,C,T)-KLH and not to the protein carrier KLH alone. A solid-phase radioimmunoassay was developed to detect nucleoside-specific antibody. SLE lymphocytes spontaneously produced high levels of anti-A,G,C,T antibody. This was further increased by antigenic stimulation, but not with pokeweed mitogen (PWM) stimulation. In contrast normal lymphocytes failed to produce anti-A,G,C,T antibody either spontaneously or in response to antigen. However, normal lymphocytes produced antibody after stimulation with PWM. More importantly, anti-A,G,C,T antibody production by SLE lymphocytes was suppressed by preincubation with A,G,C,T-IgG (A,G,C,T-HGG). The antigen-specific unresponsiveness caused by A,G,C,T-HGG was demonstrated by the observation that preincubation with A,G,C,T-HGG did not affect the production of anti-dinitrophenyl antibody response. The ability to manipulate the altered response of SLE lymphocytes to nucleic acid antigens may have therapeutic implications in these patients.
C Morimoto, A D Steinberg, S F Schlossman, Y Borel
The raised transepithelial electric potential difference (PD) across respiratory epithelia in cystic fibrosis (CF) has suggested an abnormality in ion permeation. We characterized this abnormality further by measuring in the nasal epithelia of CF and normal subjects the concentration-PD relationship for amiloride, an inhibitor of cell Na+ permeability, and PD responses to superfusion with solutions of different composition. Amiloride was more efficacious in the CF subjects but the ED50 was not different from that of normals (approximately 2 X 10(-6) M). Na+ replacement by choline induced effects similar to those of amiloride, i.e. a greater depolarization in CF subjects. A 10-fold increase in the K+ concentration of the perfusate induced a small (less than 10 mV) depolarization in both subject populations. When Cl- in the perfusate was replaced by gluconate or SO2-(4) the nasal PD of normal subjects hyperpolarized (lumen became more negative) by approximately 35 mV. A significantly smaller response (less than 17 mV) was induced in CF homozygotes but not in heterozygotes (38 mV). The smaller response of CF subjects appears to reflect an absolute decrease in luminal surface Cl- permeability because pretreatment with amiloride did not increase the response to Cl- free solution (7 mV). Accordingly, three abnormalities (decreased Cl- permeability, raised PD, greater amiloride efficacy) have been identified in CF respiratory epithelia. Whereas "excessive" active Na+ transport can account for these abnormalities and the dessication of airway surface liquid, it is possible that a lower lumenal cell membrane Cl- permeability and inhibition of a potential path of Cl- secretion can also explain the observations.
M Knowles, J Gatzy, R Boucher
The transit of 14CO2 and H14CO3- through the renal vasculature was studied in rabbit kidneys perfused without erythrocytes and in an in vivo preparation in which erythrocytes were present. In the absence of erythrocytes, the transit of 14CO2 from the renal artery to renal vein was much more rapid than that of H14CO3-. This suggests that (a) there is insufficient carbonic anhydrase (c.a.) in the vasculature between the renal artery and the exchange vessels of the kidney to ensure equilibration between CO2 and HCO3- and (b) CO2 can diffuse directly between arterial and venous vessels in the kidney. Following infusions of carbonic anhydrase, the renal venous outflow patterns of 14CO2 and H14CO3- became the same in the perfused kidneys. Although the initial recovery of 14CO2 remained greater than that of H14CO3- after infusions of acetazolamide (a c.a. inhibitor), arteriovenous diffusion of 14CO2 was diminished by this agent. This is attributed to inhibition of renal tubular c.a. The outflow patterns of H14CO3- and 14CO2 were nearly the same in the presence of erythrocytes, indicating that erythrocyte c.a. is sufficiently accessible to permit virtual equilibration of these radionuclides during the interval required for transit between the renal artery and exchange vessels. However, addition of carbonic anhydrase to the plasma seemed to accelerate transit of both 14CO2 and H14CO3- through the kidneys, and a small disequilibrium between CO2 and HCO3- may therefore normally be present in the renal interstitium and capillaries.
R M Effros, S Nioka
The binding of 125I-lipoprotein (a) [Lp(a)] to cell surface receptors was studied on cultured human fibroblasts. The results were compared with corresponding data obtained with 125I-low density lipoproteins (LDL). Equilibrium binding studies showed that Lp(a) is bound with high affinity by the cell surface receptors. The maximum binding capacity for Lp(a) was 37% lower than for LDL. For Lp(a) and LDL, the Scatchard plots displayed linearity, indicating a single category of binding sites. Half-maximal saturation occurred at a concentration of 9.52 +/- 1.04 nM for Lp(a) and 7.76 +/- 1.29 nM for LDL. Competition binding experiments revealed that Lp(a) and LDL are nearly equally potent in competing each other for the binding sites. Binding of Lp(a) and LDL were followed by suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity. Cyclohexanedione treatment of Lp(a) and LDL completely abolished receptor binding. Neither Lp(a) nor LDL were specifically bound by fibroblasts obtained from a patient with homozygous familial hypercholesterolemia (FH). The removal mechanisms for Lp(a) and LDL were further compared by in vivo studies. Radioiodinated Lp(a) and LDL were injected intravenously into 12 normolipemic individuals to measure kinetic parameters of these two lipoproteins simultaneously in each subject. Mean fractional catabolic rate (FCR) of Lp(a) was 0.260 +/- 0.060 and mean FCR of LDL was 0.377 +/- 0.077 (mean +/- SD). In each subject, FCR of Lp(a) was lower than the FCR of LDL; the mean difference was 31%. The absolute synthetic rate of Lp(a) was significantly lower than the corresponding value of LDL. In each individual, the percentage of total Lp(a) that was contained in the intravascular space was higher than the corresponding value of LDL; the mean difference was 19%. A highly significant positive correlation was found between FCR of LDL and FCR of Lp(a) (r = 0.853, P less than 0.01). No relationship was found between the serum concentration of LDL-apolipoprotein B and Lp(a). The serum level of Lp(a) was positively related to the absolute rate of Lp(a) synthesis (r = 0.979, P less than 0.01). The serum level of LDL-apolipoprotein B was inversely related to FCR of LDL (r = 0.613, P less than 0.05). In a patient with homozygous FH, FCR of LDL was 0.205 and FCR of Lp(a) was 0.210. The results of these studies show that Lp(a) is specifically bound with high affinity to the same receptors of human fibroblasts as LDL. The affinity and maximum binding capacity are slightly lower for Lp(a) than for LDL. The results of the turnover studies are consistent with the assumption that Lp(a) is removed from the plasma by similar mechanisms as LDL.
F Krempler, G M Kostner, A Roscher, F Haslauer, K Bolzano, F Sandhofer
The present study examined the respiratory responses involved in the maintenance of eucapnea during acute airway obstruction in 12 patients with chronic obstructive disease (COPD) and 3 age-matched normal subjects. Acute airway obstruction was produced by application of external flow-resistive loads (2.5 to 30 cm H2O/liter per s) throughout inspiration and expiration while subjects breathed 100% O2. Application of loads of increasing severity caused progressive increases in PCO2 in the patients, but the magnitude of the increase in PCO2 varied substantially between subjects. On a resistance of 10 cm H2O/liter per s, the highest load that could be tolerated by all COPD patients, the increase in PCO2 ranged from 1 to 11 mm Hg, while none of the normal subjects retained CO2. Based on the magnitude of the increase in PCO2 the patients could be divided into two groups: seven subjects whose PCO2 increased by less than or equal to 3 mm Hg (group I) and five subjects whose PCO2 increased by greater than 6 mm Hg (group II). Base-line ventilation and the pattern of breathing were similar in the two groups. During loading group I subjects maintained or increased tidal volume while all group II patients decreased tidal volume (VT). The smaller tidal volume in group II subjects was mainly the result of their shorter inspiratory time as the changes in mean inspiratory flow were similar in the two groups. The magnitude of CO2 retention during loading was inversely related to the magnitude of the change in VT (r = -0.91) and inspiratory time (Ti) (r = -0.87) but only weakly related to the change in ventilation (r = -0.53). The changes in PCO2, VT, and Ti during loading correlated with the subjects' maximum static inspiratory pressure, which was significantly lower in group II as compared with group I patients. These results indicate that the tidal volume and respiratory timing responses to flow loads are impaired in some patients with COPD. This impairment, presumably due to poor inspiratory muscle function, appears to lead to CO2 retention during loaded breathing.
A Oliven, S G Kelsen, E C Deal, N S Cherniack
The mechanism by which a fragment of activated Hageman factor (HFf) activates the classical pathway of complement in serum or platelet-poor plasma has been further delineated. When serum or platelet-poor plasma was incubated with various concentrations of HFf, the total complement hemolytic activity was reduced in a dose-dependent manner. This activation appears to be due to the direct interaction of HFf with macromolecular C1, since incubation of purified C1 with HFf resulted in dissociation of the subunits with concomitant reduction of C1r antigenicity that is indicative of C1 activation. HFf-dependent activation was prevented by prior treatment of HFf with the active site-directed inhibitor, H-D-proline-phenylalanine-arginine chloromethyl ketone or with a specific inhibitor of activated HF derived from corn. Incubation of HFf with highly purified C1r also resulted in activation of C1r as assessed directly using a synthetic substrate or indirectly by activation of C1s and consumption of C2. However, incubation of HFf with highly purified C1s resulted in formation of activated C1s (C1s-) but this was less efficient than HFf activation of C1r. We therefore conclude that activation of C1 in macromolecular C1 is the result of HFf conversion of C1r to C1r; activation of C1s then occurs primarily by C-1r and to a lesser degree by the direct action of HFf.
B Ghebrehiwet, B P Randazzo, J T Dunn, M Silverberg, A P Kaplan
Amiodarone was selectively perfused into the sinus node artery and atrioventricular node artery of 51 dogs. Amiodarone had an immediate negative chronotropic and dromotropic effect. Threshold concentration was 2.5 micrograms/ml. 25 and 50 micrograms/ml of amiodarone injected into the sinus node artery slowed the heart by 25.6 +/- 3.1 and 33.7 +/- 2.6 beats/min (mean +/- 1 SEM), respectively. Amiodarone 25 and 50 micrograms/ml injected into the AV node artery during AV junctional rhythm slowed the AV junctional pacemaker by 12.2 +/- 1.8 and 17.4 +/- 1.7 beats/min, respectively. Injections of amiodarone into the AV node artery during sinus rhythm regularly increased AV conduction time sometimes causing 2 degrees AV block at the highest concentration used. Impaired conduction was exclusively measured at the level of the A-H interval in the His electrogram. Neither atropine nor propranolol prevented the negative chronotropic effects of amiodarone. Amiodarone had no significant effect on sinus node response to either stellate stimulation or intranodal administration of norepinephrine. The negative chronotropic action of amiodarone was significantly enhanced when amiodarone was administered in a perfusate containing low (0.6 mM) instead of normal calcium. Taken collectively these observations indicate that amiodarone has immediate depressant electrophysiologic effects on both the sinus node and the AV junction and that these early effects might involve the blockade of the slow channel.
H O Gloor, F Urthaler, T N James
The distribution and quantitation of the iron-binding proteins of rat small intestinal mucosa was studied, in iron-deficient and replete animals, to explore their role in the absorption of iron. Adsorption (mucosal uptake) of iron in in situ ligated loops of small intestinal mucosa was found to be uniform throughout the length of the small intestine whereas absorption (carcass uptake) showed a steep decreasing gradient from the duodenum to the ileum. The disrupted, in vivo labeled mucosal cells were fractionated by isopycnic centrifugation and transferrin and ferritin were quantitated by radioimmunoassay. Transferrin derived from mucosal cells was shown to have a higher affinity for the antibody than transferrin in serum. Of the transferrin present in the mucosal extract, only a portion could be accounted for by contamination from the serum; the proteolysis resistant and intrinsic transferrin may be mucosal cell specific. Transferrin was found in similar amounts in all regions of the small intestine, was not affected by iron loading but doubled in response to iron deficiency. Mucosal ferritin was found in greater amounts in the iron-absorbing areas of the intestine, increased in the duodenum of iron-loaded animals, and decreased in iron-deficient animals. The incorporation of newly absorbed radioiron into ferritin was only found in iron absorbing regions and was completely inhibited by colchicine and cytochalasin-B, suggesting that ferritin was loaded with iron at the point of iron absorption and that the process is associated with vesicle movement and not simple diffusion. Transferrin and ferritin-specific immunoabsorption and also gel filtration established that no other soluble iron binding proteins were involved in absorption.
G Johnson, P Jacobs, L R Purves
Human platelets were studied immunochemically to determine if they contain high-molecular weight kininogen. On crossed immunoelectrophoresis with total kininogen antisera (antisera that recognizes both high- and low-molecular weight kininogen) extracts of platelets contained total kininogen antigen. Platelet total kininogen antigen showed complete antigenic identity with plasma total kininogen and displayed the same electrophoretic migration as plasma total kininogen. Using antisera monospecific to high molecular weight kininogen, a competitive enzyme-linked immunosorbent assay (CELISA) was developed to directly measure platelet high-molecular weight kininogen. By CELISA, 27-101 ng of high molecular weight kininogen antigen per 10(8) platelets was quantitated in detergent-soluble lysates of washed human platelets from nine normal donors with a mean level of 60 ng +/- 24/10(8) platelets. Plasma high-molecular weight kininogen, either in the platelet suspending medium or on the surface of the platelets, could only account for 5% of antigen measured in the solubilized platelets. On the CELISA, platelet high-molecular weight kininogen was immunochemically identical to plasma and purified high-molecular weight kininogen. Platelet high-molecular weight kininogen was secreted from platelets after exposure to ionophore A23187 (3-15 microM), collagen (5-150 micrograms/ml), and thrombin (1.6 U/ml). Secreted platelet high-molecular weight kininogen did not become a part of the platelet Triton-insoluble cytoskeleton. On cross immunoelectrophoresis secreted platelet total kininogen antigen had a similar electrophoretic migration to plasma total kininogen. Thus, human platelets contain high-molecular weight kininogen that can be secreted from platelets and that may participate in plasma coagulation reactions.
A H Schmaier, A Zuckerberg, C Silverman, J Kuchibhotla, G P Tuszynski, R W Colman
The hypocholesterolemic effect of the hydrophobic surfactant, poloxalene 2930, was studied in the rabbit to determine whether this agent prevents experimentally produced atherosclerosis. Male rabbits were divided into four groups and fed a control diet (group A) or an atherogenic diet (groups B, C, and D) for 10 wk. Diets of groups C and D were supplemented with 0.5 and 1% poloxalene 2930, respectively. Animals in group B developed significantly greater levels of cholesterol in the serum and aorta compared with group A. Addition of poloxalene 2930 to the diets of groups C and D prevented significant elevations in cholesterol concentrations of both serum and aorta compared with group B with values for group D being essentially similar to those observed in group A. Groups C and D also had significant increases of fecal excretion of both neutral fat and neutral steroids as compared with either groups A or B. There were no atherosclerotic lesions of the aortas from group D. Aortas from rabbits in group B had numerous atheromatous plaques while one rabbit each from groups A and C had several very small atheromatous lesions. These results demonstrate that poloxalene 2930 reduces the rise of serum cholesterol in rabbits in response to an atherogenic diet and prevents the development of atherosclerosis. This hypocholesterolemic effect is likely mediated by the effect of this surfactant on the small intestine.
J B Rodgers, E C Kyriakides, B Kapuscinska, S K Peng, W J Bochenek
Ketoconazole has recently been shown to interfere with steroidogenesis in patients and rat in vitro systems. In this study we attempted to elucidate the site of inhibition in the adrenal gland. Although ketoconazole impaired adrenocorticotropic hormone stimulated cyclic (c)AMP production, dibutyrl cAMP addition did not bypass the steroidogenic blockade indicating that the critical ketoconazole-inhibited step was distal to cAMP. Addition of radiolabeled substrates to isolated adrenal cells and analysis of products by high performance liquid chromatography demonstrated a ketoconazole block between deoxycorticosterone (DOC) and corticosterone. This 11-hydroxylase step is carried out by a P450-dependent mitochondrial enzyme. No restriction of progesterone or pregnenolone conversion to DOC was detected, steps carried out by non-P450-dependent microsomal enzymes. Inhibition of cholesterol conversion to pregnenolone by mitochondrial fractions indicated a second block at the side chain cleavage step, another mitochondrial P450-dependent enzyme. Adrenal malate dehydrogenase, a non-P450-dependent mitochondrial enzyme was not inhibited while renal 24-hydroxylase, a P450-dependent mitochondrial enzyme in another organ, was blocked by ketoconazole. We conclude that ketoconazole may be a general inhibitor of mitochondrial P450 enzymes. This finding suggests that patients receiving ketoconazole be monitored for side effects relevant to P450 enzyme inhibition. Further, we raise the possibility that this drug action may be beneficially exploited in situations where inhibition of steroidogenesis is a therapeutic goal.
D S Loose, P B Kan, M A Hirst, R A Marcus, D Feldman
Asymptomatic hemophilia patients receiving Factor VIII concentrate were found to have normal natural killer (NK) cells and B cells, and an inverted T helper/suppressor ratio due to an increase in cells of T suppressor phenotype. In contrast, a hemophilia patient with acquired immune deficiency syndrome (AIDS) exhibited nonfunctional NK cells, low B cells, and an inverted T helper/suppressor ratio due to very low numbers of T helper cells. Hemophilia patients on cryoprecipitate therapy exhibited normal immune parameters. A high percentage of hemophilia patients on both treatments had antibody to hepatitis B virus. The isolated finding of elevated levels of T suppressor cells in hemophilia patients receiving Factor VIII concentrate has not been recognized as an early indicator of impending AIDS, and longitudinal studies will be required to determine its clinical significance.
A Landay, M C Poon, T Abo, S Stagno, A Lurie, M D Cooper
Rabbit medullary collecting duct (MCD) acidification has been demonstrated to occur by means of a sodium-independent, aldosterone-stimulated mechanism. We have examined the anionic dependence of this process by means of the isolated perfused tubule technique. Total replacement of perfusate chloride with gluconate enhanced tubular bicarbonate reabsorption (JHCO3), from a basal rate of 10.7 +/- 1.0 pmol X mm-1 X min-1 to a rate of 15.01 +/- 1.0 pmol X mm-1 X min-1. Removal of bath chloride, with and without removal of perfusate chloride completely abolished acidification. Bath, but not luminal 4-acetamido-4' isothiocyano-2,2'-disulfonic stilbene provoked a marked decrease in JHCO3 from 10.1 +/- 1.2 pmol X mm-1 X min-1 to 2.3 +/- 0.3 pmol X mm-1 X min-1. Measurement of chloride reabsorptive rate (JCl) revealed colinearity between JHCO3 (9.18 +/- 0.9 pmol X mm-1 X min-1) and JCl (9.75 +/- 1.18 pmol X mm-1 X min-1). We propose a model of mammalian distal nephron acidification in which (a) cellular base exit is effected by means of a basolateral membrane Cl-base exchanger and (b) net electroneutrality of electrogenic proton secretion is maintained by the parallel movement of an anionic species, functionally chloride.
D K Stone, D W Seldin, J P Kokko, H R Jacobson
Cholesterol catabolism was stimulated in 6-wk-old White Carneau pigeons using a laboratory stock diet containing 1.3% cholestyramine resin. After 8 wk on this diet the animals were returned to control stock diet (no resin) for another 8-wk period. When subsequently challenged with a diet containing 0.5% cholesterol, cholestyramine-pretreated pigeons exhibited significantly lower serum cholesterol level when compared with controls and this "hyporesponder" behavior persisted throughout the study period. Furthermore, the aorta of cholestyramine-treated animals exhibited significantly (a) lower prevalence and severity of atherosclerosis and (b) lower cholesterol content. These studies demonstrate for the first time that enhancement of cholesterol catabolism in early life improves resistance to diet-induced atherosclerosis in later life in this avian model.
M T Subbiah, D Deitemeyer, R L Yunker
Tissue somatostatin-like immunoreactivity (SLI) consists of a number of molecular species including the cyclic tetradecapeptide or SRIF, an N-terminally extended form of SRIF termed somatostatin-28, as well as larger precursor peptides. The function and nature of circulating SLI is not well understood. In this report, we describe techniques for the definition of the components of plasma SLI in normal human plasma. Plasma SLI measured after gel filtration on Bio-gel P-6 columns was found to consist of from 1-3 peaks. The void volume peak was present in greatest concentration (34.2 +/- 8.9 pg/ml) and did not increase in response to a mixed meal. Very low levels of two additional peaks of SLI activity were found. To further characterize these peaks, 10-ml plasma samples were extracted and concentrated on octadecylsilyl silica (C-18) cartridges with subsequent fractionation on Bio-gel P-6 columns. The two peaks that coeluted with synthetic SRIF and S-28 markers, respectively, were present in concentrations of 5.4 +/- 1.4 and 4.8 +/- 1.9 pg/ml in fasting plasma. In response to a mixed meal, the SLI14 peak doubled (12.9 +/- 2.4 pg/ml) while the SLI28 peak increased to 29.9 +/- 7.2 pg/ml at 120 min. These results provide evidence that S-28 circulates in human plasma and its increase after feeding is consistent with a possible biological role for this peptide.
K S Polonsky, S E Shoelson, H M Docherty