Genetic polymorphism in the beta-subunit of the eighth component of human complement, C8, was defined by isoelectric focusing of serum in polyacrylamide gel in the presence of urea and development of specific patterns of hemolysis in an overlay gel containing antibody-sensitized erythrocytes and C8 beta-chain-deficient serum. Bands of hemolysis induced by serum from unrelated Caucasians suggested autosomal codominant inheritance of three structural alleles at a single locus, C82: C82 degrees A (acidic), C82 degrees B (basic), and C82 degrees A1 (very acidic) with frequencies of 0.952, 0.044, and 0.004, as well as the probable null allele C82 degrees Q0. The distribution of phenotypes agreed with the Hardy-Weinberg equilibrium. The previously described genetic polymorphism in human C8 defined with the use of "complete" C8 (C8 alpha-gamma-chain)-deficient serum was distinct from and independent of the inherited structural variation at C82. Therefore, the locus for C8 alpha-gamma-chains has been redesignated C81, and has the alleles C81 degrees A, C81 degrees A1, and C81 Q0. Linkage studies failed to show close linkage between the two loci for C8, C81, and C82, and between C82 and the major histocompatibility complex or C6.
C A Alper, D Marcus, D Raum, B H Petersen, T J Spira
Platelet-type von Willebrand disease (vWD) and pseudo-vWD are two recently described intrinsic platelet defects characterized by enhanced ristocetin-induced agglutination in platelet-rich plasma. A similar finding is also typical of type IIB vWD, where it has been related to a von Willebrand factor (vWF) rather than a platelet abnormality. Platelet aggregation induced by unmodified human vWF in the absence of other stimuli has been reported in pseudo-vWD. In this study we demonstrate that vWF induces aggregation in platelet-type but not type IIB vWD. Aggregation is observed when normal plasma cryoprecipitate or purified vWF are added to platelet-rich plasma. Cryoprecipitate also aggregates washed platelets, although at higher concentrations than required for platelet-rich plasma. Purified vWF, however, induces significant aggregation of washed platelets only when plasma is added. EDTA inhibits vWF-induced aggregation. Its effect can be overcome by calcium but much less effectively by magnesium ions. Unstimulated platelets in platelet-rich plasma from patients with platelet-type but not type IIB vWD bind 125I-vWF in a specific and saturable manner. All different sized multimers of vWF become associated with platelets. Both aggregation and binding exhibit a similar vWF concentration dependence, suggesting that a correlation exists between these two events. Removal of ADP by appropriate consuming systems is without effect upon such binding or upon vWF-induced aggregation. Thrombin-induced 125I-vWF binding to washed platelets is normal in platelet-type as well as type IIB vWD. These results demonstrate that a specific binding site for unmodified human vWF is exposed on unstimulated platelets in platelet-type vWD. The relatively high vWF concentrations required for aggregation and binding may explain the lack of significant in vivo aggregation and thrombocytopenia in these patients. Moreover, these studies provide additional evidence that platelet-type and type IIB vWD are different diseases with distinct pathogeneses.
J L Miller, J M Kupinski, A Castella, Z M Ruggeri
Using cultured skin fibroblasts, we studied the heterogeneity of inborn errors of leucine metabolism such as isovaleric acidemia (IVA), glutaric aciduria type II (GA II), and multiple carboxylase deficiency (MC). We first developed a simple macromolecular-labeling test to measure the ability of cells to oxidize [1-14C]isovaleric acid in situ in culture. Cells from two different lines were fused using polyethylene glycol, and the ability of the heterokaryons to oxidize [1-14C]isovaleric acid was tested by the macromolecular-labeling test. The MC line complemented with all 10 IVA lines tested; heterokaryons showed 99 +/- 68% more activity than the unfused mixture of component cells. GA II/IVA heterokaryons exhibited poor growth, but when the culture remained confluent, the GA II cells complemented with all six IVA lines tested, showing a 71 +/- 41% increase in activity. The relatively large standard deviations are due to a few experiments in which significant enhancement of macromolecular-labeling test activity was not observed upon fusion, but significant complementation was clearly observed in repeats of the same combinations. These results are consistent with our previous findings, which indicated that the decreased ability of GA II cells to oxidize isovaleryl-CoA involves a defective electron-transporting system rather than a defective isovaleryl-CoA dehydrogenase. IVA/IVA heterokaryons showed no complementation in any combination tested, indicating no detectable heterogeneity in isovaleric acidemia. This finding indicates that the same gene is mutated in all IVA lines. Previous results indicated that this gene codes for isovaleryl-CoA dehydrogenase.
B Dubiel, C Dabrowski, R Wetts, K Tanaka
Alloxan-diabetic rabbits develop a pronounced hypercholesterolemia and hypertriglyceridemia in response to cholesterol feeding. Despite higher levels of plasma cholesterol, these animals have much less atherosclerosis than cholesterol-fed nondiabetics. To determine whether this effect is due to properties of the lipoproteins, we compared chemical, physical, and metabolic characteristics of a very low density lipoprotein (VLDL) fraction (d less than 1.019 g/ml) from the diabetic and nondiabetic cholesterol-fed rabbits. The molar ratio of triglyceride to cholesteryl ester in the particles from diabetic animals ranged from 2:1 to 6:1, and this ratio remained constant in subfractions from individual rabbits. Triglyceride from nondiabetic control animals was a minor component. Differential scanning calorimetry showed a distinct order-disorder phase transition for cholesteryl ester at approximately 42 degrees C in the fractions from control animals, whereas in fractions from most of the diabetics no such transition was observed, indicating that both triglyceride and cholesteryl ester are present in the core of the same particle. The relative amount of apoprotein E in particles from diabetic animals was much less than that of cholesterol-fed controls. The ability of the lipoproteins from both groups to stimulate cholesteryl ester formation in mouse peritoneal macrophages also was tested. Lipoproteins from cholesterol-fed controls stimulated cholesteryl ester formation in a dose-dependent manner, but particles from the diabetic group had little or no effect. The results suggest that the presence of unusual VLDL particles in diabetic cholesterol-fed rabbits is responsible, at least in part, for the reduced incidence of atherosclerosis in this animal model.
P Brecher, A V Chobanian, D M Small, W Van Sickle, A Tercyak, A Lazzari, J Baler
In this paper we summarize our experience and report the characteristics of energy delivery in 23 patients who have undergone closed chest ablation of the normal atrioventricular (AV) conduction system for the treatment of refractory supraventricular arrhythmias. The induction of AV block was achieved by the synchronous delivery of electrical energy with a damped sinusoidal waveform utilizing a standard direct current defibrillator and a standard tripolar His bundle catheter. The procedure was well tolerated, though one patient experienced ventricular fibrillation, which was uneventfully converted with external paddles. Complete AV block was achieved in 20 of 23 patients and all were rendered arrhythmia free, though two still required antiarrhythmic drugs. A stable escape rhythm was seen in all patients with a cycle length of 1,294 +/- 243 ms. Creatine phosphokinase-MB was positive at low levels in 19 of 23 patients and cleared within 24 h. 99mTc pyrophosphate scans were faintly positive in only 2 of 22 patients. Left ventricular wall motion and ejection fractions were unchanged in 19 of 19 patients, two-dimensional echocardiography with microcavitation technique was unchanged in 12 of 12 patients, and a slight increase in pulmonary artery wedge pressure was seen in only 1 of 11 patients. Current, voltage, and their product (power) waveforms were recorded in 12 patients (12 recordings at a defibrillator setting of 200 J and 5 recordings at a defibrillator setting of 300 J) and revealed a complex voltage-current relationship due to changes occurring at the catheter electrode-tissue interface. At 200 J the peak values were 42.2 +/- 3.3 A, 2.16 +/- 0.11 kV, and 87.9 +/- 4.7 kW, while at 300 J the peak values were 58.2 +/- 2.8 A, 2.40 +/- 0.10 kV, and 134.4 +/- 6.7 kW, respectively. No instance of catheter disruption was seen, though "pitting" of the distal electrode (through which current passed) occurred in all but one catheter.
J L Trantham, J J Gallagher, L D German, A Broughton, T Guarnieri, J Kasell
Dichloroacetate (DCA) markedly reduces circulating cholesterol levels in animals and in patients with combined hyperlipoproteinemia or homozygous familial hypercholesterolemia (FH). To investigate the mechanism of its cholesterol-lowering action, we studied the effects of DCA and its hepatic metabolites, glyoxylate and oxalate, on the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase) obtained from livers of healthy, reverse light-cycled rats. Oral administration of DCA for 4 d decreased HMG CoA reductase activity 46% at a dose of 50 mg/kg per d, and 82% at a dose of 100 mg/kg per d. A 24% decrease in reductase activity was observed as early as 1 h after a single dose of 50 mg/kg DCA. The inhibitory effect of the drug was due to a fall in both expressed enzyme activity and the total number of reductase molecules present. DCA also decreased reductase activity when added to suspensions of isolated hepatocytes. With chronic administration, DCA inhibited 3H2O incorporation into cholesterol by 38% and into triglycerides by 52%. When liver microsomes were incubated with DCA, the pattern of inhibition of reductase activity was noncompetitive for both HMG CoA (inhibition constant [Ki] 11.8 mM) and NADPH (Ki 11.6 mM). Inhibition by glyoxylate was also noncompetitive for both HMG CoA (Ki 1.2 mM) and NADPH (Ki 2.7 mM). Oxalate inhibited enzyme activity only at nonsaturating concentrations of NADPH (Ki 5.6 mM). Monochloroacetate, glycollate, and ethylene glycol, all of which can form glyoxylate, also inhibited reductase activity. Using solubilized and 60-fold purified HMG CoA reductase, we found that the inhibitory effect of glyoxylate was reversible. Furthermore, the inhibition by glyoxylate was an effect exerted on the reductase itself, rather than on its regulatory enzymes, reductase kinase and reductase phosphatase. We conclude that the cholesterol-lowering effect of DCA is mediated, at least in part, by inhibition of endogenous cholesterol synthesis. The probable mechanisms are by inhibition of expressed reductase activity by DCA per se and by conversion of DCA to an active metabolite, glyoxylate, which noncompetitively inhibits HMG CoA reductase. These studies thus identify a new class of pharmacological agents that may prove useful in regulating cholesterol synthesis and circulating cholesterol levels in man.
P W Stacpoole, H J Harwood Jr, C E Varnado
Somatostatin-containing cells have been shown to be in close anatomic proximity to gastrin-producing cells in rat antral mucosa. The present studies were directed to examine the effect of secretin on carbachol-stimulated gastrin release and to assess the potential role of somatostatin in mediating this effect. Rat antral mucosa was cultured at 37 degrees C in Krebs-Henseleit buffer, pH 7.4, gassed with 95% O2-5% CO2. After 1 h the culture medium was decanted and mucosal gastrin and somatostatin were extracted. Carbachol (2.5 X 10(-6) M) in the culture medium increased gastrin level in the medium from 14.1 +/- 2.5 to 26.9 +/- 3.0 ng/mg tissue protein (P less than 0.02), and decreased somatostatin-like immunoreactivity in the medium from 1.91 +/- 0.28 to 0.62 +/- 0.12 ng/mg (P less than 0.01) and extracted mucosal somatostatin-like immunoreactivity from 2.60 +/- 0.30 to 1.52 +/- 0.16 ng/mg (P less than 0.001). Rat antral mucosa was then cultured in the presence of secretin to determine its effect on carbachol-stimulated gastrin release. Inclusion of secretin (10(-9)-10(-7) M) inhibited significantly carbachol-stimulated gastrin release into the medium, decreasing gastrin from 26.9 +/- 3.0 to 13.6 +/- 3.2 ng/mg (10(-9) M secretin) (P less than 0.05), to 11.9 +/- 1.7 ng/mg (10(-8) secretin) (P less than 0.02), and to 10.8 +/- 4.0 ng/mg (10(-7) M secretin) (P less than 0.02). Secretin (10(-7) and 10(-8) M) also increased concomitantly culture medium somatostatin concentration. To determine whether secretion inhibition of carbachol-stimulated gastrin release was mediated by somatostatin, antral mucosa was cultured with carbachol, secretin (10(-9)-10(-7) M), and antibodies to somatostatin. Inclusion of somatostatin antibodies in the culture medium abolished the capacity of secretin (10(-7) and 10(-8) M) to inhibit carbachol-stimulated gastrin release. Results of these studies indicate (a) that secretin inhibits carbachol-stimulated gastrin release and (b) that under the conditions of these experiments secretin inhibition of gastrin release is mediated, at least in part, locally through release of antral somatostatin.
M M Wolfe, G M Reel, J E McGuigan
Dogs with portal cirrhosis but without portal hypertension (end-to-side portacaval anastomosis) retain sodium and expand plasma volume before ascites formation. In our study, dogs were subjected to bile duct ligation and simultaneous side-to-side portacaval anastomosis (PCA) in order to create a canine model of hepatic cirrhosis without intrahepatic or portal hypertension. The effect of normalizing intrahepatic pressures in the face of venous outflow block on sodium handling was studied. 13 dogs survived the surgical procedures and were followed. Two dogs developed sodium retention and ascites at 5-6 wk (livers were cirrhotic) when the PCA spontaneously closed. 11 dogs were free of sodium retention and ascites for as long as 12 wks after surgery, while ingesting 35 meq/d of sodium. In this group glomerular filtration rate remained normal throughout the period of observation and there was no expansion of plasma volume. Nine of these dogs were then fed 85 meq/d of sodium; eight remained in sodium balance and one retained sodium and went on to develop ascites. When 10-15 mg i.m. of desoxycorticosterone acetate (DOCA) was given daily, five dogs developed sodium retention and ascites, while four escaped from DOCA. Dogs who developed ascites had either a partially occluded PCA (4/5) or a patent PCA, but with a significant portacaval pressure gradient of 9.5 cm H2O (1/5). In all four dogs who escaped from DOCA, the PCA was widely patent and the mean pressure gradient was only 1.6 cm H2O. Both groups were equally cirrhotic, as judged by histological and biochemical parameters. We conclude that normalizing intrahepatic pressure by providing an outflow tract for the cirrhotic liver will abolish that component of early renal tubular sodium retention not due to portal venous hypertension or ascites sequestration.
B Unikowsky, M J Wexler, M Levy
In rats, muscle glycogen depletion has been associated with increased insulin action. Whether this also occurs in man has not been reported. After 4 d rest, 13 males (E Group) had a percutaneous muscle biopsy of the vastus lateralis muscle followed by a euglycemic clamp at plasma insulin congruent to 100 microU/ml and congruent to 1,900 microU/ml, with simultaneous indirect calorimetry. This was repeated 1 wk later, but after glycogen-depleting exercise the night before the euglycemic clamp. Seven subjects underwent the same protocol but were also re-fed 100 g carbohydrate (CHO) after the exercise (EF group). In both groups, the mean muscle glycogen content was approximately 40% lower (P less than 0.01) after exercise compared with the muscle glycogen content measured after rest. In the E group, the mean muscle glycogen synthase activity (percent independent of glucose-6-phosphate) increased threefold (P less than 0.001) after exercise, but increased only twofold in the EF group (P less than 0.02 between groups). In both groups, the mean basal and insulin-stimulated CHO oxidation rates were lower in the post-exercise, glycogen-depleted condition compared with the rested, glycogen-replete condition. The mean insulin-stimulated CHO storage rate increased significantly in the E group after exercise but not in the EF group. In the E group, the total insulin-stimulated CHO disposal rate (M) was 17 (P less than 0.04) and 10% (P less than 0.03) higher after exercise during the low and high dose insulin infusion, respectively. No significant changes in M were observed in the EF group. For all subjects, after rest and exercise, the M correlated with the CHO storage rates during the low (r = 0.80, P less than 0.001) and high dose (r = 0.77, P less than 0.001) insulin infusions. After exercise, the muscle glycogen synthase activity correlated with the CHO storage rate (r = 0.73, P less than 0.002; r = 0.75, P less than 0.002) during the low and high dose insulin infusions, respectively, and also with M (r = 0.64, P less than 0.008; r = 0.57; P less than 0.02).
C Bogardus, P Thuillez, E Ravussin, B Vasquez, M Narimiga, S Azhar
Cultured human skin fibroblasts and human arterial smooth muscle cells possess high-affinity binding sites specific for high density lipoproteins (HDL). Results from the present study demonstrate that binding of HDL to these sites is up-regulated in response to cholesterol loading of cells. When fibroblasts or smooth muscle cells were preincubated with nonlipoprotein cholesterol, cellular binding of 125I-HDL3 was enhanced severalfold. This enhancement was sustained in the presence of cholesterol but was readily reversed when cells were exposed to cholesterol-free medium. The stimulatory effect of cholesterol treatment was prevented by cycloheximide, suggesting the involvement of protein synthesis. Kinetic analysis of HDL3 binding showed that prior exposure to cholesterol led to an induction of high-affinity binding sites on the cell surface. In the up-regulated state, the apparent dissociation constant (Kd) of these sites was approximately 2 micrograms protein/ml. Competition studies indicated that the HDL binding sites recognized either HDL3 or HDL2 but interacted weakly with low density lipoprotein (LDL). Exposure of cells to lipoprotein cholesterol in the form of LDL also enhanced HDL binding by a process related to delivery of sterol into cells via the LDL receptor pathway. Enhancement of HDL binding to fibroblasts by either nonlipoprotein cholesterol or LDL was associated with an increased cell cholesterol content, a suppressed rate of cholesterol synthesis, decreased LDL receptor activity, and an enhanced rate of cholesterol ester formation. A comparison of HDL3 binding with the effects of HDL3 on cholesterol transport from cells revealed similar saturation profiles, implying a link between the two processes. Thus, cultured human fibroblasts and human arterial smooth muscle cells appear to possess specific receptors for HDL that may function to facilitate cholesterol removal from cells.
J F Oram, E A Brinton, E L Bierman
The hydrolysis of sphingolipids by lysosomal enzymes requires the presence of additional proteins, which have been called activator proteins. The number of activator proteins, their specificity, exact mechanism of action, and response to a storage process all remain to be determined. In this study, antibodies to an activator protein known to bind sphingolipids and activate the enzymatic hydrolysis of GM1 ganglioside and sulfatide were used to estimate the concentration of this activator protein in small samples of liver and brain from patients with lysosomal storage diseases. By using rocket immunoelectrophoresis, the concentration of cross-reacting material (CRM) was determined. Control livers had an average of 0.95 +/- 0.18 (mean +/- 1 SD) microgram CRM/mg protein in the extracts, and control brains had an average of 0.25 +/- 0.14 microgram CRM/mg protein. Extremely high levels of CRM were found in extracts of livers from patients with type 1 GM1 gangliosidosis (15.1 and 16.9), and type A Niemann-Pick disease (10.7). Extracts of brain samples revealed a large amount of CRM in type 1 GM1 gangliosidosis (14.8), Tay-Sachs disease (5.3 and 8.7), and Sandhoff disease (13.5). Significantly elevated CRM was also measured in brain samples from patients with type 2 GM1 gangliosidosis, type A Niemann-Pick disease, metachromatic leukodystrophy, and Krabbe disease. The highest levels are found in those genetic diseases where the lipids stored, primarily or secondarily to the genetic defect, bind to this activator protein. This activator protein may have an important function in regulating intralysosomal lipid catabolism, and changes in its concentration in certain genetic diseases may be the cause of clinical, biochemical, and pathological heterogeneity found in the patients.
K Inui, D A Wenger
Lymphocyte transformation (LT) responses to Chlamydia trachomatis, to four other microbial antigens, and to phytohemagglutinin (PHA) were studied in 201 women during pregnancy and/or 3-18 wk postpartum. The LT responses to all stimulants tested were significantly depressed during pregnancy when compared with postpartum LT responses. This difference occurred whether LT assays were performed in autologous or pooled heterologous plasma collected from nonpregnant donors. Among women studied in the third trimester and again postpartum, the autologous LT stimulation index (LTSI) rose from 1.7 to 3.4 (P less than 0.001) with C. trachomatis elementary body antigen, from 3.7 to 7.9 (P less than 0.001) with Candida albicans cell wall extract, from 4.5 to 7.8 (P = 0.008) with streptokinase-streptodornase, from 1.7 to 3.0 (P = 0.007) with fluid tetanus toxoid, from 1.7 to 2.8 (P = 0.046) with mumps virus skin test antigen, from 35.5 to 87.0 (P less than 0.001) with PHA (2 micrograms/ml), and from 107.2 to 181.9 (P = 0.007) with PHA (10 micrograms/ml). LT responses to C. trachomatis were compared in 52 pregnant women and 58 nonpregnant women; all the women had C. trachomatis isolated at the time of LT assay. Using either plasma supplement, the mean LTSI with C. trachomatis antigen was significantly higher in nonpregnant women than in pregnant women, regardless of trimester (P less than 0.001). Among 12 women who were serially tested and remained culture positive for C. trachomatis throughout pregnancy and the postpartum period, the mean autologous LTSI rose from 1.9 in the third trimester to 7.8 postpartum (P = 0.0004). These data are the first to show that the immune response to an ongoing bacterial infection is depressed during pregnancy and to definitively document the depressed LT responses during human pregnancy.
R C Brunham, D H Martin, T W Hubbard, C C Kuo, C W Critchlow, L D Cles, D A Eschenbach, K K Holmes
Immunoprecipitates of human C4 from EDTA-plasma were incubated with [14C]methylamine and analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and fluorography. In addition to finding label in the alpha-chains of the secreted (C4s) and predominant plasma (C4p) forms of C4, two additional molecules with apparent molecular weights of approximately 168,000 (p168) and approximately 125,000 (p125) covalently incorporated methylamine, indicating the presence of an internal thioester bond. These two molecules were present at a concentration of approximately 5% of total plasma C4 and were not immunoprecipitated by antisera to C3 or alpha 2-macroglobulin. A human hepatoma-derived cell line (HepG2), in addition to synthesizing C4s and small quantities of the polypeptide precursor of C4 (pro-C4), was found to secrete p168 and p125 at concentrations of 14 +/- 4.8 and 21 +/- 9.2% (mean +/- SD), respectively, of total secreted C4. These molecules were not found intracellularly. Both molecules were present on reduced, but not nonreduced, SDS-polyacrylamide gels. Chido (C4B) and Rodgers' (C4A) alloantisera precipitated the C4A and C4B variants of pro-C4, p168, p125, and C4s. Both tryptic and Staphylococcus aureus V8 protease peptide analyses showed homology between p168 and the beta- and alpha-chains and between p125 and the alpha- and gamma-chains. Partial NH2-terminal sequencing revealed that the beta-chain was NH2-terminal in p168 and that the alpha-chain was NH2-terminal in p125. Taken together, these data indicate that p168 and p125 represent uncleaved beta-alpha- and alpha-gamma-fragments of pro-C4, respectively. Thus, in most individuals, plasma C4 consists of five structurally distinct molecules, the single polypeptide precursor (pro-C4), the three-subunit secreted (C4s) and predominant plasma (C4p) forms of C4, and two incompletely processed two-subunit molecules with uncleaved beta-alpha- (p168) or uncleaved alpha-gamma (p125)-subunits. In addition, all five molecules are observed for both C4A (Rodgers) and C4B (Chido) structural genes.
A C Chan, J P Atkinson
Immunological evaluations (lymphocyte markers, B cell differentiation, T cell function) were performed on peripheral blood mononuclear cells from four individuals with hyper IgM immunodeficiency. Number, proportion, and proliferation of T lymphocytes and T lymphocyte subpopulations were relatively normal in affected individuals. The percentage and number of B cells expressing surface IgM and IgD were either normal or elevated in both blood and lymph nodes. However, surface IgG- and IgA-bearing B lymphocytes were completely absent. In vitro stimulation of blood lymphocytes with both T cell-dependent and T-cell independent polyclonal B cell activators resulted in normal numbers of IgM plasma cells and IgM secretion in cultures, but failed to induce any IgG- or IgA-producing cells. This failure of isotype switching was intrinsic to the B cell population and did not involve aberrant T cell help or suppression. Therefore, individuals with this disorder possess an intrinsic B cell dysfunction that is not related to abnormal T cell regulation.
D Levitt, P Haber, K Rich, M D Cooper
Isolated microvessels and isolated and cultured microvessel endothelial cells were prepared from rabbit cardiac muscle. Pathways of arachidonic acid metabolism were determined by measurement of exogenous substrate utilization [( 1-14C]arachidonic acid incorporation and release from intact tissue and cells; [1-14C]prostaglandin H2 (PGH2) metabolism by broken cell preparations) and by quantification of endogenous products (immunoreactive 6-keto-prostaglandin F1 alpha (PGF1 alpha) and prostaglandin E (PGE) release) by selective radioimmunoassay. Rabbit coronary microvessels and derived microvascular endothelial cells (RCME cells) synthesized two major products of the cyclooxygenase pathway: 6-keto-PGF1 alpha (hydrolytic product of prostaglandin I2) and PGE2. A reduced glutathione requiring PGH-E isomerase was demonstrated in coronary microvessels and RCME cells, but not in rabbit circumflex coronary artery or aorta. In addition, a minor amount of a compound exhibiting similar characteristics to 6-keto-PGE1 was found to be produced by microvessels and RCME cells. Measurement of endogenously released prostaglandins indicated that under basal and stimulated conditions, PGE release exceeded that of 6-keto-PGF1 alpha. Microvessels and microvessel endothelial cells derived from cardiac muscle of rabbit exhibit pathways of arachidonate metabolism that are different from those of many large blood vessels and derived endothelial cells.
M E Gerritsen, C D Cheli
Elastase is released from human neutrophils during the early events of blood coagulation. Human plasma kallikrein has been shown to stimulate neutrophil chemotaxis, aggregation, and oxygen consumption. Therefore, the ability of kallikrein to release neutrophil elastase was investigated. Neutrophils were isolated by dextran sedimentation, and elastase release was measured by both an enzyme-linked immunosorbent assay, and an enzymatic assay using t-butoxy-carbonyl-Ala-Ala-Pro-Val-amino methyl coumarin as the substrate. Kallikrein, 0.1-1.0 U/ml, (0.045-0.45 microM), was incubated with neutrophils that were preincubated with cytochalasin B (5 micrograms/ml). The release of elastase was found to be proportional to the kallikrein concentration. Kallikrein released a maximum of 34% of the total elastase content, as measured by solubilizing the neutrophils in the nonionic detergent Triton X-100. A series of experiments was carried out to determine if kallikrein was a major enzyme involved in neutrophil elastase release during blood coagulation. When 10 million neutrophils were incubated in 1 ml of normal plasma in the presence of 30 mM CaCl2 for 90 min, 2.75 micrograms of elastase was released. In contrast, neutrophils incubated in prekallikrein-deficient or Factor XII-deficient plasma released less than half of the elastase, as compared with normal plasma. The addition of purified prekallikrein to prekallikrein-deficient plasma restored neutrophil elastase release to normal levels. Moreover, release of elastase was enhanced in plasma deficient in C1-inhibitor, the major plasma inhibitor of kallikrein. This release was not dependent upon further steps in the coagulation pathway, or on C5a, since levels of elastase, released in Factor XI- or C5-deficient plasma, were similar to that in normal plasma, and an antibody to C5 failed to inhibit elastase release. These data suggest that kallikrein may be a major enzyme responsible for the release of elastase during blood coagulation.
Y T Wachtfogel, U Kucich, H L James, C F Scott, M Schapira, M Zimmerman, A B Cohen, R W Colman
Using a recently developed model of nasal challenge, we have obtained data that clearly demonstrate, for the first time, kinin generation during a local allergic reaction in vivo. Allergic individuals (n = 8) and matched nonallergic controls (n = 8) were challenged intranasally with the appropriate antigen and nasal washes were taken before and after challenge. Washes were assayed for kinin, histamine, and [3H]-N-alpha-tosyl-L-arginine methyl ester (TAME)-esterase activity. Increased kinin generation was found by radioimmunoassay (RIA) in the nasal washes of all the allergics (5,560 +/- 1,670 pg/ml) but in none of the controls (38 +/- 16 pg/ml). The presence of kinin was highly correlated with that of histamine and TAME-esterase activity and with the onset of clinical symptoms (P less than 0.001). Serial dilutions of nasal washes produced RIA displacement curves that paralleled the standard curve, and recovery of standard kinins that were added to nasal washes was 100 +/- 4% (n = 14). Kinin recovery was identical in both allergics and controls and did not vary significantly with antigen challenge. The immunoreactive kinin in nasal washes was stable to boiling and not precipitated by ethanol, but completely destroyed by carboxypeptidase B. It was evenly distributed between the sol and gel phases of nasal washes. High performance liquid chromatography analysis of the immunoreactive kinin in nasal washes showed it to be a mixture of lysylbradykinin and bradykinin. We conclude that kinins are produced during local allergic reactions in the nose and may contribute to the symptomatology of the allergic response.
D Proud, A Togias, R M Naclerio, S A Crush, P S Norman, L M Lichtenstein
It is currently unclear whether the T cell dysfunctions observed during active systemic lupus erythematosus (SLE) reflect a disorder intrinsic to the T cell or defects that result from interaction with anti-T cell autoantibody. To determine whether a disorder intrinsic to the T cell exists in SLE, the T cell capping mechanism was selected as a model of cellular function. The normal T cell capping mechanism is a rapid, energy-dependent and coordinated sequence of membrane events that consists of microaggregation, capping, endocytosis, and regeneration of the surface molecule. The monoclonal antibodies OKT3, OKT4, and OKT8, directed against the T cell-specific membrane glycoproteins T-3, T-4, T-8, served as specific probes of the glycoproteins' mobility within the membrane and membrane glycoprotein regeneration. When compared with greater than 91% T cell capping in normal and control subjects with active Sjögren's syndrome, active rheumatoid arthritis and active tuberculosis, only 49-60% of T cells from active SLE patients completed the capping sequence (SLE vs. healthy controls; T-3, P less than 0.002; T-4, P less than 0.004; T-8, P less than 0.002). Colchicine (10(-5) M), which inhibits microtuble polymerization and augments the rate of normal T cell capping, failed to restore the abnormal capping. However, as judged by the elapsed time intervals to half-maximal capping, the capping kinetics of the T cells able to initiate capping were not significantly different from controls. Fluorescence microscopy demonstrated an abnormal staining pattern characterized by microaggregation of ligand-glycoprotein complexes on resting T cells, coarse aggregation of ligand-glycoprotein complexes over the surfaces of cells that failed to cap, and cleaved or disrupted caps. After clearance of determinants by capping, greater than 94% of T cells from healthy controls regenerated T-3, -4, and -8 within 24 h. In contrast, only 20-40% of capped T cells from active SLE patients reexpressed new determinants. With improving disease activity, the proportion of cells capping and regenerating T-3, -4, and -8 increased, but remained significantly below control levels. In conclusion, this study has identified a disorder of T cell surface glycoprotein mobility and regeneration affecting the majority (60-80%) of both the T-3+, T-4+, (inducer/helper), and T-3+, T-8+ (suppressor) subsets during active SLE. Although the impaired capping and reexpression improve with disease remission, a residual defect persists. The data support the concept of a disorder intrinsic to the T cell in SLE.
G M Kammer
Our plan was to evaluate the potentially important role of phospholipids in erythrocyte shape alterations by determining if their orientation was altered during endocytosis. Stomatocytosis and endocytosis were induced in normal intact human erythrocytes by incubation with three agents: primaquine, vinblastine, and chlorpromazine, each of which has its own requirements and time course for producing endocytosis. The organization of the phospholipid bilayer was assessed by measuring the extent of degradation of phophatidylcholine (PC), phophatidylethanolamine (PE), phosphatidylserine (PS), and sphingomyelin (SM) produced by exposure of erythrocytes to a nonpenetrating protease-free phospholipase A2 alone or in combination with a purified sphingomyelinase as well. The induction of stomatocytosis did not change this orientation. However, correlating with the onset of endocytosis but not its extent, there was an increase in PE degradation, which could be detected regularly only by use of phospholipase A2 alone. Use of the combination of phospholipase A2 and sphingomyelinase showed that the extent and course of endocytosis was paralleled by an apparent movement of PC and SM from the outer to the inner half of the lipid bilayer. Since no further PE was hydrolyzed and because no PS was ever degraded, this inward movement of PC and SM did not represent the establishment of complete symmetry in the membrane. By adjusting the experimental design it was possible to implicate the endocytic process, and not insertion of drug in the membrane, as the cause of the alterations in phospholipid organization seen. Our findings indicate that the phospholipid orientation is very closely involved in the endocytosis process and that specific states of phospholipid asymmetry may be related to identifiable membrane events.
S L Schrier, D T Chiu, M Yee, K Sizer, B Lubin
Radioiodinated Factor IX was cleaved by a crude sonicate from leukocytes. In the absence of calcium, fragments of less than 15,000 mol wt were seen from reduced samples on gel electrophoresis. After digestion in 2 mM calcium, however, electrophoresis of reduced samples showed, in addition to low molecular weight fragments, protein bands corresponding in size to heavy and light chains of Factor XIa-activated Factor IX. The cleaving activity in leukocyte sonicates was inhibited by soybean trypsin inhibitor, but only to a small extent by aprotinin. Granulocyte elastase was isolated from purified polymorphonuclear leukocyte granules by affinity chromatography on soybean trypsin inhibitor-agarose and further chromatography on carboxymethyl cellulose. The purified fraction contained two isozymes on acidic gels which cleaved both an ester sensitive to elastase and radiolabeled Factor IX. These two activities were inhibited by elastase-specific chloromethyl ketone. The isolated protease fraction rapidly inactivated apparent Factor IX activity in a coagulant assay system. The degree of inactivation correlated with the amount of intact, radiolabeled Factor IX cleaved. As with the crude sonicate, generation of the larger heavy and light chain-sized fragments was dependent upon calcium. To assess directly the effect of elastase on Factor IX, an immunospecific, active site-directed assay was developed. In this assay, the sample was incubated with solid-phase antibody to Factor IX and the amount of activated product was detected as that which had complexed with radioiodinated antithrombin III. In this system, exposure of Factor IX to Factor XIa showed progressive increase in the ability to bind antithrombin III, whereas after elastase, Factor XIa was unable to generate antithrombin III binding. The elastase-degraded Factor IX did not inhibit activation of additional Factor IX in clotting assays. When Factor IXa was incubated with elastase, binding of antithrombin III was decreased, corresponding to appearance of low molecular weight fragments on parallel samples that were reduced and electrophoresed. These data are consistent with elastase inactivating Factor IX by cleaving bonds near, but distinct from, bonds cleaved by Factor XIa.
A Takaki, D L Enfield, A R Thompson
The rate of blood flow entering a capillary network can, in some vascular systems, regulate capillary surface area and the rate of fluid and solute transfer. To determine whether such a mechanism exists in the renal peritubular capillary, we performed micropuncture studies in 28 rats during relatively low and high efferent arteriolar blood flow (EABF). High EABF was achieved by intravenous infusion of isoncotic plasma (group 1: from 120 +/- 11 to 301 +/- 49 nl/min [+/- SE]); whole blood with high hematocrit (approximately 75 vol %) (group 2: from 141 +/- 14 to 252 +/- 31 nl/min); or acetylcholine (group 3: from 193 +/- 20 to 266 +/- 26 nl/min). In group 1 rats, plasma infusion caused an increase in single nephron glomerular filtration rate (SNGFR), on average, from 23.2 +/- 2.4 to 45.2 +/- 3.9 nl/min, owing primarily to increased glomerular plasma flow rate (from 63 +/- 5 to 210 +/- 21 nl/min). The rate of fluid uptake by the peritubular capillary, assessed by the absolute rate of proximal fluid reabsorption (APR), also rose significantly, on average from 10.5 +/- 1.2 to 17.5 +/- 2.4 nl/min. This rise in APR was associated with near constancy in mean transcapillary hydraulic (delta Pc) and oncotic (delta IIc) pressure differences, and was therefore attributed to a significant increase in peritubular capillary reabsorption coefficient (Kr), with the mean from 0.017 +/- 0.003 to 0.030 +/- 0.005 nl/(s . mmHg). In group 2 rats, high hematocrit blood infusion led to a significant rise in APR; on average, from 10.7 +/- 0.7 to 15.0 +/- 1.2 nl/min, without changing SNGFR. This rise in APR occurred despite unfavorable changes in the physical forces, namely a significant increase in delta Pc and constancy in delta IIc. Instead, an increase in EABF was again associated with a significant rise in Kr (on average, from 0.016 +/- 0.002 to 0.030 +/- 0.06 nl/[s . mmHg]), which accounted entirely for the rise in APR, independently of SNGFR. In group 3 rats, in which an increase of EABF was induced pharmacologically with acetylcholine, a rise in EABF was also accompanied by a significant increase in Kr, on average, from 0.019 +/- 0.002 to 0.026 +/- 0.004 nl/(s . mmHg). The results indicate that: (a) Kr is modulated by EABF. (b) In view of plasma flow dependence of GFR, blood flow dependence of Kr and APR provides an important basis for glomerulotubular balance.
V Kon, M L Hughes, I Ichikawa
The insulin-binding isotherms and the structural composition of human liver insulin receptors were examined by using plasma membranes that were prepared from liver biopsies of nine non-obese and 10 obese subjects undergoing elective surgery. The insulin-binding characteristics of liver membranes from non-obese subjects were quite similar to those previously described in rat liver membranes. However, when the membranes from obese subjects were compared with the non-obese group, insulin-binding activity was reduced by 50% (P less than 0.01). The reduction in obesity resulted primarily from a decrease in total receptor number, although a small decrease in receptor affinity was also observed. Insulin binding was not correlated with sex or with the fasting plasma insulin level. The insulin-binding sites of liver membranes were affinity-labeled with 125I-insulin and the cross-linking reagent, disuccinimidyl suberate. The liver membranes from both the non-obese and the obese group had heterogenous (nonreduced) insulin-binding species of 300,000, 260,000, and 150,000 mol wt, which were again comparable to the findings reported in rat liver. Sulfhydryl reduction demonstrated a major sub-unit of 125,000 and a minor component of 40,000-45,000 in both groups. These results indicate a close similarity between the hepatic insulin receptor of man and the more intensely studied rat hepatic receptor. Obesity in human subjects is associated with a loss of hepatic insulin receptors. This alteration may contribute to the insulin resistance reported in this organ as well as to obesity-mediated glucose tolerance.
P Arner, K Einarsson, L Backman, K Nilsell, K M Lerea, J N Livingston
Since the initial proposal of the glucose fatty acid cycle, considerable controversy has arisen concerning its physiologic significance in vivo. In the present study, we examined the effect of acute, physiologic elevations of FFA concentrations on glucose production and uptake in normal subjects under three controlled experimental conditions. In group A, plasma insulin levels were raised and maintained at approximately 100 microU/ml above base line by an insulin infusion, while holding plasma glucose at the fasting level by a variable glucose infusion. In group B, plasma glucose concentration was raised by 125 mg/100 ml and plasma insulin was clamped at approximately 50 microU/ml by a combined infusion of somatostatin and insulin. In group C, plasma glucose was raised by 200 mg/100 ml above the fasting level, while insulin secretion was inhibited with somatostatin and peripheral glucagon levels were replaced with a glucagon infusion (1 ng/min X kg). Each protocol was repeated in the same subject in combination with a lipid-heparin infusion designed to raise plasma FFA levels by 1.5-2.0 mumol/ml. With euglycemic hyperinsulinemia (study A), lipid infusion caused a significant inhibition of total glucose uptake (6.3 +/- 1.3 vs. 7.4 +/- 0.6 mg/min X kg, P less than 0.02). Endogenous glucose production (estimated by the [3-3H]glucose technique) was completely suppressed both with and without lipid infusion. With hyperglycemic hyperinsulinemia (study B), lipid infusion also induced a marked impairment in glucose utilization (6.2 +/- 1.1 vs. 9.8 +/- 1.9 mg/min X kg, P less than 0.05); endogenous glucose production was again completely inhibited despite the increase in FFA concentrations. Under both conditions (A and B), the percentage inhibition of glucose uptake by FFA was positively correlated with the total rate of glucose uptake (r = 0.69, P less than 0.01). In contrast, when hyperglycemia was associated with relative insulinopenia and hyperglucagonemia (study C), thus simulating a diabetic state, lipid infusion had no effect on glucose uptake (2.9 +/- 0.2 vs. 2.6 +/- 0.2 mg/min X kg) but markedly stimulated endogenous glucose production (1.4 +/- 0.5 vs. 0.5 +/- 0.4 mg/min X kg, P less than 0.005). Under the same conditions as study C, a glycerol infusion producing plasma glycerol levels similar to those achieved with lipid-heparin, enhanced endogenous glucose production (1.5 +/- 0.5 vs. 0.7 +/- 0.6 mg/min X kg, P less than 0.05). We conclude that, in the well-insulinized state raised FFA levels effectively compete with glucose for uptake by peripheral tissues, regardless of the presence of hyperglycemia. When insulin is deficient, on the other hand, elevated rates of lipolysis may contribute to hyperglycemia not by competition for fuel utilization, but through an enhancement of endogenous glucose output.
E Ferrannini, E J Barrett, S Bevilacqua, R A DeFronzo
We infused tracer-labeled l-[3H]-norepinephrine, d-[14C]norepinephrine, and d,l-[3H]-isoproterenol simultaneously into patients with essential hypertension and into normotensive control subjects, in order to determine whether abnormalities in the disappearance kinetics of these substances characterized the hypertensive patients. The mean preinfusion venous plasma norepinephrine concentration was somewhat higher in the hypertensive group (260 vs. 194 pg/ml, P = 0.06), but the groups did not differ in the disappearance kinetics of l- or d-norepinephrine or of isoproterenol. Preinfusion plasma norepinephrine was significantly positively correlated with calculated spillover rates in both the hypertensive and normotensive groups, but not with norepinephrine clearances. The d/l ratio in plasma norepinephrine was the same as in the infusate during and after the infusion, even after pretreatment with the neuronal norepinephrine uptake blocker, desipramine. Because isoproterenol is not taken up by nerve endings, the ratio of [3H]isoproterenol to l-[3H]norepinephrine increased after the infusion ended. This increase was almost completely abolished by pretreatment with desipramine. These results indicate that (a) increased plasma norepinephrine levels seen in some patients with essential hypertension result from increased sympathetic neural activity and not from decreased clearance of norepinephrine, (b) changes in the isoproterenol/norepinephrine ratio after simultaneous infusion of both provide an index of neuronal norepinephrine uptake in man, and (c) neuronal norepinephrine uptake is not stereospecific.
D S Goldstein, D Horwitz, H R Keiser, R J Polinsky, I J Kopin
Platelet-derived growth factor (PDGF) stimulates both proliferation of fibroblasts and chemotaxis of leukocytes. In this study we compared the mitogenic and chemotactic activities of native PDGF and reduced PDGF. Reduction of PDGF (Mr = 32,000) to its constituent polypeptides (Mr = 14,000 and 17,000) caused a loss of the ability to stimulate proliferation of Balb/c 3T3 cells. However, reduced PDGF retained virtually all of its activity as a chemotactic agent for human neutrophils and monocytes. A half-maximal chemotactic response to both native and reduced PDGF occurred at a concentration of approximately 0.08 nM for neutrophils and 0.1 nM for monocytes. The maximal chemotactic response to reduced PDGF was at least as great as the maximal response to native PDGF. Both native and reduced PDGF stimulated the release of the lysosomal enzyme, beta-glucosaminidase, from neutrophils with a half-maximal response at less than 0.1 nM. However, the net maximum release of this enzyme by PDGF (and reduced PDGF) was significantly less than that stimulated by a maximal concentration of the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine. These results indicate that different structural determinants are required for the proliferative response of 3T3 cells to PDGF and for the chemotactic response of leukocytes to PDGF.
L T Williams, H N Antoniades, E J Goetzl
Rapid dissociation of organic anions from plasma albumin maximizes the presentation of free ligand to the cell surface and thus favors its efficient hepatic extraction. Even assuming these optimal conditions, however, taurocholate and rose bengal have hepatic extraction fractions that are higher than can be accounted for by spontaneous dissociation of their albumin-ligand complexes. In this study we developed a transport model that attributes this behavior to sites on the hepatocyte plasma membrane that bind the albumin-ligand complexes, promoting the transport of ligand into the hepatocyte. Fitting this model to rose bengal removal rates measured over a wide range of albumin concentrations yields estimates of the number of cell surface sites and their affinity for albumin. These estimates are in good agreement with those reported by Weisiger, Gollan, and Ockner for the binding of ligand-free albumin to isolated hepatocytes. We conclude that both experiments measure the same phenomenon and, accordingly, that the binding of albumin to the cell surface is the functional equivalent of albumin-mediated transport.
E L Forker, B A Luxon
The dihydrotestosterone content of normal peripheral and benign hyperplastic prostates was measured in tissue obtained at open surgical procedures on 29 men of ages 36 to 82 yr. The dihydrotestosterone content in normal prostates (mean +/- SE, 5.1 +/- 0.4 ng/g tissue) and in benign hyperplastic prostates (5.0 +/- 0.4) was similar. In 11 patients in whom both normal and hyperplastic prostatic tissue was harvested simultaneously at the same operation, there was no significant difference in the content of dihydrotestosterone in the two types of tissue. These findings fail to confirm the widespread belief that dihydrotestosterone content is elevated in benign hyperplastic prostates. Our data differ from the reported literature in one major respect: the dihydrotestosterone content of normal peripheral prostate in this study is three to four times higher than previously reported. This difference between the present and earlier studies was resolved by experiments performed on cadavers, which were the source of normal prostatic tissue used by other investigators. Dihydrotestosterone content was measured in seven cadavers ranging in age from 19 to 82 yr of age. The results of this experiment indicate that the dihydrotestosterone content of prostatic tissue removed at autopsy is factitiously low (0.7-1.0 ng/g tissue). This finding was confirmed by in vitro incubations of fresh prostatic tissue at 37 degrees C that demonstrated reduction of dihydrotestosterone content to low levels within 2 h. When taken together, these results indicate that when prostatic tissue is harvested appropriately, the dihydrotestosterone content of normal peripheral and hyperplastic tissues is the same. This finding should influence future research into the etiology of benign prostatic hyperplasia.
P C Walsh, G M Hutchins, L L Ewing
The local conversion of thyroxine (T4), which is an important source of intracellular 3,5,3'-triiodothyronine (T3) in several rat tissues, has been subject of recent investigations. In the present study the regulation of this phenomenon in vivo was investigated in various peripheral tissues of the rat. Intact euthyroid and radiothyroidectomized (Tx) rats received a continuous intravenous infusion of [125I]T4 and [131I]T3 until isotope equilibrium was attained. In addition to the labeled iodothyronines, Tx rats received a continuous intravenous infusion of 0.2 or 1.0 microgram carrier T4/100 g body wt per d, to create hypothyroid or slightly hypothyroid conditions, respectively. After the animals were bled and perfused the contribution of T3 derived from local conversion of T4 to T3 [Lc T3(T4)] to the total T3 in homogenates from several tissues and subcellular fractions from the liver, kidney, and anterior pituitary gland could be calculated. In all experiments T3 in muscle was derived exclusively from the plasma. In the cerebral cortex and cerebellum, however, most of the intracellular T3 was derived from the intracellular conversion of T4 to T3. It is demonstrated that for hypothyroid rats an increased relative contribution of Lc T3(T4) reduced the loss of total T3 in the brain. This phenomenon was also encountered for the anterior pituitary gland, although in this tissue the proportion of the total tissue T3, contributed by locally produced T3 was considerably lower than the values found for the cerebral cortex and cerebellum in all experiments. The present findings, regarding the source and quantity of pituitary nuclear T3 strongly suggest that both plasma T3 and T4 (through its local conversion into T3) play a role in the regulation of thyrotropin secretion. The contribution of Lc T3(T4) to the total pituitary nuclear T3 was of minor importance in euthyroid rats (approximately 20%), compared with that found for both groups in T4-supplemented athyreotic rats (approximately 40%). The total T3 concentration in the liver decreased from euthyroid to hypothyroid rats and was associated with a decrease in the tissue/plasma T3 concentration gradient. A minor proportion of hepatic T3 was contributed by Lc T3(T4), which in fact decreased significantly from the euthyroid to the hypothyroid state. In contrast to other subcellular fractions from the liver, no Lc T3(T4) could be demonstrated in the nuclear fraction. It is suggested that the liver plays an important role with respect to regulation of the circulating T3 concentration. In the kidney, a very small proportion of the total T3 was derived from locally produced T3 in all experiments (4-7%). As found in the liver, all nuclear T3 appeared to be derived from the plasma. In contrast to the liver, subcellular T3 pools in the kidney seemed to be exchangeable.
J van Doorn, D van der Heide, F Roelfsema
Degranulation of lysosomes is one of the consequences of neutrophil activation. Regulatory mechanisms of lysosomal secretion are thought to be localized largely in the plasma membrane and cytosol, with the lysosome playing a passive role in secretion. Recent evidence indicates that the intralysosomal pH is highly acidic (pH congruent to 5.5) and is maintained by active transport of H+. We investigated whether changes in the intralysosomal pH altered the availability of lysosomes for exocytosis. Intralysosomal pH in intact neutrophils was monitored with the weakly basic fluorescent probe, 9-aminoacridine (9AA). The weak bases, methylamine, chloroquine, clindamycin, propanolol, and ammonium chloride (0.1-50 mM), caused an alkalinization of the intralysosomal pH as determined by reversal of quenching of 9AA fluorescence. Similarly, each of the weak bases, including ammonium chloride, methylamine, chloroquine, ethylamine, propylamine, propanolol, clindamycin, and dansylcadaverine, inhibited neutrophil degranulation in response to the calcium ionophore A23187, phorbol myristate acetate, or the chemotactic peptide, formyl-methionine-leucine-phenylalanine plus cytochalasin B. These studies indicate that an acid intralysosomal pH is important to the neutrophil secretory response and suggest that the lysosome may play an active part in control of degranulation.
M S Klempner, B Styrt
Interstitial lung disorders are characterized both by a chronic inflammation of the lower respiratory tract that includes increased numbers of activated alveolar macrophages and by increased numbers of fibroblasts within the alveolar wall. Since alveolar macrophages from normal individuals can be activated to release a growth factor for lung fibroblasts (alveolar macrophage-derived growth factor [AMDGF]), we hypothesized that the activated alveolar macrophages within the lower respiratory tract of patients with fibrotic lung disorders might be spontaneously releasing AMDGF. To evaluate this hypothesis, alveolar macrophages (suspension culture, 4 h, 37 degrees) from 65 patients with interstitial lung disorders and 30 control subjects were examined for the spontaneous release of fibroblast growth-promoting activity, with human lung fibroblasts as the target. Whereas none of the controls had macrophages spontaneously releasing a growth-promoting activity for fibroblasts, 82% of the patients with interstitial lung disease had alveolar macrophages that were spontaneously releasing a growth-promoting activity for fibroblasts. In common with AMDGF, the fibroblast growth-promoting activity released by these macrophages eluted from DEAE cellulose at 270 mM NaCl, had a partition coefficient of 0.3 by gel filtration on Sephadex G-50, was distinct from other characterized growth factors, and acted as a progression factor for fibroblast replication in a serum-free complementation test. These data suggest that the expansion of fibroblast numbers within the alveolar structures in interstitial lung disorders may result, in part, from the release of AMDGF by alveolar macrophages stimulated in vivo.
P B Bitterman, S Adelberg, R G Crystal
Prednisone-induced insulin resistance may depend on either reduced sensitivity (receptor defect) or reduced response to insulin (postreceptor defect). To clarify the mechanism of prednisone-induced insulin resistance, a [3H]glucose infusion (1 microCi/min) was performed for 120 min before and during a euglycemic clamp repeated at approximately 100, approximately 1,000, and approximately 10,000 microU/ml steady state plasma insulin concentration in 10 healthy, normal weight subjects, aged 35 +/- 7 yr. Each test was repeated after 7-d administration of placebo or prednisone (15 plus 15 mg/d per subject), in a randomized sequence with an interval of 1 mo between the two tests. Mean fasting blood glucose (89.5 +/- 2.1 vs. 83.7 +/- 1.9 mg/dl) and mean fasting plasma insulin values (17.8 +/- 1.2 vs. 14.3 +/- 0.8 microU/ml) were significantly higher (P less than 0.01) after prednisone. The insulin sensitivity index (glucose metabolic clearance rate in ml/kg per min) was significantly lower (P less than 0.001) after prednisone at all three steady state plasma insulin levels: 2.8 +/- 0.3 vs. 7.4 +/- 1.1 at approximately 100 microU/ml; 6.0 +/- 0.5 vs. 12.2 +/- 1.1 at approximately 1,000 microU/ml; 7.4 +/- 0.6 vs. 14.4 +/- 0.5 at approximately 10,000 microU/ml. Fasting glucose production (in mg/kg per min) was significantly higher after prednisone: 3.7 +/- 0.2 vs. 2.9 +/- 0.2, P less than 0.001. Suppression of glucose production at steady state plasma insulin level of approximately 100 microU/ml was less after prednisone (1.01 +/- 0.35 vs. 0.14 +/- 0.13, NS), and total at approximately 1,000 and approximately 10,000 microU/ml after both prednisone and placebo. The metabolic kinetic parameters of insulin after prednisone were not significantly different from those after placebo. In addition, insulin binding and 3-ortho-methyl-glucose transport were studied in vitro on fat cells from 16 normal-weight surgical candidates aged 40 +/- 8 yr (10 treated with placebo and 6 with prednisone as above). No significant difference was observed with regard to specific insulin binding (tested with 1 ng/ml hormone only), whereas significant transport differences were noted at the basal level (0.40 +/- 0.10 vs. 0.54 +/- 0.12 pmol/10(5) cells, P less than 0.05), and at increasing concentrations up to the maximum stimulation values (5 ng/ml): 0.59 +/- 0.04 vs. 0.92 +/- 0.12 pmol/10(5) cells, P less than 0.005. These results suggest that (a) administration of an anti-inflammatory dose of prednisone for 7 d induces insulin resistance in man; (b) this is more dependent on depressed peripheral glucose utilization than on increased endogenous production; (c) total insulin binding on isolated adipocytes is not significantly affected; (d) insulin resistance is primarily the outcome of postreceptor defect (impaired glucose transport).
G Pagano, P Cavallo-Perin, M Cassader, A Bruno, A Ozzello, P Masciola, A M Dall'omo, B Imbimbo
Although alcoholism is a leading cause of morbidity and mortality of middle-aged Americans, there are no data available pertaining to the consequences of Laennec's cirrhosis on total body energy requirements or mechanisms for maintaining fuel homeostasis in this patient population. Therefore, we simultaneously used the techniques of indirect calorimetry and tracer analyses of [14C]palmitate to measure the nature and quantity of fuels oxidized by patients with biopsy-proven alcoholic cirrhosis and compared the results with values obtained from health volunteers. Cirrhotic patients were studied after an overnight fast (10-12 h). Normal volunteers were studied after an overnight fast (12 h) or after a longer period of starvation (36-72 h). Total basal metabolic requirements were similar in overnight fasted cirrhotic patients (1.05 +/- 0.06 kcal/min per 1.73 m2), overnight fasted normal subjects (1.00 +/- 0.05 kcal/min per 1.73 m2), and 36-72-h fasted normal volunteers (1.10 +/- 0.06 kcal/min per 1.73 m2). Indirect calorimetry revealed that in cirrhotic patients the percentages of total calories derived from fat (69 +/- 3%), carbohydrate (13 +/- 2%), and protein (17 +/- 4%) were comparable to those found in 36-72-h fasted subjects, but were clearly different from those of overnight fasted normal individuals who derived 40 +/- 6, 39 +/- 4, and 21 +/- 2% from fat, carbohydrate, and protein, respectively. These data are strikingly similar to data obtained through tracer analyses of [14C]palmitate, which showed that in overnight fasted patients with alcoholic cirrhosis, 63 +/- 4% of their total CO2 production was derived from oxidation of 287 +/- 28 mumol free fatty acids (FFA)/min per 1.73 m2. In contrast, normal overnight fasted humans derived 34 +/- 6% of their total CO2 production from the oxidation of 147 +/- 25 mumol FFA/min per 1.73 m2. On the other hand, values obtained from the normal volunteers fasted 36-72 h were similar to the overnight fasted cirrhotic patients. These results show that after an overnight fast the caloric requirements of patients with alcoholic cirrhosis are normal, but the nature of fuels oxidized are similar to normal humans undergoing 2-3 d of total starvation. Thus, patients with alcoholic cirrhosis develop the catabolic state of starvation more rapidly than do normal humans. This disturbed but compensated pattern for maintaining fuel homeostasis may be partly responsible for the cachexia observed in some patients with alcoholic cirrhosis. This study also showed remarkably good agreement between the results obtained with indirect calorimetry and those obtained with 14C tracer analyses.
O E Owen, V E Trapp, G A Reichard Jr, M A Mozzoli, J Moctezuma, P Paul, C L Skutches, G Boden
Prolonged cold storage of plasma may induce the conversion of plasma prorenin (inactive renin) to renin. This phenomenon is exaggerated in oral contraceptive (OC) users; the titer of Hageman factor (HF, Factor XII) in OC users is higher than in nonusers. The present study relates these observations. The increment in plasma renin activity (PRA) during cold storage, as measured by generation of angiotensin I, correlated strongly with the initial plasma titer of HF. Increasing the HF titer of nonusers to that observed in OC users by addition of purified HF increased cold-induced PRA at least twofold, while reducing the plasma HF titer of OC users correspondingly decreased cold-induced PRA. Thus, in OC users, the enhanced conversion of plasma prorenin to renin during cold storage reflects the elevated plasma titer of HF.
E M Gordon, J Douglas, O D Ratnoff
Polymorphonuclear leukocytes (PMNL) isolated from two patients with chronic granulomatous disease (CGD) were tested for their ability to metabolize arachidonic acid to lipoxygenase products including 5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid (LTB4). Analyses of incubations of these PMNL with arachidonic acid and the calcium ionophore A23187 did not differ from simultaneous controls in the production of LTB4, other 5,12-dihydroxy-eicosatetraenoic acids, or monohydroxy-eicosatetraenoic acids. The clinical diagnosis of CGD was confirmed in both cases by determination of PMNL chemiluminescence. Leukocytes from both patients failed to generate active oxygen species in response to either LTB4 or formyl-methionyl-leucyl-phenylalanine. The observation of arachidonic acid oxidation in the absence of superoxide anion precludes a role for the active oxygen species in this metabolic process. These studies clearly dissociate the ionophore-induced leukocyte respiratory burst from the oxidation of arachidonate to the leukotrienes. In addition, the defect of CGD appears to be unrelated to the ability of PMNL to carry out arachidonate oxygenation.
S J Feinmark, A M Udén, J Palmblad, C Malmsten
Using a monoclonal antibody to bromodeoxyuridine (BUdR) and immunohistochemistry, we measured the incorporation of this thymidine analogue into the DNA of human normal and malignant cells exposed in vivo. BUdR given as a constant intravenous infusion for 12 or 24 h daily for up to 13 d resulted in a steady-state plasma level of 10(-6) M during the infusion. We demonstrated extensive incorporation of BUdR into both normal skin, normal bone marrow, and malignant melanoma cells. In addition, this infusion of BUdR was adequate to identify sister chromatid exchanges from human marrow chromosomes exposed in vivo. Using this constant infusion, significant but reversible (acute) toxicity was observed with myelosuppression and skin photosensitivity. These techniques, which are considerably less cumbersome and time-consuming than the use of radioactive isotopes of thymidine, can be used for further human studies of cell kinetics and chromosomal replication in both normal and malignant cells.
G Morstyn, S M Hsu, T Kinsella, H Gratzner, A Russo, J B Mitchell
DNA complementary to bovine preproparathyroid hormone mRNA was cloned and labeled by nick translation in order to measure mRNA by molecular hybridization. Bovine parathyroid cells were maintained in primary tissue culture for periods up to 96 h at 0.5 mM, 1.25 mM, and 2.5 mM calcium, which was followed by extraction of cellular RNA. Levels of mRNA showed no differences at 0.5 or 1.25 mM calcium, but at high calcium levels, there was a reversible decrease that began at 16 h to a plateau at 30% of control after 72 h. These studies suggest that the glandular capacity to synthesize hormone may be at or near maximal at normal calcium, but at high calcium, there is a decrease over time in steady state levels of mRNA.
J Russell, D Lettieri, L M Sherwood
Metabolism of [3H]25-hydroxyvitamin D3(25-OH-D3) was studied in primary cultures of pulmonary alveolar macrophages (PAM) from seven patients with sarcoidosis and two patients with idiopathic pulmonary fibrosis. Production of a [3H]1,25-dihydroxyvitamin D3 (1,25-[OH]2-D3)-like metabolite of [3H]25-OH-D3 was detected in lipid extracts of cells from five patients with sarcoidosis. Synthesis of this compound in vitro was limited to viable PAM and was greatest in cells derived from a patient with hypercalcemia and an elevated serum concentration of 1,25-dihydroxyvitamin D. The tritiated PAM metabolite coeluted with authentic 1,25-(OH)2-D3 in three different solvent systems on straight-phase high performance liquid chromatography (HPLC) and demonstrated binding to extracted receptor for 1,25-(OH)2-D3, which was identical to that of commercially available [3H]1,25-(OH)2-D3 of comparable specific activity. Incubation of PAM with high concentrations of 25-OH-D3 resulted in production of an unlabeled metabolite that co-chromatographed with the 3H-PAM metabolite on HPLC and that was bound with high affinity by both the specific receptor for 1,25-(OH)2-D3 and antiserum to 1,25-(OH)2-D3.
J S Adams, O P Sharma, M A Gacad, F R Singer