Changes in the oral intake of phosphorus could induce the reported changes in the serum concentration of 1,25-dihydroxyvitamin D (1,25-(OH)2D) by inducing changes in its production rate (PR) or metabolic clearance rate (MCR), or both. To investigate these possibilities, we employed the constant infusion equilibrium technique to measure the PR and MCR of 1,25-(OH)2D in six healthy men in whom the oral intake of phosphorus was initially maintained at 1,500 mg/70 kg body weight per d for 9 d, then restricted to 500 mg/d (coupled with oral administration of aluminum hydroxide) for 10 d, and then supplemented to 3,000 mg/d for 10 d. With phosphorus restriction, the serum concentration of 1,25-(OH)2D increased by 80% from a mean of 38 +/- 3 to 68 +/- 6 pg/ml, P less than 0.001; the PR increased from 1.8 +/- 0.2 to 3.8 +/- 0.6 micrograms/d, P less than 0.005; the MCR did not change significantly. The fasting serum concentration of phosphorus decreased from 3.5 +/- 0.2 to 2.6 +/- 0.2 mg/dl, P less than 0.01. With phosphorus supplementation, the serum concentration of 1,25-(OH)2D decreased abruptly, reaching a nadir within 2 to 4 d; after 10 d of supplementation, the mean concentration of 27 +/- 4 pg/ml was lower by 29%, P less than 0.01, than the value measured when phosphorus intake was normal. The PR decreased to 1.3 +/- 0.2 micrograms/d, P less than 0.05; the MCR did not change significantly. The fasting serum concentration of phosphorus increased significantly, but only initially. These data demonstrate that in healthy men, reductions and increases in the oral intake of phosphorus can induce rapidly occurring, large, inverse, and persisting changes in the serum concentration of 1,25-(OH)2D. Changes in the PR of 1,25-(OH)2D account entirely for the phosphorus-induced changes in serum concentration of this hormone.
A A Portale, B P Halloran, M M Murphy, R C Morris Jr
The expression of HLA-DR antigens by normal myeloid progenitor cells (CFU-GM) has been linked to inhibition of colony growth by prostaglandin E (PGE), while resistance to the inhibitory effects of PGE in chronic myeloid leukemia (CML) has been attributed to a lower fraction of HLA-DR+ CFU-GM in this disease. However, we have previously shown that virtually all CFU-GM in normal bone marrow (NBM) as well as CML peripheral blood express HLA-DR antigens, which raises the possibility that these surface molecules may not be the sole determinants of a progenitor cell's sensitivity to PGE. In order to evaluate the relationship between HLA-DR expression and prostaglandin inhibition, we partially purified NBM progenitor cells using fluorescence-activated cell sorting to prepare cell fractions with high and low HLA-DR antigen density. Normal progenitor cells with high DR density tended to form monocyte colonies in agar culture, whereas the low DR density fraction was enriched for granulocyte colony-forming cells. Inhibition by PGE was greatest in the high DR+ fraction and was largely restricted to monocyte progenitor cells. Inhibition of CFU-GM by PGE was less in CML than in NBM, but this decreased inhibition correlated with a significantly lower number of monocyte-CFU in CML. These data suggest that high HLA-DR antigen density may select for normal progenitor cells that are committed to monocyte differentiation and are, therefore, more likely to be inhibited by PGE. The relative deficit of monocyte progenitor cells in CML may partially explain the phenomenon of PGE resistance in this disease.
S A Cannistra, F Herrmann, R Davis, K Nichols, J D Griffin
Calcium precipitation in bile is a requisite event in the initiation and growth of all pigment gallstones. Calcium solubility in bile is thus of great importance. This is the first attempt to define the ion-product of CaCO3 in bile in any species. If the ion-product: [Ca++] X [CO = 3] exceeds solubility product (K'sp), the sample is supersaturated and CaCO3 precipitation is thermodynamically possible. We have recently determined K'sp of calcite to be 3.76 X 10(-8) mol/liter at 37 degrees C and total ionic strength = 0.16 M. Gallbladder (GB) bile was obtained from 15 anesthetized dogs after 12-24-h fasts. Duct bile was obtained from three dogs (n = 12) during variable taurocholate infusion. Samples were assayed for pH, partial pressure of carbon dioxide (PCO2), total bile salt concentration ([TBS]), total calcium concentration ([Ca]), and free calcium ion concentration ([Ca++]). With increasing [TBS] in both GB and duct bile, there was a linear decline in pH, a curvilinear decline in [HCO-3] and [CO = 3], and linear increase in [Ca++] and [Ca]. All ductular samples were supersaturated with CaCO3, with saturation indices (SI) as high as 17.5 and a mean of 8.36 +/- 1.43 (SE). In sharp contrast, none of the GB samples were supersaturated, due to the marked decline in [CO = 3] upon concentration and acidification of bile. In GB bile, the SI ranged from 0.006 to 0.126, with a mean of 0.039 +/- 0.011. The gallbladder thus produced a change in the SI from a value as high as 17.5 to a value as low as 0.006 in concentrated GB bile, which is a nearly 3,000-fold change. The average change in the SI was approximately 215-fold. Since all duct samples were supersaturated, and since the dog does not normally form gallstones, the data support our previous hypotheses that: (a) in canine bile, as in canine pancreatic juice, a nucleating factor is necessary for CaCO3 precipitation; (b) bile salts are important buffers for Ca++ in bile; and (c) normal GB mucosal function (concentration and acidification of bile) plays an important role in reducing CaCO3 lithogenicity in GB bile.
R V Rege, E W Moore
A large proportion of sickle erythrocytes is removed from the circulation by the macrophages of the reticuloendothelial system. In view of the proposed role for natural antibodies in the destruction of normal senescent erythrocytes, we looked for a possible similarity in the antibodies that bind in situ to senescent and sickle cells. Bound IgG molecules were detected by a highly sensitive rosetting antiglobulin test, using K562 myeloid cells. After separation on Stractan density gradients, the 0.6% most dense (senescent) normal cells and the most dense 40% sickle cells displayed membrane-bound IgG as reflected by the high proportion of rosettes formed. No antibody was found on low-density cells of either type. The bound antibodies were readily eluted from both sickle and normal senescent cells by carbohydrates containing alpha-galactosyl residues. These antibodies appear identical to the recently discovered human natural anti-alpha-galactosyl IgG (anti-Gal), an IgG antibody present in high titers in normal sera. Moreover, affinity-purified anti-Gal interacted specifically with sickle and normal cells depleted of the autologous antibodies. A similar pattern of binding to the various erythrocyte subpopulations was observed when the radiolabeled lectin with anti-alpha-galactosyl specificity, Bandeiraea simplicifolia, was used. In vitro phagocytosis of normal and sickle erythrocyte subpopulations correlated with the presence of anti-Gal on these cells. The in situ binding of anti-Gal to a large proportion of sickle erythrocytes may reflect an accelerated physiologic aging process by which immune recognition of prematurely exposed alpha-galactosyl-bearing antigenic sites contributes to shortened cell survival.
U Galili, M R Clark, S B Shohet
Biologically active fragments from polymorphonuclear leukocytes (PMN) are simplified systems that can be used to elucidate specific pathways by which cell function is altered. In the current study we have found that cytokineplasts, which are motile fragments derived from the leading front (protopod, lamellipodium) of human PMN, rapidly increase their intracellular free calcium concentration when stimulated by chemotactic formyl peptide or by leukotriene B4, as measured by Quin-2 acetoxymethyl ester fluorescence. As in the parent cell, extracellular EGTA blunts this response only partially. Hence, cytokineplasts retain a mobilizable internal calcium pool, despite a general lack of intracellular organelles. In addition, formyl peptide more than doubles the amount of cytoskeleton-associated (polymerized) actin. In contrast, cytoplasts made by high-speed, discontinuous gradient centrifugation of cytochalasin B-treated leukocytes also increase their intracellular free calcium on stimulation, but cytoskeleton-associated actin increases by only approximately 14%. Thus, defective motile function in the latter cytoplast is associated with compromised effector function (actin polymerization).
D E Dyett, S E Malawista, P H Naccache, R I Sha'afi
Glutathione synthetase (GSH-S) is one of the two known hereditary causes of glutathione deficiency. We describe a family whose two children have hemolytic anemia. The children's erythrocytes lack GSH and are severely deficient in GSH-S activity. No neurologic findings or 5-oxoprolinuria were present. A concurrent deficiency of glutathione-S-transferase (GST) was also detected in the erythrocytes. Residual glutathione could be detected in the erythrocytes using a sensitive cycling assay. The deficiency was found to be most severe in reticulocyte-depleted preparations. The GSH-S activity of the erythrocytes of the parents was one-half normal, while the glutathione S-transferase activity was normal. We conclude that the primary defect is one of GSH-S. Glutathione stabilizes GST in vitro, and it is assumed that the deficiency of GST in the erythrocytes of the patients is due to the instability of this enzyme in the absence of adequate intracellular GSH levels.
E Beutler, T Gelbart, C Pegelow
Using a continuous intravenous infusion of D-(-)-3-hydroxy[4,4,4-2H3]butyrate tracer, we measured total ketone body transport in 12 infants: six newborns, four 1-6-mo-olds, one diabetic, and one hyperinsulinemic infant. Ketone body inflow-outflow transport (flux) averaged 17.3 +/- 1.4 mumol kg-1 min-1 in the neonates, a value not different from that of 20.6 +/- 0.9 mumol kg-1 min-1 measured in the older infants. This rate was accelerated to 32.2 mumol kg-1 min-1 in the diabetic and slowed to 5.0 mumol kg-1 min-1 in the hyperinsulinemic child. As in the adult, ketone turnover was directly proportional to free fatty acid and ketone body concentrations, while ketone clearance declined as the circulatory content of ketone bodies increased. Compared with the adult, however, ketone body turnover rates of 12.8-21.9 mumol kg-1 min-1 in newborns fasted for less than 8 h, and rates of 17.9-26.0 mumol kg-1 min-1 in older infants fasted for less than 10 h, were in a range found in adults only after several days of total fasting. If the bulk of transported ketone body fuels are oxidized in the infant as they are in the adult, ketone bodies could account for as much as 25% of the neonate's basal energy requirements in the first several days of life. These studies demonstrate active ketogenesis and quantitatively important ketone body fuel transport in the human infant. Furthermore, the qualitatively similar relationships between the newborn and the adult relative to free fatty acid concentration and ketone inflow, and with regard to ketone concentration and clearance rate, suggest that intrahepatic and extrahepatic regulatory systems controlling ketone body metabolism are well established by early postnatal life in humans.
P F Bougneres, C Lemmel, P Ferré, D M Bier
Intraluminal fat inhibits gastric secretion through as yet undetermined mechanisms which involve release of one or more hormonal enterogastrones. As intraluminal fat releases Peptide YY (PYY) in amounts sufficient to inhibit meal-stimulated acid secretion, this ileo-colonic peptide exhibits the characteristics required of an enterogastrone. The present study seeks to determine the mechanism by which PYY inhibits acid secretion by examining the effects of PYY on gastric acid stimulated by pentagastrin, histamine, and bethanechol. In addition, effects of PYY on the acid response to sham feeding and distention of a denervated gastric pouch were examined. A dose of PYY (400 pmol X kg-1 X h-1) was employed that reproduced blood levels observed after intestinal perfusion with oleic acid and inhibited the acid secretory response to an intragastric meal by 35 +/- 6%. This same dose of PYY maximally inhibited histamine- and pentagastrin-stimulated acid secretion by 28 +/- 7% (P less than 0.05), and 17 +/- 4% (P less than 0.05), respectively. Although PYY had no effect on bethanechol-stimulated secretion it markedly inhibited the secretory response to sham feeding, maximally reducing secretion by 90 +/- 4% (P less than 0.01). We speculate that PYY acts by inhibiting acetylcholine release from vagal nerve fibers rather than by inhibiting acetylcholine's action on the parietal cell. The demonstration that PYY virtually abolishes cephalic phase acid secretion while having little if any effect on the response to exogenous secretogogues gives PYY unique characteristics among the known hormonal inhibitors of gastric secretion.
T N Pappas, H T Debas, I L Taylor
Recent studies have shown that alveolar macrophages (AM) are able to release leukotrienes (LTs). Since cigarette smoking inhibits the cyclooxygenase pathway of arachidonic acid metabolism in the AM, we evaluated the LT production by AM from smokers and nonsmokers. AM were obtained from 35 volunteers, 16 nonsmokers, and 19 smokers. The cells were incubated under various conditions including stimulation with 30 microM arachidonic acid, 2 microM ionophore A23187, or both. Each experiment was performed in parallel using cells from a smoker and a nonsmoker. Lipoxygenase products were analyzed by reverse-phase high performance liquid chromatography. After stimulation, nonsmokers' AM produced LTB4 and 5-hydroxy-eicosatetraenoic acid (5-HETE). In incubations of AM with arachidonic acid and ionophore, the amounts of products formed were: LTB4, 317 +/- 56 pmol/10(6) cells and 5-HETE, 1,079 +/- 254, mean +/- SEM. No metabolites were generated under control conditions (no stimulation). In all incubations performed, the peptido-LTs (LTC4, LTD4, and LTE4) were undetectable. In comparison with AM from nonsmokers, those from smokers showed a 80-90% reduction of 5-HETE and LTB4 synthesis (P less than 0.05 to P less than 0.001 according to stimulatory conditions). This defective lipoxygenase metabolite production in AM from smokers was observed over a wide range of stimuli concentrations and incubation times; AM from smokers also had lower levels of intracellular (esterified) 5-HETE than nonsmokers' AM. We also studied blood polymorphonuclear leukocytes (PMNL) and no difference in the synthesis of 5-lipoxygenase products in these cells was noticed between smokers and nonsmokers. These data show that cigarette smoking causes a profound inhibition of the 5-lipoxygenase pathway in AM but not in blood PMNL.
M Laviolette, R Coulombe, S Picard, P Braquet, P Borgeat
Isoquinoline sulfonamides have recently been shown to exert novel inhibitory effects on mammalian protein kinases by competitively binding to the ATP substrate site (Hidaka, H., M. Inagaki, S. Kawamoto, and Y. Sasaki, 1984, Biochemistry, 23: 5036-5041). We synthesized a unique analog of the previously reported compounds, 1-(5-isoquinolinesulfonyl) piperazine (C-I), in order to assess the role of protein kinases in modulating the agonist-stimulated oxidative burst of human polymorphonuclear leukocytes (PMN). Compound C-I, at micromolar concentration, markedly inhibited the release of superoxide anion from human PMN stimulated with phorbol myristate acetate or the synthetic diacylglycerol, 1-oleoyl-2-acetyl glycerol. These data are consonant with previously reported data which indicate that the calcium and phospholipid-dependent protein kinase, protein kinase C, serves as the intracellular receptor for these agonists. In contrast, superoxide anion production stimulated by the complement anaphylatoxin peptide C5a or the synthetic chemotaxin formyl-methionyl-leucyl-phenylalanine were not inhibited by C-I. These data suggest that parallel pathways exist for the agonist-stimulated respiratory burst of human neutrophils, only one of which utilizes the calcium and phospholipid-dependent protein kinase.
C Gerard, L C McPhail, A Marfat, N P Stimler-Gerard, D A Bass, C E McCall
To determine whether regional myocardial dysfunction occurring after exercise-induced ischemic might be caused by continued abnormalities of myocardial blood flow in the post-exercise period, nine dogs were instrumented with ultrasonic microcrystals for determination of circumferential segment shortening, circumflex artery electromagnetic flow probes, and hydraulic coronary artery occluders. Dogs performed treadmill exercise during partial inflation of the coronary artery occluder. When the stenosis was maintained after exercise (persistent stenosis), subendocardial flow = 0.79 +/- 0.42 ml/min per g vs. 1.39 +/- 0.43 ml/min per g control), and this was associated with continued dysfunction in the ischemic zone (segment shortening 45.4 +/- 36.9% of resting control). When the stenosis was released immediately after exercise (temporary stenosis), however, flow was markedly increased 1 min post-exercise (mean transmural flow 4.24 +/- 1.22 ml/min per g; subendocardial flow 4.18 +/- 1.52 ml/min per g), and this was associated with a transient increase in segment shortening to 104.5 +/- 9.3% of resting control. 5 min after exercise, however, moderate reductions in ischemic segment shortening were noted after both temporary stenosis and persistent stenosis runs, and these persisted for 30 min post-exercise. It is concluded that regional left ventricular dysfunction may persist for a significant period of time after exercise-induced ischemia. Furthermore, early after exercise, dysfunction is related to persistent abnormalities of myocardial blood flow, whereas late after exercise it is independent of primary reductions in myocardial blood flow.
D C Homans, E Sublett, X Z Dai, R J Bache
Human bone marrow cells were sequentially fractionated by three negative selection steps to remove adherent cells and Fc receptor-bearing cells, followed by immune adsorption (panning) to deplete maturing cells that react with a panel of monoclonal antibodies. This nonadherent Fc receptor and antibody negative fraction could be further enriched by a positive selection "panning" step, using an antibody to HLA-DR antigen; 12-27% of the cells formed erythroid burst-forming unit (BFU-E), erythroid colony-forming unit, granulocyte-monocyte colony-forming unit, and erythroid and granulocyte and/or monocyte colony-forming unit-derived colonies with recovery of 0.5-1% of the cells and 20-100% of the colony-forming cells. Sequential fractionation resulted in increasing dependence of a subset of BFU-E-derived colonies on exogenous burst-promoting activity (BPA) for proliferation in culture, but the most enriched progenitor fraction still contained a proportion of accessory cell or BPA-independent BFU-E that responded to either natural or biosynthetic erythropoietin when added to cultures on day 0 in the absence of BPA. If the addition of erythropoietin was delayed until day 3, the data suggest that this population of BFU-E either died or became unresponsive to erythropoietin. Delayed addition of erythropoietin to cultures of enriched progenitors provided a sensitive BPA assay, since BPA-independent but erythropoietin-responsive BFU-E were eliminated. The surviving BFU-E that were dependent for their proliferation on the presence of both BPA and erythropoietin showed a characteristic dose response to increasing BPA concentrations.
C A Sieff, S G Emerson, A Mufson, T G Gesner, D G Nathan
Primate erythrocytes appear to play a role in the clearance of potentially pathogenic immune complexes (IC) from the circulation. This study was undertaken to compare the clearance from the circulation and tissue uptake of two monoclonal IC probes: one of which, IgG1-IC, was bound well by erythrocytes, the other of which, IgA-IC, was bound relatively poorly by erythrocytes. The IC probes were labeled with different iodine isotopes and infused either concomitantly or sequentially into the arterial circulation. The results indicate that, compared with IgG1-IC, IgA-IC bind less well to primate erythrocytes, are cleared from the circulation more quickly despite their smaller size, and show increased uptake in kidney and lung but decreased uptake in liver and spleen. Evidence is presented which suggests that this pattern of clearance from the circulation and systemic uptake of IgA-IC is the result of decreased binding of IgA-IC to circulating erythrocytes. These findings support the hypothesis that the primate erythrocyte-IC clearing mechanism may be critically important for the safe removal of IC from the circulation.
F J Waxman, L A Hebert, F G Cosio, W L Smead, M E VanAman, J M Taguiam, D J Birmingham
In these experiments, we assessed the role of hepatocyte lysosomes in biliary excretion of iron. We loaded rats with iron by feeding 2% carbonyl iron and collected bile for 24 h via bile fistulae from iron-loaded and control rats. In additional rats, bile was collected before and after the administration of colchicine. Rats were then killed and their livers were homogenized and fractionated for biochemical analyses or processed for electron microscopy and x-ray microanalysis. Inclusion of 2% carbonyl iron in the diet caused a 45-fold increase (P less than 0.001) in hepatic iron concentration compared with controls (1,826 +/- 159 vs. 38 +/- 6.7 micrograms/g liver, mean +/- SE). Electron microscopy with quantitative morphometry and x-ray microanalysis showed that the excess iron was sequestered in an increased number of lysosomes concentrated in the pericanalicular region of the hepatocyte. Iron loading was also associated with a twofold increase in biliary iron excretion (4.06 +/- 0.3 vs. 1.75 +/- 0.1 micrograms/g liver/24 h; P less than 0.001). In contrast, the biliary outputs of three lysosomal enzymes were significantly lower (P less than 0.0005) in iron-loaded rats compared with controls (mean +/- SE) expressed as mU/24 h/g liver: N-acetyl-beta-glucosaminidase, 26.7 +/- 4.6 vs. 66.2 +/- 13.4; beta-glucuronidase, 10.1 +/- 1.3 vs. 53.2 +/- 17.9; beta-galactosidase, 8.9 +/- 1.0 vs. 15.4 +/- 2.3. In iron-loaded rats but not in controls, biliary iron excretion was coupled to the release into bile of each of the three lysosomal hydrolases as assessed by linear regression analysis (P less than 0.001). In contrast, no relationships were found between biliary iron excretion and the biliary outputs of a plasma membrane marker enzyme (alkaline phosphodiesterase I) or total protein. After administration of colchicine, there was a parallel increase in biliary excretion of iron and lysosomal enzymes in iron-loaded rats, but not controls. We interpret these data to indicate that, in the rat, biliary iron excretion from hepatocyte lysosomes is an important excretory route for excess hepatic iron.
G D LeSage, L J Kost, S S Barham, N F LaRusso
We undertook this study to examine the accuracy of plasma C-peptide as a marker of insulin secretion. The peripheral kinetics of biosynthetic human C-peptide (BHCP) were studied in 10 normal volunteers and 7 insulin-dependent diabetic patients. Each subject received intravenous bolus injections of BHCP as well as constant and variable rate infusions. After intravenous bolus injections the metabolic clearance rate of BHCP (3.8 +/- 0.1 ml/kg per min, mean +/- SEM) was not significantly different from the value obtained during its constant intravenous infusion (3.9 +/- 0.1 ml/kg per min). The metabolic clearance rate of C-peptide measured during steady state intravenous infusions was constant over a wide concentration range. During experiments in which BHCP was infused at a variable rate, the peripheral concentration of C-peptide did not change in proportion to the infusion rate. Thus, the infusion rate of BHCP could not be calculated accurately as the product of the C-peptide concentration and metabolic clearance rate. However, the non-steady infusion rate of BHCP could be accurately calculated from peripheral C-peptide concentrations using a two-compartment mathematical model when model parameters were derived from the C-peptide decay curve in each subject. Application of this model to predict constant infusions of C-peptide from peripheral C-peptide concentrations resulted in model generated estimates of the C-peptide infusion rate that were 101.5 +/- 3.4% and 100.4 +/- 2.8% of low and high dose rates, respectively. Estimates of the total quantity of C-peptide infused at a variable rate over 240 min based on the two-compartment model represented 104.6 +/- 2.4% of the amount actually infused. Application of this approach to clinical studies will allow the secretion rate of insulin to be estimated with considerable accuracy. The insulin secretion rate in normal subjects after an overnight fast was 89.1 pmol/min, which corresponds with a basal 24-h secretion of 18.6 U.
K S Polonsky, J Licinio-Paixao, B D Given, W Pugh, P Rue, J Galloway, T Karrison, B Frank
Human hybridomas have been produced by fusing peripheral blood lymphocytes from patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) with the GM 4672 human cell line. 262 hybridoma clones from the fusions of four RA and five SLE patients were screened for binding to denatured DNA (dDNA), native DNA, and the Fc fragment of human IgG (HIgG). Of the 17 hybridoma antibodies (nine RA, eight SLE) selected for strong binding to denatured DNA, Fc, or both, five reacted with dDNA only, one with Fc only, and eight with both dDNA and Fc. Hybridoma supernatants exhibiting dual reactivity were absorbed over HIgG and bovine serum albumin (BSA)-Sepharose immunoabsorbent columns. The reactivities to both DNA and HIgG were completely removed by the HIgG column but unaffected by passage over the BSA column, and both DNA binding and rheumatoid factor activities were recovered in the acid eluates from the Sepharose-IgG column. The binding of dual reactive hybridoma autoantibodies to the Fc fragment of HIgG was specifically competed by dDNA and HIgG, providing additional evidence that one antibody may be capable of reacting both as a rheumatoid factor and as an anti-DNA antibody.
J Rauch, H Tannenbaum, K Straaton, H Massicotte, J Wild
Proton secretion in the renal medullary collecting duct is thought to occur via a luminal proton-ATPase. In order to determine what mechanism(s) participate in proton transport across medullary collecting duct (MCD) cells membranes, intracellular pH (pHi) regulation and proton extrusion rates were measured in freshly prepared suspensions of rabbit outer MCD cells. Cells were separated by protease digestion and purified by Ficoll gradient centrifugation. pHi was estimated fluorometrically using the entrapped intracytoplasmic pH indicator, 6-carboxyfluorescein. Proton extrusion rates were measured using a pH stat. The resting pHi of MCD cells was 7.19 +/- 0.05 (SE) in a nonbicarbonate medium of pH 7.30. When cells were acidified by exposure to acetate salts or by abrupt withdrawal of ammonium chloride, they exhibited pHi recovery to the resting pHi over a 5-min time-course. Depletion of greater than 95% of cellular ATP content by poisoning with KCN in the absence of glucose inhibited pHi recovery. ATP depletion inhibited proton extrusion from MCD cells. Treatment with N-ethylmaleimide also inhibited pHi recovery. In addition, cellular ATP content was dependent on transmembrane pH gradients, suggesting that proton extrusion stimulated ATP hydrolysis. Neither removal of extracellular sodium nor addition of amiloride inhibited pHi recovery. These results provide direct evidence that a plasma membrane proton-ATPase, but not a Na+/H+ exchanger, plays a role in proton transport and pHi regulation in rabbit MCD.
M L Zeidel, P Silva, J L Seifter
During puberty the effects of adrenal androgens upon skeletal maturation are obscured by the influence of gonadal steroids. Suppression of gonadarche with an analogue of luteinizing hormone releasing hormone (LHRHa) affords an opportunity to examine the onset and progression of adrenarche in the absence of pubertal levels of gonadal steroids in a controlled fashion and to explore the relationship between adrenal androgens and the rate of epiphyseal maturation. In 29 children with central precocious puberty, gonadarche was suppressed with LHRHa administration for 1-4 yr. During LHRHa exposure, dehydroepiandrosterone sulfate (DHAS) levels, as an index of adrenal maturation, were constant or increased in an age-expected manner. The change in bone age for change in chronologic age decreased from 1.7 +/- 0.1 to 0.49 +/- 0.05 (P = 0.00005), indicating that the LHRHa-induced return to a prepubertal gonadal steroid environment was associated with a slowing of skeletal maturation. DHAS levels were correlated with the rate of skeletal advancement before (r = 0.57, P = 0.001) and during 12 to 48 mo of exposure to LHRHa (r = 0.52, P = 0.003). A negative correlation of DHAS values with subsequent increases in predicted mature height was observed (r = -0.49, P = 0.007). Thus, in children with central precocious puberty, adrenarche progressed normally during LHRHa suppression of gonadarche. In children with the onset of progression of adrenarche during maintenance of a prepubertal gonadal steroid milieu, there was less evidence than in preadrenarchal children of a restraint upon skeletal maturation. These data suggest that adrenal androgens contribute importantly to epiphyseal advancement during childhood.
M E Wierman, D E Beardsworth, J D Crawford, J F Crigler Jr, M J Mansfield, H H Bode, P A Boepple, D C Kushner, W F Crowley Jr
Trypanosoma cruzi causes Chagasic heart disease, a major public health problem in Latin America. The mechanism of interaction of this protozooan parasite with host cells is poorly understood. We recently found that the infective trypomastigote form a T. cruzi exhibits neuraminidase activity and can desialylate mammalian erythrocytes. However, it is not known if T. cruzi can also modify the surfaces of cardiovascular cells that are directly involved in the most important clinical manifestations of this disease. Accordingly, this study determined whether T. cruzi can remove sialic acid from cultured rat myocardial or human vascular endothelial cells. Sialic acid was labeled metabolically with the precursor 3H-N-acetyl-D-mannosamine. Soluble neuraminidase, isolated from intact T. cruzi trypomastigotes, caused significant release of labeled material from myocardial cells (e.g., 2,174 +/- 27 dpm/h vs. spontaneous release of 306 +/- 30 dpm/h, n = 4, P less than 0.001). Chromatographic analysis showed that the bulk of the radioactivity released by T. cruzi neuraminidase was sialic acid. Intact T. cruzi trypomastigotes also released sialic acid from metabolically labeled myocardial cells in a concentration-dependent manner. In contrast, a noninfective form of T. cruzi, the amastigote, did not desialylate these cells. Galactose oxidase labeling demonstrated newly desialylated glycoproteins on the surface of myocardial cells treated with T. cruzi neuraminidase. Desialylation of myocardial cells was confirmed histochemically by the appearance of binding sites for peanut agglutinin, a lectin that binds to complex oligosaccharide moieties after removal of the terminal sialyl residue. T. cruzi neuraminidase also removed sialic acid from adult human saphenous vein endothelial cells, as determined by both histochemical and metabolic labeling studies. Thus, infective forms of T. cruzi can chemically modify the surfaces of myocardial and vascular endothelial cells by desialylation. This alteration may play a role in the initial interaction of this parasite with these important target cells of the host cardiovascular system.
P Libby, J Alroy, M E Pereira
Our previous studies in cortical collecting ducts isolated from rat kidneys have shown that vasopressin increases both sodium absorption and potassium secretion, while bradykinin inhibits sodium absorption without affecting potassium transport. To determine which anions are affected by these agents, we perfused cortical collecting ducts from rats treated with deoxycorticosterone and measured net chloride flux, net bicarbonate flux (measured as total CO2), transepithelial voltage, and the rate of fluid absorption. Arginine vasopressin (10(-10) M in the peritubular bath) caused a sustained sixfold increase in net chloride absorption and a two- to threefold increase in the magnitude of the lumen negative transepithelial voltage. Before addition of vasopressin, the tubules secreted bicarbonate. Vasopressin abolished the bicarbonate secretion, resulting in net bicarbonate absorption (presumably due to proton secretion) in many tubules. Bradykinin (10(-9) M added to the peritubular bath) caused a reversible 40% inhibition of net chloride absorption, but did not affect the transepithelial voltage or the bicarbonate flux. We concluded: (a) that arginine vasopressin stimulates absorption of chloride and inhibits bicarbonate secretion (or stimulates proton secretion) in the rat cortical collecting duct; and (b) that bradykinin inhibits net chloride absorption in the rat cortical collecting duct without affecting transepithelial voltage or bicarbonate flux. Combining these results with the previous observations on cation fluxes described above, we conclude that bradykinin inhibits electroneutral NaCl absorption (or stimulates electroneutral NaCl secretion) in the rat cortical collecting duct.
K Tomita, J J Pisano, M B Burg, M A Knepper
We investigated nephritogenic potential of antibodies to heparan sulfate-proteoglycan of glomerular basement membrane. Glomeruli were isolated, basement membranes were prepared, proteoglycans extracted, and purified core protein was obtained. We immunized rabbits with the core protein, IgG fraction prepared from the antisera and specificity of the antibody determined. A single immunoprecipitin line in agar diffusion plate and a single band (approximately 18,000 mol wt) on the immunoblot autoradiograms were visualized. The antibody showed precise reactivity with the glomerular basement membranes. The clearance studies indicated that approximately 75% of the radioiodinated antibody disappeared from circulation within 1 h and 1-2% bound to the kidney. For nephritogenicity experiments, the antibody was intravenously administered into rats and we examined their kidneys at 1 h to 24 d later. A linear immunofluorescence of glomerular basement membranes was observed with rabbit IgG at all times while that of C3 until the 10th day. Early morphologic changes included glomerular infiltration of polymorphonuclear leukocytes with focal exfoliation of endothelium. The leukocytic infiltration subsided by the third day and was followed by progressive thickening of basement membranes, focal mesangial cell proliferation, increase in mesangial matrix, and accumulation of monocytes. Focal knob-like thickening of glomerular basement membrane was observed from the 15th day onward. Regularly-spaced electrondense deposits were seen in the lamina rara interna and externa of glomerular basement membranes and persisted throughout the investigatory period. No significant proteinuria was observed at any stage of the experiment. These findings suggest that the antibodies to the basement membrane heparan sulfate-proteoglycan are nephrotoxic but possess weak nephritogenic potential.
H Makino, J T Gibbons, M K Reddy, Y S Kanwar
Impaired platelet aggregation, normal shape change, and agglutination and normal ATP secretion and thromboxane synthesis in response to high concentrations of thrombin or arachidonic acid were found in a patient with multiple myeloma and hemorrhagic tendency. The purified IgG1 kappa or its F(ab1)2 fragments induced similar changes when added in vitro to platelet-rich plasma from normal subjects. In addition, the paraprotein inhibited adhesion to glass microbeads, fibrin clot retraction, and binding of radiolabeled fibrinogen or von Willebrand factor to platelets exposed to thrombin or arachidonic acid without affecting intraplatelet levels of cAMP. The radiolabeled para-protein bound to an average of 35,000 sites on normal platelets but it bound to less than 2,000 sites on the platelets from a patient with Glanzmann's thrombasthenia. Immunoprecipitation studies showed that the platelet antigen identified by the paraprotein was the glycoprotein IIIa. Furthermore, binding of radiolabeled prostaglandin E1 (PGE1) to resting platelets as well as binding of von Willebrand factor to platelets stimulated with ristocetin were entirely normal in the presence of patient's inhibitor. These studies indicate that bleeding occurring in dysproteinemia may be the result of a specific interaction of monoclonal paraproteins with platelets. In addition, our data support the concept that the interaction of fibrinogen and/or von Willebrand factor with the platelet glycoprotein IIb-IIIa complex is essential for effective hemostasis.
G DiMinno, F Coraggio, A M Cerbone, A M Capitanio, C Manzo, M Spina, P Scarpato, G M Dattoli, P L Mattioli, M Mancini
We have examined the effect of thrombin on the activity of plasminogen activator (PA) and plasminogen activator-inhibitor (PA-I) in medium conditioned by primary cultures of human umbilical vein endothelial cells. PA activity was measured by fibrinolytic and esterolytic assays, and total tissue-type PA (tPA) antigen by radioimmunoassay. Net PA-I activity was assayed by titration of human urokinase esterolytic activity. Incubation of confluent endothelial cell cultures with thrombin for 24 h caused a sixfold increase in PA-I activity. The effect of thrombin was half-maximal at approximately 0.4 U/ml (less than 4 nM), and required concomitant RNA and protein synthesis. The stimulation of PA-I activity required active alpha-thrombin and was not obtained with gamma-thrombin nor with thrombin catalytically inactivated with hirudin. Because of the excess of PA-I, PA activity was not measurable in either control or thrombin-treated cells. Thrombin did, however, increase medium concentration of tPA antigen by approximately fourfold. The thrombin-induced PA-I inhibited both tPA and urokinase, did not lose activity upon acidification, and was stable to sodium dodecyl sulfate and thiol reduction. We conclude that physiologic concentrations of thrombin increase both PA-I activity and tPA antigen in medium conditioned by human umbilical vein endothelial cells. Because there was always a several-fold increase in the net activity of free PA-I, these observations suggest that the net effect of thrombin is to decrease fibrinolytic activity in human endothelial cells. Thus, thrombin, in addition to its role in coagulation, may protect clots from premature lysis by increasing the amount of a specific fibrinolytic inhibitor.
T D Gelehrter, R Sznycer-Laszuk
This study demonstrates the presence of oxalate transporters on the brush border membrane of rabbit ileum. We found that an inside alkaline (pH = 8.5 inside, 6.5 outside) pH gradient stimulated [14C]oxalate uptake 10-fold at 1 min with a fourfold accumulation above equilibrated uptake at 5 min. 1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonate (disodium salt; DIDS) profoundly inhibited the pH-gradient stimulated oxalate uptake. Using an inwardly directed K+ gradient and valinomycin, we found no evidence for potential sensitive oxalate uptake. In contrast to Cl:HCO3 exchange, HCO3 did not stimulate oxalate uptake more than was seen with a pH gradient in the absence of HCO3. An outwardly directed Cl gradient (50 mM inside, 5 mM outside) stimulated oxalate uptake 10-fold at 1 min with a fivefold accumulation above equilibrated uptake. Cl-stimulated oxalate uptake was largely inhibited by DIDS. Addition of K+ and nigericin only slightly decreased the Cl gradient-stimulated oxalate uptake, which indicates that this stimulation was not primarily due to the Cl gradient generating an inside alkaline pH gradient via Cl:OH exchange. Further, an outwardly directed oxalate gradient stimulated 36Cl uptake. These results suggested that both oxalate:OH and oxalate:Cl exchange occur on the brush border membrane. To determine if one or both of these exchanges were on contaminating basolateral membrane, the vesicle preparation was further fractionated into a brush border and basolateral component using sucrose density gradient centrifugation. Both exchangers localized to the brush border component. A number of organic anions were examined (outwardly directed gradient) to determine if they could stimulate oxalate and Cl uptake. Only formate and oxaloacetate were found to stimulate oxalate and Cl uptake. An inwardly directed Na gradient only slightly stimulated oxalate uptake, which was inhibited by DIDS.
R G Knickelbein, P S Aronson, J W Dobbins
Previous studies from this laboratory and by others in rats, monkeys, and humans support the concept that growth hormone (GH) can regulate its own secretion through an autofeedback mechanism. With the availability of human growth hormone-releasing factor (GRF), the possible existence of such a mechanism was reexplored by examining the effect of exogenous GH on the GH response induced by GRF-44-NH2 in six normal men (mean age, 32.4 yr). In all subjects the plasma GH response evoked by GRF-44-NH2 (1 microgram/kg i.v. bolus) was studied before and after 5 d of placebo (1 ml normal saline i.m. every 12 h), and then before and 12 h after 5 d of biosynthetic methionyl human GH (5 U i.m. every 12 h). The GH response to GRF (maximal increment over time 0 value) was significantly inhibited after GH treatment (0-1.3 vs. 2.3-11.2 ng/ml before treatment, P = 0.05), but was not significantly affected by placebo. This impaired pituitary response to GRF persisted for at least 24 h following exogenous GH treatment in two subjects who underwent further study. Serum somatomedin-C concentrations were significantly increased after 5 d of GH treatment (2.66-5.00 vs. 0.92-1.91 U/ml before treatment, P = less than 0.01). The impaired pituitary response to GRF may be mediated indirectly through somatomedin, somatostatin, by a direct effect of GH on the pituitary somatotropes, or by all of these mechanisms. These data suggest that after GH treatment, the blunted GH response to synthetic GRF is not solely a consequence of the inhibition of hypothalamic GRF secretion.
S M Rosenthal, J A Hulse, S L Kaplan, M M Grumbach
Abnormal regulation of vitamin D metabolism is a feature of X-linked hypophosphatemic rickets in man and of the murine homologue of the disease in the hypophosphatemic (Hyp)-mouse. We previously reported that mutant mice have abnormally low renal 25-hydroxyvitamin D-1 alpha-hydroxylase (1 alpha-hydroxylase) activity for the prevailing degree of hypophosphatemia. To further characterize this defect, we examined whether Hyp-mouse renal 1 alpha-hydroxylase activity responds normally to other stimulatory and inhibitory controls of enzyme function. We studied stimulation by parathyroid hormone (PTH) using: (a) a calcium-deficient (0.02% Ca) diet to raise endogenous PTH; or (b) 24-h continuous infusion of 0.25 IU/h bovine PTH via osmotic minipump. In both cases enzyme activity of identically treated normal mice increased to greater levels than those attained by Hyp-mice. The relative inability of PTH to stimulate 1 alpha-hydroxylase activity is not a function of the hypophosphatemia in the Hyp-mouse since PTH-infused, phosphate-depleted normal mice sustained a level of enzyme activity greater than that of normal and Hyp-mice. In further studies we investigated inhibition of enzyme activity by using: (a) a calcium-loaded (1.2% Ca) diet to suppress endogenous PTH; or (b) 24-h continuous infusion of 0.2 ng/h 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). The 1 alpha-hydroxylase activity of normal and Hyp-mice was significantly reduced to similar absolute levels following maintenance on the calcium-loaded diet. Further, infusion of 1,25(OH)2D3 caused a comparable reduction of 1 alpha-hydroxylase activity in normal, Hyp-, and phosphate-depleted normal mice. These observations indicate that the inhibitory control of 1 alpha-hydroxylase by reduced levels of PTH or increased 1,25(OH)2D3 concentrations is intact in the mutants. However, the inability of PTH and hypophosphatemia to stimulate enzyme activity in a manner analogous to that in normal and phosphate-depleted mice indicates that a generalized defect of 1 alpha-hydroxylase regulation is manifest in Hyp-mice.
T Nesbitt, M K Drezner, B Lobaugh
We characterized 24 unrelated patients with a deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) in an attempt to better understand the nature and spectrum of mutations that underlie this prototype-inherited disease. Lymphoblast cell lines derived from each patient were analyzed at multiple molecular levels including the structure and function of the residual HPRT enzyme, messenger RNA (mRNA), and gene. Our studies demonstrate the following: (a) at least 16 of the 24 patients represent unique and independent mutations at the HPRT structural gene; (b) the majority of cell lines have normal quantities of mRNA but undetectable quantities of enzyme; (c) 33% of patients retain significant quantities of structurally altered, functionally abnormal, HPRT enzyme variants; and (d) a minority of patients are void of both enzyme and mRNA, possibly representing examples of aberrations in gene expression. Our studies provide direct evidence for marked genetic heterogeneity in this disorder and illustrate the kinds of mutations and mutational consequences that underlie inherited disease in humans.
J M Wilson, J T Stout, T D Palella, B L Davidson, W N Kelley, C T Caskey
We have used a high performance liquid chromatography assay, which detects chymotryptic cleavage of the phe8-his9 bond of angiotensin I to yield angiotensin II, in order to examine human lung mast cells for the presence of chymotryptic activity. Mast cells, purified from human lung by enzymatic dispersion, countercurrent elutriation, and Percoll gradient centrifugation, were lysed or challenged with goat anti-human IgE. In multiple experiments angiotensin II-converting activity was detected in lysates of 10-99% pure mast cell preparations. Regression analysis of net percent release values of histamine and the angiotensin I-converting activity from dose-response experiments demonstrated a correlation between the two parameters, indicating that the chymotrypsin-like enzyme is a constituent of the mast cell secretory granule. The chymotryptic activity was completely inhibited by 10(-3) M phenylmethylsulfonylfluoride but not by 10(-3) M Captopril, and the pH optimum of activity was 7.5-9.5. Gel filtration of released material separated the activity from tryptase and demonstrated an approximate molecular weight of 30-35,000. The mast cell enzyme, like a human skin chymotrypsin-like proteinase, can be distinguished from leukocyte cathepsin G by lack of susceptibility to inhibition by bovine pancreatic trypsin inhibitor. Thus, an enzyme with limited chymotryptic specificity is present in human lung mast cells. The Michaelis constant of the enzyme for angiotensin I of 6.0 X 10(-5) M is similar to that of endothelial cell angiotensin-converting enzyme and is consistent with a reaction of physiologic importance.
B U Wintroub, C E Kaempfer, N M Schechter, D Proud
Human umbilical arteries converted arachidonic acid to three hydroxyeicosatetraenoic acids (HETEs) as well as prostaglandins. The mono-HETEs have been identified by reverse-phase high pressure liquid chromatography and gas chromatography-mass spectroscopy as 15-HETE and 11-HETE. 15-HETE in arterial segments appears to be derived mainly via the 15-lipoxygenase pathway, whereas 11-HETE, and the presumed di-HETE(s) were products of cyclooxygenase. Nordihydroguaiaretic acid, a lipoxygenase inhibitor, stimulated prostanoid production with a concomitant inhibition of 15-HETE formation. These results suggested that 15-HETE may function as an endogenous regulator of prostacyclin. In human umbilical arterial microsomes, 15-HETE was found to inhibit 6-keto-prostaglandin F1 alpha and total prostanoid production in a concentration-dependent manner (median inhibition constant [IC50] of 52 +/- 3 and 63 +/- 4 microM respectively). The relative distribution of prostaglandins, however, remained unaffected, indicating that the site of action was cyclooxygenase. Kinetic analysis revealed that 15-HETE was a competitive inhibitor of the enzyme. Although no changes in maximum velocity occurred, the apparent Km was significantly different (9.3 +/- 6.9 microM [1 SD] for control vs. 37.6 +/- 17.7 microM for the 15-HETE-treated enzyme). Furthermore, the inhibitory effect of 15-HETE on prostacyclin production was confirmed using cultured bovine endothelial cells. In this cell system, not only did 15-HETE inhibit endogenous prostacyclin production, but also the conversion of exogenous [1-14C]arachidonic acid to prostacyclin (IC50 of 40 +/- 17 microM). No effect on arachidonic acid release was noted. To investigate whether our in vitro finding that 15-HETE inhibited prostacyclin production could be relevant to the in vivo situation, our final studies were performed on vasculature obtained from the diabetic milieu. We found that the production of 15-HETE was significantly increased in vasculature obtained from the infant of the diabetic mother (1.14 +/- 0.26 pmol/mg) when compared to control neonates (0.77 +/- 0.22; P less than 0.01). A concomitant decrease in prostacyclin production was seen (51.6 +/- 12.6 pmol/mg in infants of diabetic mothers vs. 71 +/- 22.3 in controls). Moreover, an inverse correlation between these two eicosanoids was also noted. Our results suggest a potential in vivo regulatory role for 15-HETE on prostacyclin production.
B N Setty, M J Stuart
During mild or moderate nonexhausting exercise, glucose utilization increases sharply but is normally matched by increased glucose production such that hypoglycemia does not occur. To test the hypothesis that redundant glucoregulatory systems including sympathochromaffin activation and changes in pancreatic islet hormone secretion underlie this precise matching, eight young adults exercised at 55-60% of maximal oxygen consumption for 60 min on separate occasions under four conditions: (a) control study (saline infusion); (b) islet clamp study (insulin and glucagon held constant by somatostatin infusion with glucagon and insulin replacement at fixed rates before, during and after exercise with insulin doses determined individually and shown to produce normal and stable plasma glucose concentrations prior to each study); (c) adrenergic blockage study (infusions of the alpha- and beta-adrenergic antagonists phentolamine and propranolol); (d) adrenergic blockade plus islet clamp study. Glucose production matched increased glucose utilization during exercise in the control study and plasma glucose did not fall (92 +/- 1 mg/dl at base line, 90 +/- 2 mg/dl at the end of exercise). Plasma glucose also did not fall during exercise when changes in insulin and glucagon were prevented in the islet clamp study. In the adrenergic blockade study, plasma glucose declined initially during exercise because of a greater initial increase in glucose utilization, then plateaued with an end-exercise value of 74 +/- 3 mg/dl (P less than 0.01 vs. control). In contrast, in the adrenergic blockade plus islet clamp study, exercise was associated with glucose production substantially lower than control and plasma glucose fell progressively to 58 +/- 7 mg/dl (P less than 0.001); end-exercise plasma glucose concentrations ranged from 34 to 72 mg/dl. Thus, we conclude that: (a) redundant glucoregulatory systems are involved in the precise matching of increased glucose utilization and glucose production that normally prevents hypoglycemia during moderate exercise in humans. (b) Sympathochromaffin activation, perhaps sympathetic neural norepinephrine release, plays a primary glucoregulatory role by limiting glucose utilization as well as stimulating glucose production. (c) Changes in pancreatic islet hormone secretion (decrements in insulin, increments in glucagon, or both) are not normally critical but become critical when catecholamine action is deficient. (d) Glucoregulation fails, and hypoglycemia can develop, both when catecholamine action is deficient and when changes in islet hormones do not occur during exercise in humans.
D R Hoelzer, G P Dalsky, W E Clutter, S D Shah, J O Holloszy, P E Cryer
Caffeine consumption causes significant physiologic effects due to its antagonism of adenosine receptors. The A1 adenosine receptor is coupled in an inhibitory manner to adenylate cyclase. To study the effects of chronic caffeine ingestion, rats were provided with 0.1% caffeine drinking solution for 28 d. The A1 adenosine receptor agonist radioligand [3H]phenylisopropyladenosine identifies two affinity states in control rat cerebral cortex membranes with a high affinity dissociation constant (KH) of 0.40 +/- 0.08 nM and low affinity dissociation constant (KL) of 13.7 +/- 3.9 nM, with 33% of the receptors in the high affinity state. In membranes from caffeine-treated animals, all of the A1 receptors are shifted to the high affinity state with a dissociation constant (KD) of 0.59 +/- 0.06 nM. Guanylyl-imidodiphosphate (10(-4) M) decreases binding by 43% in control membrane, with no change in KH or KL, while membrane binding in caffeine-treated animals decreases by 45% with a threefold shift in KD to 1.5 +/- 0.3 nM. Concomitant with the enhanced high affinity A1 receptor state and increased sensitivity to guanine nucleotides, membranes from treated animals show a 35% enhancement in (-)-N6-(R-phenylisopropyl)adenosine-mediated inhibition of adenylate cyclase compared with controls (P less than 0.03). Photoaffinity crosslinking the receptors with [125I]N6-2-(3-iodo-4-aminophenyl)ethyladenosine reveals that A1 receptors from both groups migrate as Mr 38,000 proteins. beta-adrenergic receptor binding with [125I]iodocyanopindolol shows a decrease in the number of beta-receptors from 233 +/- 7 fmol/mg protein in control membranes to 190 +/- 10 fmol/mg protein in treated membranes (P = 0.01). These data indicate that the adenosine receptor antagonist, caffeine, induces a compensatory sensitization of the A1 receptor-adenylate cyclase system and downregulation of beta-adrenergic receptors, and provides a molecular mechanism for the caffeine withdrawal syndrome.
R M Green, G L Stiles
The model of sodium and chloride transport proposed for the colon is based on studies performed in the distal segment and tacitly assumes that ion transport is similar throughout the colon. In rat distal colon, neutral sodium-chloride absorption accounts for the major fraction of overall sodium absorption and aldosterone stimulates electrogenic, amiloride-sensitive sodium absorption. Since we have demonstrated qualitative differences in potassium transport in proximal and distal segments of rat colon, unidirectional 22Na and 36Cl fluxes were performed under short-circuit conditions across isolated proximal colon of control and sodium-depleted rats with secondary hyperaldosteronism. In the control group, net sodium absorption (JNanet) (7.4 +/- 0.5 mu eq/h . cm2) was greater than Isc (1.4 +/- 0.1 mu eq/h . cm2), and JClnet was 0 in Ringer solution. Residual flux (JR) was -5.2 +/- 0.5 mu eq/h . cm2 consistent with hydrogen ion secretion suggesting that neutral sodium absorption may represent sodium-hydrogen exchange. 1 mM mucosal amiloride, which inhibits sodium-hydrogen exchange in other epithelia, produced comparable decreases in JNanet and JR (4.1 +/- 0.6 and 3.2 +/- 0.6 mu eq/h . cm2, respectively) without a parallel fall in Isc. Sodium depletion stimulated JNanet, JClnet, and Isc by 7.0 +/- 1.4, 6.3 +/- 1.9, and 0.8 +/- 0.2 mu eq/h . cm2, respectively, and 1 mM amiloride markedly inhibited JNanet and JClnet by 6.0 +/- 1.1 and 4.0 +/- 1.6 mu eq/h . cm2, respectively, with only a minimal reduction in Isc. Conclusions: the predominant neutral sodium-absorptive mechanism in proximal colon is sodium-hydrogen exchange. Sodium depletion stimulates electroneutral chloride-dependent sodium absorption (most likely as a result of increasing sodium-hydrogen and chloride-bicarbonate exchanges), not electrogenic chloride-independent sodium transport. The model of ion transport in the proximal colon is distinct from that of the distal colon.
E S Foster, M E Budinger, J P Hayslett, H J Binder
Renal hemodynamics increase dramatically during pregnancy, and pressor responsiveness to exogenous administration of vasoconstrictors is attenuated. We investigated whether or not vasodilatory prostaglandins mediate these phenomena. Trained, chronically instrumented, conscious pregnant rats were used. Control values of glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) were elevated at midgestation (P less than 0.01 and P = 0.05 from prepregnant means, respectively), and effective renal vascular resistance was decreased (P = 0.05). Indomethacin (4.5-6.5 mg/kg body weight [BW]) failed to decrease renal hemodynamics at this stage of pregnancy; in fact, it raised GFR somewhat further (P less than 0.05). Systemic pressor responsiveness to bolus administration of norepinephrine and angiotensin II (AII) was significantly attenuated by at least gestational day 20. Neither indomethacin (7 mg/kg BW) or meclofenamate (6 mg/kg BW) affected the refractory response. The renal vasculature was also relatively unresponsive to an intravenous infusion of AII (5 ng X kg-1 X min-1) during late gestation (day 19); in particular, the fall in ERPF in response to AII (16 +/- 3%) was markedly less than that observed in the prepregnant condition (34 +/- 3%; P less than 0.05). Indomethacin (6 mg/kg BW) failed to restore this blunted response, and further attenuation was evident, despite the presence of the inhibitor (gestational day 21). We conclude that vasodilatory prostaglandins do not appear to mediate the rise in renal hemodynamics, and the attenuation of the systemic and renal pressor responsiveness observed during pregnancy, insofar as these phenomena were unaffected by acute cyclooxygenase inhibition in unstressed, conscious rats.
K P Conrad, M C Colpoys
To understand the relationship between the proliferation of epidermis and its arachidonic acid metabolism, we studied human keratinocytes grown in vitro at confluent or nonconfluent densities. Keratinocyte cultures incubated with [14C]arachidonic acid synthesized prostaglandin (PG)E2 PGD2, PGF2 alpha, and small quantities of 6-keto-F1 alpha. Nonconfluent cultures, however, synthesized fourfold more PGE2 than did confluent cultures. When proliferation was studied using [3H]thymidine incorporation into DNA, it was found that this increased synthesis of PGE2 was accompanied by a fourfold increase in the rate of proliferation. When PGE2 synthesis was inhibited by indomethacin, the rate of proliferation of nonconfluent cultures was decreased 40%, while the rate of proliferation of confluent cultures was unchanged. Addition of 1 ng/ml of PGE2, but not PGF2 alpha, PGD2, or a stable analog of PGI2 to the indomethacin-treated nonconfluent cultures restored the initial rate of proliferation. These results suggest that PGE2 is a growth-promoting autocoid for epidermis. The synthesis of PGE2 by epidermis may be enhanced in wound healing and disease states where epidermal continuity is disrupted.
A P Pentland, P Needleman
To determine whether the amount of cyclooxygenase metabolites correlates with the development of lupus nephritis, intrarenal eicosanoid production was measured in autoimmune mice. Disease progression was related to the renal biosynthesis of prostaglandin (PGE2), prostacyclin (6 keto PGF1 alpha), and thromboxane (TXB2) using the MRL-lpr and NZB X NZW F1 hybrid mouse strains with predictably progressive forms of renal disease that mimic the human illness. Mice were evaluated for renal disease by measuring urinary protein excretion and renal immunopathological conditions and these features were related to renal eicosanoid production. These studies show that: (a) intrarenal synthesis of TXB2 increased incrementally in MRL-lpr and NZB X NZW F1 hybrid mice as renal function deteriorated and renal pathologic events progressed; (b) there were no consistent increases in the levels of two other cyclooxygenase metabolites, PGE2 or 6 keto PGF1 alpha; (c) increased TXB2 production occurred in the renal medulla, cortex, and within enriched preparations of cortical glomeruli; (d) when renal disease was prevented by pharmacologic doses of PGE2, intrarenal TXB2 did not increase; (e) administration of a dose of ibuprofen (9 mg/kg), a cyclooxygenase inhibitor capable of reducing 90% of platelet TXB2 without affecting intrarenal levels, did not retard the progression of renal damage. Taken together, these data indicate that the intrarenal level of TXB2 rises in relation to the severity of murine lupus nephritis. Furthermore, because of the potential deleterious effects of TXA2, enhanced production of this eicosanoid may be an important mediator of renal injury.
V E Kelley, S Sneve, S Musinski
The effect of diabetes on the structure and function of insulin receptors was studied in rats 7 d after streptozotocin injection, using solubilized, partially purified receptors from rat hindlimb muscles. Diabetes increased the number of insulin receptors per gram of muscle 60-70% without apparent change in insulin binding affinity. Incubation of receptors at 4 degrees C with [gamma-32P]ATP and insulin resulted in dose-dependent autophosphorylation of the beta-subunit on tyrosine residues; receptors from diabetic rats showed decreased base-line phosphorylation, as well as a decrease in autophosphorylation at maximally stimulating insulin concentrations. These receptors also showed diminished exogenous substrate kinase activity using histone H2b and angiotensin II as phosphoacceptors. The electrophoretic mobility (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of a subpopulation of beta-subunits derived from diabetics was slightly decreased; differences in electrophoretic mobility between control- and diabetic-derived beta-subunits were enhanced by generating fragments by partial Staphylococcus aureus V8 protease digestion. Endoglycosidase-H or neuraminidase treatment increased the electrophoretic mobility of beta-subunits in both groups, but only neuraminidase appeared to decrease or abolish differences in electrophoretic mobility between controls and diabetics, suggesting that excess sialilation may account, in part, for the altered mobility of diabetic derived beta-subunits. All structural and functional alterations in insulin receptors were prevented by treating diabetic rats with insulin for 60 h. Peripheral insulin resistance associated with insulinopenic diabetes may be related to modifications in insulin receptor structure, resulting in impaired signal transmission.
C F Burant, M K Treutelaar, M G Buse
C-myc proto-oncogene transcripts from serially harvested, colony-stimulating activity (CSA)-stimulated, normal progenitor-enriched human bone marrow cells were compared to those of the promyelocytic leukemia cell line HL-60 and to those of freshly obtained human myeloid leukemic cells. During the early culture period both normal and leukemic cells expressed the c-myc oncogene. In normal cells maximal expression occurred after 24 h of culture and did not occur in the absence of CSA. At this time, progranulocytes predominated in the cultured cells. Although cellular proliferation occurred for 96 h in vitro, c-myc expression ceased after 24-36 h. Terminally differentiated cells predominated in these cultures by 120 h. In contrast, although leukemic cells also expressed c-myc in vitro, transcription persisted throughout the culture period and, in the case of HL-60 cells, occurred in the absence of exogenous CSA. We also noted that normal cells with only one diploid gene copy exhibited, after 24 h of culture, only twofold fewer transcripts than did HL-60 cells in which there were 16 myc copies. Furthermore, c-myc mRNA degradation rates were similar in normal cells and in HL-60 cells. We conclude that c-myc transcription is a normal event in granulopoiesis linked to proliferative activity as well as to primitive developmental stage. Furthermore, the most consistent abnormality in leukemic cells in vitro is their failure to suppress transcriptional activity of this gene. We suggest that c-myc transcription in HL-60 cells may be appropriate for cells arrested at that developmental stage and that the amplified genes in HL-60 cells are quiescent relative to c-myc in normal cells at the same differentiation stage. The techniques described herein may be of value in identifying mechanisms by which normal hematopoietic cells suppress c-myc expression and aberrancies of these mechanisms in leukemic cells.
S D Gowda, R D Koler, G C Bagby Jr
The influence of dietary fatty acid composition on intestinal active and passive transport function, brush border membrane composition, and morphology was examined in rats. Animals fed a semisynthetic diet high in saturated fatty acids demonstrated enhanced in vitro jejunal uptake of decanoic, dodecanoic, palmitic, stearic, and linoleic acid, as well as cholesterol and chenodeoxycholic and taurochenodeoxycholic acid, as compared with uptake in animals fed a semisynthetic diet high in polyunsaturated fatty acids but equivalent in total content of fat and other nutrients, or as compared with Purina chow. Feeding the saturated fatty acid diet was also associated with reduced jejunal uptake of a range of concentrations of glucose, enhanced ileal uptake of leucine, unchanged uptake of galactose, and lower uptake of decanol. The semisynthetic diets did not alter brush border membrane protein, sucrase or alkaline phosphatase activities, cholesterol, or total phospholipids, although the percentage of jejunal amine phospholipids was higher than in rats fed chow. The morphologic differences between the jejunum and ileum were abolished in animals fed the high polyunsaturated fatty acid diet; in rats fed the high saturated fatty acid diet, there was reduced mean ileal villus height, width, thickness, surface area, cell size, and villus density, as well as reduced mucosal surface area. The changes in jejunal transport were not correlated with the alterations in morphology, unstirred layer resistance, food intake, or body weight gain. It is proposed that small changes in the percentage of total dietary lipids composed of essential and nonessential fatty acids (without concurrent alterations in dietary total fat, carbohydrate, or protein) influence active and passive intestinal transport processes in the rat.
A B Thomson, M Keelan, M T Clandinin, K Walker
A strain of Streptococcus faecalis with plasmid-mediated penicillinase production was studied further. Partially purified penicillinase from the S. faecalis strain hydrolyzed penicillin, ampicillin, and ureido-penicillins but not penicillinase-resistant semisynthetic penicillins, cephalosporins, or imipenem; hydrolysis was inhibited by clavulanic acid. Hydrolysis of a given antibiotic correlated with a marked increase in the minimal inhibitory concentration (MIC) of that drug when a high inoculum was used. As with most enterococci, the MICs of cephalosporins and penicillinase-resistant semisynthetic penicillins were too high for clinical usefulness, although these agents did not show an inoculum effect. Based upon hybridization under stringent conditions of plasmid DNA from the S. faecalis strain to cloned penicillinase genes from Staphylococcus aureus, it appears that these resistance determinants are highly homologous and suggests that this enzyme was introduced into streptococci from staphylococci.
B E Murray, B Mederski-Samoraj, S K Foster, J L Brunton, P Harford
Human B cell-triggering mechanisms were investigated using the polyclonal activators Staphylococcus aureus Cowan I (SAC) and pokeweed mitogen (PWM). When the cultures of B cells, T cells, and monocytes were stimulated for 5 d by SAC or PWM, B cells could be activated by both mitogens to proliferate and secrete Ig. Even when T cells were substituted by T cell-derived soluble factors, SAC-stimulated B cells could differentiate into Ig-secreting cells. In contrast, interactions of B and T cells for at least the first 6 h of culture were necessary for the B cell triggering by PWM. Experiments that allow a more precise delineation of the B cell-triggering mechanisms by PWM demonstrated that interactions of B cells with T4+ but not T8+ cells are required for the B cell triggering; anti-Ia or anti-T4 antibody can block this triggering; in contrast, anti-T3 or anti-T8 antibody do not exert any effects on the B cell triggering. However, all these monoclonal antibodies could not modulate the ability of B cells that had been already activated by PWM to respond to T cell-derived factors. These data suggest that SAC can directly activate B cells, while cognate interactions between Ia-like antigens on B cells and T4+ cells are essential for B cell triggering by PWM. Furthermore, once B cells are triggered, they will proliferate, differentiate, and secrete Ig in response to T cell-derived factors; Ia-like antigens or T cell differentiation antigens may not be involved in the processes in this cascade.
N Suzuki, T Sakane, Y Ueda, Y Murakawa, T Tsunematsu
This clinical study was based on experimental results obtained in nude mice grafted with human colon carcinoma, showing that injected 131I-labeled F(ab')2 and Fab fragments from high affinity anti-carcinoembryonic antigen (CEA) monoclonal antibodies (MAb) gave markedly higher ratios of tumor to normal tissue localization than intact MAb. 31 patients with known colorectal carcinoma, including 10 primary tumors, 13 local tumor recurrences, and 21 metastatic involvements, were injected with 123I-labeled F(ab')2 (n = 14) or Fab (n = 17) fragments from MAb anti-CEA. The patients were examined by emission-computerized tomography (ECT) at 6, 24, and sometimes 48 h after injection using a rotating dual head scintillation camera. All 23 primary tumors and local recurrences except one were clearly visualized on at least two sections of different tomographic planes. Interestingly, nine of these patients had almost normal circulating CEA levels, and three of the visualized tumors weighed only 3-5 g. Among 19 known metastatic tumor involvements, 14 were correctly localized by ECT. Two additional liver and several bone metastases were discovered by immunoscintigraphy. Altogether, 86% of the tumor sites were detected, 82% with F(ab')2 and 89% with Fab fragments. The contrast of the tumor images obtained with Fab fragments suggests that this improved method of immunoscintigraphy has the potential to detect early tumor recurrences and thus to increase the survival of patients. The results of this retrospective study, however, should be confirmed in a prospective study before this method can be recommended for the routine diagnosis of cancer.
B Delaloye, A Bischof-Delaloye, F Buchegger, V von Fliedner, J P Grob, J C Volant, J Pettavel, J P Mach
1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is known to stimulate osteoclastic bone resorption in vivo and whole organ bone culture systems in vitro. It has not been established whether 1,25(OH)2D3 acts directly on osteoclasts or whether its action on osteoclasts is mediated via other bone cells (e.g., osteoblasts) or recruitment of osteoclast precursor cells. Circulating monocytes have been characterized as osteoclast precursors. In the present study, vitamin D3-replete chicken on a calcium-deficient diet were studied. Circulating monocytes, whole bone cell preparations, and isolated osteoclasts (differential sedimentation) were examined for presence of 1,25(OH)2D3 receptors. Reversible, specific, and saturable binding of [3H]-1,25(OH)2D3 to a 3.5 S macromolecule was demonstrated in nuclear fractions of monocytes (maximal binding capacity, 48 fmol/mg protein; dissociation constant, 1.3 X 10(-10) M) and of whole bone cell preparations. 1,25(OH)2D3 receptors were not demonstrable in osteoclast preparations (70% pure; detection threshold, 2 fmol/mg protein). Data are consistent with indirect action of 1,25(OH)2D3 on osteoclastic bone resorption.
J Merke, G Klaus, U Hügel, R Waldherr, E Ritz
The sites of action of angiotensin II along the nephron are not well defined and both proximal and distal effects are suggested. Using a microassay that permits measurement of hormone binding in discrete tubule segments, we determined the binding sites of 125I-angiotensin II along the nephron of Sprague-Dawley rats. Specific binding in proximal convoluted tubule (PCT) (at 25 degrees C, pH 7.4) was linearly related to tubule length and saturable, with an apparent maximal binding capacity of approximately 300 amol X cm-1. Binding specificity was verified in competition experiments that revealed significant (P less than 0.001) and comparable competition for radioligand binding by angiotensin II and angiotensin precursor, metabolite, and analogues, whereas unrelated peptides of similar size (bradykinin, ACTH [1-10]) were without effect. The profile of specific angiotensin II binding along the nephron was: PCT, 216 +/- 13; pars recta, 86 +/- 14; medullary thick ascending limb of Henle's loop, 46 +/- 8; cortical thick ascending limb of Henle's loop, 77 +/- 8; distal convoluted tubule, 49 +/- 10; cortical collecting tubule, 15 +/- 1; medullary collecting tubule, 32 +/- 7 amol X cm-1. These results indicate the presence of specific angiotensin II binding sites in all tubule segments studied, but binding capacity was highest in the proximal convoluted tubule, in agreement with transport studies that localize the effects of the hormone in this segment.
S K Mujais, S Kauffman, A I Katz
Mammalian erythrocytes have large amounts of catalase, an enzyme which catabolizes hydrogen peroxide (H2O2). Because catalase has a low affinity for H2O2, others have suggested that glutathione peroxidase clears most H2O2 within the erythrocyte and that catalase is of little import. We hypothesized that erythrocyte catalase might function to protect heterologous somatic cells against challenge by high levels of exogenous H2O2 (e.g., in areas of inflammation). We find that, whereas nucleated cells (L1210 murine leukemia) are readily killed by an enzymatically generated flux of superoxide (and, therefore, H2O2), the addition of human and murine erythrocytes blocks lethal damage to the target cells. Inhibition of erythrocyte superoxide dismutase, depletion of glutathione, and lysis of the erythrocytes do not diminish this protection. However, inhibition of erythrocyte catalase abrogates the protective effect and the addition of purified catalase (but not superoxide dismutase) restores it. Furthermore, erythrocytes derived from congenitally hypocatalasemic mice (in which other antioxidant systems are intact) do not protect L1210 cells. Our results raise the possibility that the erythrocyte may serve as protection against by-products of its own cargo, oxygen.
N S Agar, S M Sadrzadeh, P E Hallaway, J W Eaton
Morphologic and functional abnormalities of vascular endothelium are well recognized in diabetes. In view of our previous finding that high glucose concentrations accelerate death and hamper replication of cultured human endothelial cells, we have investigated in the same model the possibility that exposure to high glucose may result in DNA damage. DNA from human endothelial cells--but not from fibroblasts--exposed to 30 mM glucose for 9-14 d manifested an accelerated rate of unwinding in alkali indicative of an increased number of single strand breaks (P less than 0.001 vs. control). Endothelial cells exposed to high glucose also manifested an increased amount of hydroxy-urea-resistant thymidine incorporation (333 +/- 153 cpm/10(5) cells vs. 88 +/- 42 in control cells, mean +/- SD, P = 0.04), which is indicative of increased DNA repair synthesis. Neither DNA damage nor repair synthesis were increased by medium hypertonicity achieved with 30 mM mannitol. These findings suggest the possibility that, under conditions of high ambient glucose, excess glucose entry in cells that are insulin independent for glucose transport may, directly or indirectly, perturb DNA function. Further, they suggest the possibility that different individual capabilities to repair DNA damage--a process that is under genetic control--may represent a mechanism for different individual susceptibilities to development of diabetic vascular complication.
M Lorenzi, D F Montisano, S Toledo, A Barrieux
We have investigated the secretory function of cell suspensions from bone eosinophilic granulomas surgically collected in two patients with histiocytosis X. Unseparated cell preparations spontaneously produced interleukin 1 (IL-1) and prostaglandin E2 (PGE2). In order to ascertain that this secretion was due to the characteristic Langerhans cell-like histiocytosis X cells predominantly found in the bone lesions, we have purified T6+ cells by the use of a fluorescence-activated cell sorter. Such highly purified cell preparations were found to secrete IL-1 and PGE2 spontaneously in culture. Stimulation with endotoxins and treatment with interferon gamma (IFN gamma) revealed an intense IL-1 secretory function of histiocytosis X cells. Since both IL-1 and PGE2 are able to induce bone resorption in vitro, our findings are compatible with the hypothesis that histiocytosis X cells are responsible for the typical osteolytic lesion observed in histiocytosis X through the local secretion of these two mediators.
F Arenzana-Seisdedos, S Barbey, J L Virelizier, M Kornprobst, C Nezelof