A rabbit model of pneumococcal meningitis was used to examine the importance of bactericidal vs. bacteriostatic antimicrobial agents in the therapy of meningitis 112 animals were infected with one of two strains of type III Streptococcus pneumoniae. Both strains were exquisitely sensitive to ampicillin, minimum inhibitory concentration (MIC)/minimum bactericidal concentration (MBC)<0.125 μg/ml. The activity of chloramphenicol against the two strains varied: strain1—MIC 2 μg/ml, MBC 16 μg/ml; strain2—MIC 1 μg/ml, MBC 2 μg/ml. Animals were treated with either ampicillin or chloramphenicol in dosages that achieved a peak bactericidal effect in cerebrospinal fluid (CSF) for ampicillin against both strains. Two different dosages were used for chloramphenicol. The first dosage achieved a peak CSF concentration of 4.4±1.1 μg/ml that produced a bacteriostatic effect against strain1 and bactericidal effect against strain2. The second dosage achieved a bactericidal effect against both strains (mean peak CSF concentration 30.0 μg/ml). All animals were treated intramuscularly three times a day for 5 d. CSF was sampled daily and 3 d after discontinuation of therapy for quantitative bacterial cultures. Results demonstrate that only antimicrobial therapy that achieved a bactericidal effect in CSF was associated with cure. Over 90% of animals treated with one of the bactericidal regimens (i.e., animals in which the bacterial counts in CSF dropped >5 log10 colony-forming units [cfu]/ ml after 48 h) had sterile CSF after 5 d of treatment. On the other hand, the regimen that achieved bacteriostatic concentrations (CSF drug concentrations between the MIC and MBC) produced a drop of 2.4 log10 cfu/ml by 48 h; however, none of the animals that survived had sterile CSF after 5 d. These studies clearly demonstrate in a strictly controlled manner that maximally effective antimicrobial therapy of experimental pneumococcal meningitis depends on achieving a bactericidal effect in CSF.
W. Michael Scheld, Merle A. Sande
2-Methylthio-ADP and its radioactive analogue [beta-32P]2-methylthio-ADP were synthesized and used to investigate platelet receptors for ADP. 2-Methylthio-ADP induced platelet aggregation and shape change, and inhibited cyclic AMP accumulation in platelets exposed to prostaglandin E1. Compared with ADP, 2-methylthio-ADP was 3-5 times as active as an aggregating agent and 150-200 times as active as an inhibitor of cyclic AMP accumulation. Binding of [beta-32P]2-methylthio-ADP to platelets was measured after centrifuging them through silicone oil to separate platelets from their suspension medium. Binding was reversible, saturable, and specific, with between 400 and 1,200 sites/cell in different platelet preparations. There was no evidence for a second class of binding sites with different affinity. The second order association rate constant was approximately 3.5 X 10(6) M-1 S-1, and the first order dissociation rate was 0.024 s-1, both measured at 23 degrees C. The dissociation equilibrium constant (approximately 15 nM) was about three times higher than the concentration giving half-maximal inhibition of prostaglandin E1-stimulated cyclic AMP accumulation in platelet-rich plasma. Binding was inhibited by ADP (Ki = 3.5 microM), ATP (7 microM), 2-azido-ADP (0.12 microM), inosine diphosphate (IDP, 150 microM), guanosine diphosphate (GDP, 350 microM), and AMP (800 microM). Binding of 2-methylthio-ADP was also blocked by the non-cell-penetrating thiol reagent, p-mercuribenzene sulphonate, a reagent that blocks the inhibition of adenylate cyclase by ADP, but which does not block the ability of ADP to induce aggregation or platelet shape change. The amount of 2-methylthio-ADP bound at saturation was independent of pH in the range 6-8, but the affinity was reduced at pH 6 compared with pH 6.5-8.0. The dissociation constant was not temperature dependent in the range 32 degrees -40 degrees C, whereas the rate of dissociation of 2-methylthio-ADP from platelets after the addition of an excess of ADP approximately doubled over this range. The activation energy for dissociation was approximately 15 kcal/mol. Our results support the conclusion that platelets have a receptor for ADP, which inhibits cyclic AMP accumulation, and which has a sulphydryl group in the binding pocket.
D E Macfarlane, P C Srivastava, D C Mills
Peroxidative decomposition of cellular membrane lipids is a postulated mechanism of hepatocellular injury in parenchymal iron overload. In the present study, we looked for direct evidence of lipid peroxidation in vivo (as measured by lipid-conjugated diene formation in hepatic organelle membranes) from rats with experimental chronic iron overload. Both parenteral ferric nitrilotriacetate (FeNTA) administration and dietary supplementation with carbonyl iron were used to produce chronic iron overload. Biochemical and histologic evaluation of liver tissue confirmed moderate increases in hepatic storage iron. FeNTA administration produced excessive iron deposition throughout the hepatic lobule in both hepatocytes and Kupffer cells, whereas dietary carbonyl iron supplementation produced greater hepatic iron overload in a periportal distribution with iron deposition predominantly in hepatocytes. Evidence for mitochondrial lipid peroxidation in vivo was demonstrated at all three mean hepatic iron concentrations studied (1,197, 3,231, and 4,216 micrograms Fe/g) in both models of experimental chronic iron overload. In contrast, increased conjugated diene formation was detected in microsomal lipids only at the higher liver iron concentration (4,161 micrograms Fe/g) achieved by dietary carbonyl iron supplementation. When iron as either FeNTA or ferritin was added in vitro to normal liver homogenates before lipid extraction, no conjugated diene formation was observed. We conclude that the presence of conjugated dienes in the subcellular fractions of rat liver provide direct evidence of iron-induced hepatic mitochondrial and microsomal lipid peroxidation in vivo in two models of experimental chronic iron overload.
B R Bacon, A S Tavill, G M Brittenham, C H Park, R O Recknagel
To investigate the fate of intrinsic factor and cobalamin during cobalamin absorption, we incubated enterocytes isolated from guinea pig ileum for periods of up to 30 min with 57Co-labeled cyano-cobalamin bound either to human intrinsic factor or to rabbit intrinsic factor biosynthetically labeled with [35S]methionine. When the labeled complex was incubated for 30 min with isolated ileal cells under conditions that block cellular metabolism, virtually all cellular radioactivity could be removed by washing the cell surface with EDTA or acid. In contrast, washing removed only half the radioactivity from cells incubated at 37°C in O2. When residual cellular radioactivity was extracted and analyzed by gel filtration, 80-94% of both the 35S and 57Co radioactivity eluted in the same fractions as the original complex. The remaining 6-20% eluted as free [57Co]cobalamin or [35S]methionine. To examine events occurring after 30 min, we instilled into tied-off ileal loops of intact guinea pigs radiolabeled intrinsic factor-cobalamin complex and extracted nondissociable radioactivity 2-4.5 h later. The proportion of extracted 57Co eluting as free cobalamin increased to 39-46%, that eluting as intrinsic factor-cobalamin complex declined to 22-45%, and 9-34% now eluted as a macromolecule that reacted with antitranscobalamin II antibody but not antiintrinsic factor antibody. Extracted 35S radioactivity eluted in several peaks in addition to the intrinsic factor peak. These findings suggest that (a) after reversible attachment of intrinsic factor-cobalamin complex to its ileal surface receptor, an energy-dependent process prevents removal of the complex from the cell surface by EDTA or acid; (b) cobalamin dissociates from intrinsic factor and, as suggested by previous workers, binds to a molecule antigenically similar to transcobalamin II; and (c) intrinsic factor is slowly degraded and forms breakdown products that are detectable in ileal extracts.
Cyrus R. Kapadia, Del Serfilippi, Kurt Voloshin, Robert M. Donaldson Jr.
Cholesterol esterification, cholesteryl ester transfer between lipoproteins, and cholesterol transport between lipoproteins and cultured cells have been measured in the plasma of 22 patients with primary hyperlipidemia and 10 normolipidemic subjects. In hyperbetalipoproteinemia, increase in plasma low density lipoprotein levels was associated with a reduction of cholesteryl ester transfer rates, and with a reversal of the normal direction of sterol transport between fibroblasts and their plasma culture medium. Instead of net transport from cells to medium there was a net uptake of sterol from plasma by the cells, despite a level of plasma lecithin/cholesterol acyltransferase activity that was within the normal range. In dysbetalipoproteinemia, esterification rates were increased above normal levels, but cholesteryl ester transfer was reduced and the direction of sterol transport between the cells and plasma medium was reversed, as in the hyperbetalipoproteinemic group. In hypertriglyceridemia, those subjects with cardiovascular disease showed a metabolic pattern similar to the hyperbetalipoproteinemic group. The subjects in this group without symptoms of cardiovascular disease showed a normal direction of sterol transport, normal or raised rates of cholesteryl ester transfer between lipoproteins, and an increased rate of sterol esterification in plasma that decreased towards normal levels as plasma triglyceride levels decreased. Despite their quite distinct metabolic patterns there was no consistent difference between the two hypertriglyceridemic groups in triglyceride or cholesterol levels, very low density lipoprotein composition, or electrophoretic or isoelectric focussing patterns. All hypertriglyceridemic subjects with documented cardiovascular disease showed reversed cell-plasma sterol transport and all subjects without such disease showed a normal direction of cell-plasma sterol transport. The results of this study indicate major and reproducible abnormalities in plasma cholesterol metabolism in several groups of subjects with genetically distinct hyperlipidemias, who are at risk for atherosclerotic vascular disease. The possible predictive value of sterol metabolic measurements in the analysis of cardiovascular disease is discussed.
P E Fielding, C J Fielding, R J Havel, J P Kane, P Tun
In a previous study, molecular cloning of the alpha-globin genes from a patient with nondeletion Hb-H disease (genotype--/alpha alpha) showed that a single nucleotide mutation (CTG to CCG) in one of the genes resulted in a leucine to proline substitution. This paper describes the approach we used to detect the abnormal alpha-globin chain. The chain was identified using a cell-free translation system. It turned over rapidly both in vitro and in vivo in the patient's reticulocytes. The unusual feature of this unstable alpha-globin is that the alpha-globin deficiency causes alpha-thalassemia. Simple heterozygotes for this lesion (alpha Pro alpha/alpha alpha) resemble alpha-thalassemia carriers and do not exhibit the hemolytic anemia usually associated with unstable hemoglobins.
S A Liebhaber, Y W Kan
In adults, glucose infusion results in a decreased glucose production rate (GPR) as a mechanism for maintaining euglycemia. To document the development of glucose homeostasis, we derived the GPR in 23 preterm appropriate for gestational age infants, 14 term appropriate for gestational age infants, and in 6 adults. After a 3-h fast, the average plasma glucose and insulin concentration was measured and the GPR was derived. During glucose infusion (5.6 +/- 0.3 mg X kg-1 min-1), compared with saline controls, the preterms had a rise in plasma glucose and plasma insulin, and the GPR was 1.4 mg X kg-1 min-1 (range, 0-4.4) vs. 3.0 mg X kg-1 min-1 (range, 1.8-4.1) (saline controls). In the term infants, only the plasma insulin concentration was elevated when the glucose infused (5.7 +/- 0.3 mg X kg-1 min-1) infants were compared with the saline controls and GPR was 0.4 X kg-1 min-1 (range, 0-2.6) vs. 3.4 mg X kg-1 min-1 (range, 2.8-5.7) (saline controls). In comparison to saline infused adults, glucose infusion (3.2 +/- 0.1 mg X kg-1 min-1) resulted in a significant rise in plasma glucose and in plasma insulin; and the GPR was reduced to 0.1 mg X kg-1 min-1 (range, 0-0.3) from 2.0 mg X kg-1 min-1 (range, 1.5-2.4). 5 of 13 preterms and 2 of 7 term infants had persistent GPR during glucose infusion; in contrast, the GPR in all adults was unmeasurable. There was no correlation between the plasma glucose concentration and the GPR in the newborn or in the adult. Both newborns and adults did have a correlation between plasma insulin concentration and the GPR; however, there was considerable variability in the neonate. We conclude that there are significant developmental differences in neonatal glucose homeostasis and that insulin is important in neonatal hormonal control of glucose production.
R M Cowett, W Oh, R Schwartz
The effects of extrahepatic cholestasis upon adrenergic regulation of glycogenolysis and upon the numbers of adrenoceptors in rat liver were studied using isolated hepatocytes and plasma membranes, respectively. A 60% decrease in the number of alpha 1 adrenoceptors (285 vs. 680 fmol/mg protein) and a simultaneous 2.7-fold increase in the number of beta adrenergic sites (67 vs. 25 fmol/mg protein) were observed beginning 36 h after bile flow obstruction and persisted for at least 68 h. The reciprocal modification of the numbers of alpha 1 and beta adrenoceptors was accompanied by a change in the manner of stimulation of glycogen phosphorylase by catecholamines in hepatocytes; originally alpha 1 adrenergic in normal rats (phenylephrine Ka = 0.9 microM, isoproterenol Ka = 7.1 microM), the stimulation became predominantly beta adrenergic in cholestatic animals (phenylephrine Ka = 3.7 microM, isoproterenol Ka = 0.06 microM). In normal rats, activation of the enzyme by epinephrine was inhibited by the alpha blocker phentolamine, without inhibition by the beta blocker propranolol. In contrast, propranolol was more effective than phentolamine in cholestatic rat hepatocytes. Modification of the regulation of glycogenolysis after cholestasis did not seem to be secondary to an alteration in the metabolism of thyroid hormones or in the action of glucocorticoids. However, cholestasis provoked a 10-fold increase in the number of hepatic mitoses and in the incorporation of thymidine into liver DNA of cholestatic animals. Similar changes were observed in regenerating livers, following two-thirds hepatectomy. We propose that the changes following extrahepatic cholestasis might, as well, be explained by a regenerative process.
M Aggerbeck, N Ferry, E S Zafrani, M C Billon, R Barouki, J Hanoune
Intravenous cationic bovine serum albumin (BSA, pI > 9.5) induces membranous nephropathy in immunized rabbits. In this study, unimmunized rabbits received intravenous injections of cationic (n = 3) or native (n = 3) or native (n = 3) BSA, followed by ex vivo isolated left renal perfusions with sheep anti-BSA antibody. Capillary wall deposits of IgG and C3 were seen exclusively in the group receiving cationic BSA, confirming an in situ pathogenesis for cationic, BSA-induced membranous nephropathy, and demonstrating the importance of a cationic antigen for its production. We then explored whether membranous nephropathy in this model is prevented by the concomitant injection of protamine sulfate, a filterable, relatively non-immunogenic polycation. An in vitro study demonstrated that protamine sulfate incubated with glomerular basement membrane (GBM) decreased the subsequent binding of radiolabeled cationic BSA (P < 0.05). In vivo, protamine sulfate was shown to bind to anionic sites in the glomerular capillary wall after intravenous injection.
Sharon G. Adler, Haiyen Wang, Harry J. Ward, Arthur H. Cohen, Wayne A. Border
The whole body clearance of norepinephrine (NE) was measured in seven patients pre- and postoperatively. L[3H]NE was infused intravenously for 90 min and steady-state concentrations of L[3H]NE were measured at 75 and 90 min in both arterial and peripheral venous blood. Preoperatively, in the resting supine position, the clearance values based on arterial and venous sampling averaged 1.4 and 2.5 liter/min, respectively (P < 0.02). The difference in clearance values was due to a peripheral uptake of NE averaging 45%.
Jannik Hilsted, Niels J. Christensen, Sten Madsbad
An inactive form of renin has been isolated from human plasma. It has been suggested that this may represent renin precursor secreted from the kidney. However, early studies failed to isolate inactive renin from human renal tissue. In this investigation, rapid processing of human kidney cortex at temperatures below 4 degrees C in the presence of protease inhibitors followed by cibacron-blue affinity chromatography allowed us to extract a totally inactive form of renal renin. Furthermore, we found that in kidney inactive renin constituted from 10 to as much as 50% of the total renin concentration. Biochemical characterization of the inactive renin from plasma and from kidney indicates that they are structural homologues and, when activated, have enzymatic properties that resemble active renal renin. Renal and plasma inactive renin were found to have the following properties in common: (a) a pH optimum of activation of 3.3; (b) reversible activation by acid dialysis on return to pH 7.4, 37 degrees C; (c) pH optima of enzyme activity of 7.8 with sheep angiotensinogen and 5.5 and 6.7 (biphasic) with human angiotensinogen; (d) Michaelis-Menten constants, Km, of 0.29-0.34 microM with sheep angiotensinogen, and 0.99-1.25 microM with human angiotensinogen; (e) an antibody to human renal renin mean inhibitory titer of 1:30,000 with 1 X 10(-4) Goldblatt units of activated renal or plasma inactive renin; (f) gel filtration profiles consisting of two peaks with apparent molecular weights of 56,000 +/- 1,500 and 49,200 +/- 1,000. Activation of plasma and kidney inactive renin by acid plus renal kallikrein was not accompanied by a change in gel filtration elution patterns. To determine whether inactive renin is released by the kidney, we measured inactive renin in samples obtained simultaneously from both the renal veins and inferior vena cava below the origin of the renal veins. In eight consecutive patients, inactive renin concentration was significantly higher in renal venous blood than in inferior vena caval blood. These data indicate that human kidney contains and secretes significant quantities of inactive renin. Thus, the kidney appears to be a major source of inactive renin in human plasma.
W A Hsueh, E J Carlson, V J Dzau
Cultured, spontaneously beating heart cells were used to study the role of calmodulin in regulating cardiac contraction. Trifluoperazine (TFP), an antipsychotic drug that binds to calmodulin, reversibly inhibited myocardial contraction. This effect occurred over a TFP concentration range of 5-100 μM with half maximal activity at ∼15 μM TFP. When the phosphoprotein profile of TFP-treated cells was compared with control cultures, there was a significant decrease in 32P content of the 20,000-D myosin light chain. As measured by two-dimensional gel electrophoresis, the fraction of phosphorylated myosin light chain decreased from 0.31±0.06 in control to 0.16±0.05 in TFP-treated cells (P < 0.05). This inhibition of protein phosphorylation was relatively selective, as two other phosphoproteins (∼ 41,000 and 36,000 D) were unaffected, and a third protein (∼ 28,000 D) showed an increase in 32P activity. In contrast, the cessation of spontaneous beating by 50 mM KCl did not reproduce these changes. This inhibition of contraction mediated by TFP associated with alterations in the phosphorylation of certain cardiac phosphoproteins suggests a role for calmodulin and for the myosin light chain in the regulation of cardiac cell contraction.
Binding of human high density lipoproteins (HDL, d = 1.063-1.21) to cultured human fibroblasts and human arterial smooth muscle cells was studied using HDL subjected to heparin-agarose affinity chromatography to remove apoprotein (apo) E and B. Saturation curves for binding of apo E-free 125I-HDL showed at least two components: low-affinity nonsaturable binding and high-affinity binding that saturated at approximately 20 micrograms HDL protein/ml. Scatchard analysis of high-affinity binding of apo E-free 125I-HDL to normal fibroblasts yielded plots that were significantly linear, indicative of a single class of binding sites. Saturation curves for binding of both 125I-HDL3 (d = 1.125-1.21) and apo E-free 125I-HDL to low density lipoprotein (LDL) receptor-negative fibroblasts also showed high-affinity binding that yielded linear Scatchard plots. On a total protein basis, HDL2 (d = 1.063-1.10), HDL3 and very high density lipoproteins (VHDL, d = 1.21-1.25) competed as effectively as apo E-free HDL for binding of apo E-free 125I-HDL to normal fibroblasts. Also, HDL2, HDL3, and VHDL competed similarly for binding of 125I-HDL3 to LDL receptor-negative fibroblasts. In contrast, LDL was a weak competitor for HDL binding. These results indicate that both human fibroblasts and arterial smooth muscle cells possess specific high affinity HDL binding sites. As indicated by enhanced LDL binding and degradation and increased sterol synthesis, apo E-free HDL3 promoted cholesterol efflux from fibroblasts. These effects also saturated at HDL3 concentrations of 20 micrograms/ml, suggesting that promotion of cholesterol efflux by HDL is mediated by binding to the high-affinity cell surface sites.
R Biesbroeck, J F Oram, J J Albers, E L Bierman
We have recently provided evidence suggesting that the action of purified cationic bactericidal/permeability-increasing protein (BPI) from neutrophils on susceptible gram-negative bacteria requires saturation binding to negatively charged surface sites (Weiss, J., S. Beckerdite-Quagliata, and P. Elsbach, 1980, J. Clin. Invest., 65: 619-628.)
Jerrold Weiss, Michael Victor, Peter Elsbach
I tested the hypothesis that chronic hyperglycemia alters fetal lung maturation by continuous infusion of glucose (14±2 mg/kg per min, mean±SE) from 112 up to 145 d gestation into six chronically catheterized fetal lambs from which tracheal fluid could be collected. Serum glucose levels (32±2 mg/dl) and serum insulin levels (38±4 μU/ml) in these glucose-treated fetuses were significantly higher than serum glucose levels (18±2 mg/dl, P < 0.001) and serum insulin levels (12±3 μU/ml, P < 0.001) in six chronically catheterized control fetuses of the same gestational ages. Glucose infusion to the fetuses did not alter maternal serum glucose (60±3 mg/dl) or serum insulin levels (35±5 μU/ml). Arterial blood gases (pH 7.34±0.01, Po2 24.3±0.5 mmHg, Pco2 41.5±0.9 mmHg), oxygen saturation (73±2%), hematocrit (31±1%), and tracheal fluid flow (2.4±0.1 ml/g per h) in the glucose-treated fetuses were not significantly different from controls. Among the control fetuses, surface active material (SAM) began to appear in tracheal fluid at 123 d gestation and was present in all six fetuses by 129 d gestation, whereas SAM did not appear at all in tracheal fluid of four of the glucose-treated fetuses, and appeared in two at low levels after 142 d gestation. SAM flux in the glucose-treated fetuses (<1 μg/g per h) was statistically lower than SAM flux in the control fetuses (60±9 μg/kg per h, P < 0.001). Between 130 and 140 d gestation, tracheal fluid phospholipid content rose fourfold, mixed lecithin content rose ninefold, disaturated phosphatidylcholine content rose fourfold in the control fetuses, whereas little or no increase in these measurements occurred in the glucose-treated fetuses (all differences significant). I conclude that chronic hyperglycemia with secondary hyperinsulinemia reduces SAM flux in tracheal fluid of fetal lambs. The reduction in SAM flux is attributed to low surface active phospholipid content of the SAM. A similar mechanism may operate in utero to cause respiratory distress in infants of diabetic mothers whose maternal glucose homeostasis is poorly controlled.
Immunoglobulin (Ig) E synthesis was studied in vitro in eight patients who had received transplants of allogeneic bone marrow. Seven of these patients developed acute graft vs. host disease (GVHD) and elevated serum IgE levels, whereas the eighth did not. In vitro synthesis of IgE, but not of IgG, was elevated in cultures of lymphocytes obtained during acute GVHD (17,923 +/- 14,607 pg/10(6) cells) but not in cultures of lymphocytes obtained after resolution of the acute GVHD when the serum IgE had returned to normal (106 +/- 31 pg/10(6) cells). In contrast, lymphocytes from the patient with no acute GVHD, like normal lymphocytes, failed to synthesize IgE in vitro. The increased in vitro IgE synthesis in acute GVHD was suppressed by normal allogeneic lymphocytes and by autologous lymphocytes obtained after the resolution of the acute GVHD, but not by allogeneic lymphocytes obtained from patients undergoing acute GVHD. The deficiency in functional IgE-specific suppressor cells in acute GVHD occurred in the face of normal or increased percentages of circulating T8+ cells, which in normal subjects contain the IgE-specific suppressor cells. In two patients studied, there was evidence of activated IgE-specific, circulating helper T cells. T cells from these two patients, but not normal T cells, secreted spontaneously upon culture in vitro a factor that induced IgE, but not IgG, synthesis by normal B cells. Finally, a survey of 21 bone marrow transplant recipients revealed that acute GVHD was a necessary requirement for the development of elevated serum IgE levels in recipients of bone marrow transplants. These results suggest that acute GVHD is accompanied by an imbalance in IgE-specific immunoregulatory T cells consisting of activated helper T cells and deficient suppressor cells.
J A Saryan, J Rappeport, D Y Leung, R Parkman, R S Geha
The receptor alterations involved in catecholamine-induced desensitization of adenylate cyclase in human neutrophils have been investigated as has the ability of hydrocortisone to modify such alterations. Incubation of human neutrophils with isoproterenol for 3 h in vitro resulted in an 86% reduction in the ability of isoproterenol to stimulate cyclic AMP accumulation in the cells. Two types of receptor alterations were documented. There was a 40% reduction in the number of beta adrenergic receptors (42 vs. 25 fmol/mg protein, P < 0.005) present after desensitization as assessed by [3H]dihydroalprenolol ([3H]DHA) binding. In addition the receptors appeared to be relatively uncoupled from adenylate cyclase. This uncoupling was assessed by examining the ability of the agonist isoproterenol to stabilize a high-affinity form of the receptor, detected by computer modelling of competition curves for [3H]DHA binding. Desensitized receptors were characterized by rightward-shifted agonist competition curves. When hydrocortisone was added to the desensitizing incubations (combined treatment) there was a statistically significant attenuation in the desensitization process as assessed by the ability of isoproterenol to increase cyclic AMP levels in the cells. Although combined treatment did not prevent the decline in receptor number, it did attenuate the uncoupling of the receptors. Combined treatment resulted in competition curves intermediate between the control and the rightward-shifted desensitization curves. Prednisolone was similar to hydrocortisone in attenuating isoproterenol-induced uncoupling. Thus, steroids appeared to attenuate agonist-induced desensitization of the beta adrenergic receptor-adenylate cyclase system by dampening the ability of agonists to uncouple receptors without modifying their ability to promote down-regulation of beta adrenergic receptors.
Albert O. Davies, Robert J. Lefkowitz
The physiologic effects of epinephrine on mineral metabolism are not known. In six healthy men, insulin-induced hypoglycemia, a potent stimulus to endogenous epinephrine secretion, resulted in a decrement of 0.9±0.1 mg/dl (mean±SE, P < 0.001) in serum inorganic phosphorus and smaller increments in magnesium and total and ionized calcium. Plasma immunoreactive parathyroid hormone (iPTH) decreased and plasma immunoreactive calcitonin (iCT) increased appropriately with the increments in calcium and magnesium. We wished to determine to what extent these changes in mineral metabolism might be attributable to epinephrine. Therefore, in the same protocol, we infused the hormone over 60 min in these six men, in doses that resulted in steady-state plasma epinephrine concentrations ranging from 52 to 945 pg/ml (levels that span the physiologic range), for a total of 25 studies. Serum ionized calcium, iPTH, and iCT concentrations were unaltered by these physiologic elevations of plasma epinephrine. However, epinephrine resulted in dose-dependent decrements in serum inorganic phosphorus of 0.6±0.1 mg/dl (P < 0.005) for the highest epinephrine infusion rate. The plasma epinephrine concentration threshold for this hypophosphatemic effect was ∼50-100 pg/ml. Thus, the sensitivity of the hypophosphatemic response to epinephrine is comparable to that of the cardiac chronotropic, systolic pressor, and lipolytic responses to epinephrine, and considerably greater than that of the diastolic depressor, glycogenolytic, glycolytic, and ketogenic responses to the hormone in human beings. In view of its rapidity, the hypophosphatemic effect of epinephrine is probably the result of a net shift of phosphate from the extracellular compartment to intracellular compartments. We suggest that it is a direct effect of epinephrine, in that it is not mediated by changes in availability of the primary regulatory hormones PTH and CT, although indirect effects mediated by changes in other hormones, such as insulin, cannot be excluded. The hypophosphatemic response is also not attributable to increments in plasma calcium. These data indicate that epinephrine in physiologic concentrations is a hypophosphatemic hormone in man.
Jean-Jacques Body, Philip E. Cryer, Kenneth P. Offord, Hunter Heath III
Because human erythrocyte membrane Ca2+-ATPase is a calmodulin-dependent enzyme, and because physiological levels of thyroid hormone stimulate this enzyme system in vitro, we have studied the role of calmodulin in this model of extranuclear thyroid hormone action. Ca2+-ATPase activity in the absence of thyroid hormone ("basal activity") was increased by inclusion in the preassay incubation mixture of purified calmodulin or hypothyroid erythrocyte hemolysate that contained calmodulin (39 micrograms calmodulin/ml packed cells, determined by radioimmunoassay); addition of L-thyroxine or 3,5,3'-triiodo-L-thyronine (10(-10)M) significantly enhanced (P less than 0.001) enzyme activity in the presence of calmodulin or hemolysate. The stimulatory effects of thyroid hormone, calmodulin, and hemolysate were additive. At 5-10 microM, trifluoperazine, an antagonist of calmodulin, inhibited thyroid hormone stimulation of Ca2+-ATPase activity. Higher concentrations of trifluoperazine (50-100 microM) inhibited basal and hormone-stimulated enzyme activity, with or without added calmodulin. Anti-calmodulin antibody (10-50 micrograms antibody/mg membrane protein) inhibited basal, calmodulin-stimulated and thyroid hormone-stimulated Ca2+-ATPase activity. Membrane preparations were shown by radioimmunoassay to contain residual endogenous calmodulin (0.27 +/- 0.02 micrograms/mg membrane protein). The latter accounts for the effect of trifluoperazine and calmodulin antibody on membrane Ca2+-ATPase activity in the absence of added purified calmodulin. These results support the conclusion that the in vitro action of physiological levels of iodothyronines on human erythrocyte Ca2+-ATPase activity requires the presence of calmodulin.
F B Davis, P J Davis, S D Blas
Synthetic ovine corticotropin-releasing factor (CRF) was administered to normal male volunteer subjects as an intravenous bolus or 30-s infusion. Doses of CRF ranging from 0.001 to 30 micrograms/kg body wt were administered, and plasma immunoreactive (IR)-ACTH and IR-cortisol concentrations were measured. The threshold dose appeared to be 0.01-0.03 micrograms/kg, the half-maximal dose 0.3-1 micrograms/kg, and the maximally effective dose 3-10 micrograms/kg. Basal concentrations of IR-ACTH and IR-cortisol were 14 +/- 7.6 pg/ml (mean +/- SD) and 5.6 +/- 2.2 micrograms/dl, respectively. IR-ACTH rose as early as 2 min after CRF injection, reached peak levels in 10-15 min, and declined slowly thereafter. IR-cortisol rose at 10 min or later and reached peak levels in 30-60 min. At a dose of 30 micrograms/kg, neither IR-ACTH nor IR-cortisol fell from peak levels of 82 +/- 21 pg/ml (mean +/- SE) and 23 +/- 1.4 micrograms/dl, respectively, during the 2-h course of the experiment, indicating that CRF has a sustained effect on ACTH release and/or a prolonged circulating plasma half-life. There was little or no increase in the levels of other anterior pituitary hormones. At doses of 1 microgram/kg and higher, facial flushing, tachycardia, and, in some subjects, a 15-29-mmHg decline in systemic arterial blood pressure were observed, even though blood volume was replaced and the subjects remained supine. These data indicate that synthetic ovine CRF is a very potent and specific ACTH secretagogue in man. Administered with caution until its vasomotor effects are more fully defined, CRF promises to be a safe and very useful investigative, diagnostic, and, possibly, therapeutic agent in man.
D N Orth, R V Jackson, G S DeCherney, C R DeBold, A N Alexander, D P Island, J Rivier, C Rivier, J Spiess, W Vale
The gallbladder and small intestine are reservoirs for the bile acid pool during its enterohepatic circulation and, as such, may regulate biliary secretion of bile acid. During studies of biliary bile acid secretion, a stimulus to gallbladder contraction is continuously infused into the duodenum. Under these conditions, it is assumed that the gallbladder is tonically contracted and that the rate of bile acid secretion into the duodenum equals the hepatic bile acid secretion rate. However, secretion rates vary by as much as 100%, depending upon which of two standard stimuli is used. Therefore, we studied the role of gallbladder emptying and small intestinal transit in determining biliary lipid secretion rate and composition during infusion of these stimuli in five healthy subjects. Each subject was studied with a liquid formula containing 40% of calories as fat, and with an amino acid solution for 10 h. Bile acid, phospholipid, cholesterol, and markers were measured in duodenal bile and hourly secretion rates were calculated by marker dilution technique. Real-time gallbladder sonographs and serum pancreatic polypeptide levels were obtained every 30 min. Small bowel transit time was estimated levels were obtained every 30 min. Small bowel transit time was estimated by the breath hydrogen response after giving lactulose intraduodenally.
Gregory T. Everson, Michael J. Lawson, Carol McKinley, Radene Showalter, Fred Kern Jr.
We determined the plasma half-life of the acute phase protein C-reactive protein (CRP) both in normal rabbits and in rabbits that had received inflammatory stimuli. Rabbit CRP was purified from acute phase serum by Cx-polysaccharide affinity chromatography, radiolabeled, and rendered pyrogen-free. Six unstimulated rabbits were injected intravenously with 1251-CRP prepared by the lactoperoxidase method and four were injected with CRP labeled by methylation using [14C]formaldehyde. Blood samples were obtained at 0.25 h and at intervals thereafter. Plasma half-life of CRP was calculated from the data generated during the first 12 h, by which time an average of 86% of labeled protein had disappeared from the blood stream. The mean half-life for CRP was 4.45±0.2 h, with no significant difference (0.40 < P < 0.45) between 1251- and 14C-labeled CRP. In six animals stimulated with either endotoxin or turpentine 24 h before injection of labeled CRP, a mean half-life of 5.8±0.6 h was found, not significantly different (0.30 < P < 0.35) from unstimulated rabbits. We equated fractional catabolic rate to fractional disappearance rate, since the rate constant for passage of CRP from vascular to extravascular compartment can be assumed to be relatively small compared to the observed fractional disappearance rate. Fractional catabolic rate was independent of serum CRP concentration; average fractional catabolic rate in all 16 animals was 14±0.8% h-1 of the plasma pool. We were able to estimate rate of CRP synthesis, based on steady-state assumptions of pool sizes in those rabbits whose serum CRP levels did not change substantially during the period of study. Values as low as 6.7 μg/kg per h in the unstimulated animals and as high as 560 μg/kg per h in the stimulated animals were found.
Mohanathasan Chelladurai, Stephen S. Macintyre, Irving Kushner
The binding of human 125I-labeled lactoferrin (LF) to a population of adherent mononuclear cells (ADMC) and nonrosetting lymphocytes (E-) was abolished by prior treatment of the cells with deoxyribonuclease (DNase), but not ribonuclease (RNase). When DNase-treated ADMC were incubated with exogenous DNA, the binding of 125I-LF was restored. Enzymatic digestion with other enzymes, trypsin, phospholipase D, and neuraminidase, did not significantly influence 125I-LF binding. Saturable binding of LF at 0 degrees C was demonstrated for both E- and ADMC, with equilibrium dissociated constants of 0.76 x 10(-6) M and 1.8 x 10(-6) M, respectively. E- cells bound 2.5 x 10(7) and ADMC bound 3.3 x 10(7) molecules of Lf at saturation. Cell membranes were isolated from ADMC, E- and E+ and reacted with 125I-labeled LF; significant binding was only seen with ADMC and E-. Prior treatment of the membranes with DNase abolished the binding. Immunofluorescence studies indicated that a population of ADMC and E-, but not E+, exhibited a peripheral staining pattern for LF. Prior treatment of ADMC and E- with DNase abolished the surface immunofluorescence. This study provides evidence that cell membrane DNA acts as a binding site for exogenous LF. This is a novel role for DNA that has not been previously reported. Furthermore, it points to a basic difference between E+ cells vs. ADMC and E- cells in respect to their possession of cell surface DNA.
R M Bennett, J Davis, S Campbell, S Portnoff
Thrombin stimulation results in increased surface expression of endogeneous fibronectin and binding of plasma fibronectin to human platelets. Platelets of patients with Glanzmann's thrombasthenia, a bleeding disorder, exhibit reduced thrombin-induced platelet aggregation, little or no clot retraction, and abnormal platelet spreading on glass surfaces. Thrombin stimulation of patient platelets from four thrombasthenic kindreds resulted in little fibronectin binding. Nevertheless, thrombin did induce serotonin secretion from these cells, indicating that stimulation was occurring. Thrombasthenic platelets did not inhibit thrombin-stimulated fibronectin binding to coincubated normal cells, suggesting that their defect was not due to the presence of a soluble inhibitor of fibronectin binding. Thrombin-stimulated afibrinogenemic platelets bound similar quantities of fibronectin to normal cells, indicating that the thrombasthenic deficit is not secondary to reduced fibrinogen content or binding. The thrombasthenic cells had an endogenous fibronectin content of 2.9 +/- 0.7 micrograms/10(9) platelets, whereas cells simultaneously prepared from five normal individuals contained 1.8 +/- 0.7 micrograms/10(9) platelets, a statistically insignificant difference. Nevertheless, thrombin stimulation did not increase expression of endogeneous fibronectin antigen on the surface of the thrombasthenic platelets as judged by immunofluorescence. These defects in platelet fibronectin binding and surface expression may account for some of the manifestations of Glanzmann's thrombasthenia.
M H Ginsberg, J Forsyth, A Lightsey, J Chediak, E F Plow
Succinylacetone (4,6-dioxoheptanoic acid) is an abnormal metabolite produced in patients with hereditary tyrosinemia as a consequence of an inherited deficiency of fumarylacetoacetate hydrolase. It is known that patients with this hereditary disease excrete excessive amounts of δ-aminolevulinic acid (ALA) in urine and that certain patients have an accompanying clinical syndrome resembling that of acute intermittent porphyria (AIP). In order to elucidate the relation of succinylacetone to the heme biosynthetic pathway, we have examined the effects of this metabolite on the cellular heme content of cultured avian hepatocytes and on the activity of purified ALA dehydratase from normal human erythrocytes and from mouse and bovine liver. Our data indicate that succinylacetone is an extremely potent competitive inhibitor of ALA dehydratase in human as well as in animal tissues. By using purified preparations of the enzyme from human erythrocytes and mouse and bovine liver, an inhibitor constant ranging from 2 × 10-7 M to 3 × 10-7 M was obtained. In cultured hepatocytes, succinylacetone also inhibited ALA dehydratase activity, decreased the cellular content of heme and cytochrome P-450, and greatly potentiated the induction response of ALA synthase to drugs such as phenobarbital, chemicals such as allylisopropylacetamide and 3,5-dicarbethoxy-1,4-dihydrocollidine, and natural steroids such as etiocholanolone. Four patients with hereditary tyrosinemia have been studied and all were found to have greatly depressed levels of erythrocyte ALA dehydratase activity and elevated concentrations of this inhibitor in urine. These findings indicate that tyrosinemia is a disorder of special pharmacogenetic interest because succinylacetone, an abnormal product of the tyrosine metabolic pathway, resulting from the primary gene defect of the disease, profoundly inhibits heme biosynthesis in normal cells through a blockade at the ALA dehydratase level, leading to clinical and metabolic consequences that mimic another genetic disease, AIP.
Shigeru Sassa, Attallah Kappas
Intermediate-sized filaments have been studied in human malignant melanomas and in normal melanocytes by immunofluorescence microscopy with antibodies directed against keratin, vimentin, desmin, neurofilament protein, and glial filament protein. Both human melanotic and amelanotic tumor cells and tumor metastases as well as normal melanocytes in human skin and in the rat eye contain exclusively intermediate filaments of the vimentin type. No reaction was seen with antibodies to keratin, desmin, neurofilaments, or glial filaments. These latter four antisera, however, gave strong reactions in epidermis and other epithelial tissues, muscle, or neural tissues, respectively. The results favor a mesenchymal character of melanocytes, although a neuroectodermal origin in an early developmental stage is possible. The finding that melanomas contain exclusively vimentin intermediate filaments may prove useful in differential diagnosis of melanomas from other tumor types.
F C Ramaekers, J J Puts, O Moesker, A Kant, G P Vooijs, P H Jap
The purpose of this study was to correlate the fasting enterohepatic circulation (EHC) of bile acids with the migrating myoelectric complex. Four dogs were surgically provided with a functional cholecystectomy, a duodenal cannula for direct vision cannulation of the common bile duct, and 12 bipolar electrodes implanted from stomach to terminal ileum. Bile was collected in equal-volume, timed aliquots over 6 to 10 h. Aliquots were sampled and either returned to the duodenum for study of the intact EHC, or collected and retained in order to study the time course of the bile acid pool washout. In the washout experiments boluses of radiolabeled taurocholic acid were instilled into the duodenum before and after duodenal phase III of the migrating motor or myoelectric complex (MMC). In another group of experiments the bile acid pool was washed out and during a continuous duodenal infusion of taurocholic acid bile was collected to study the pattern of hepatic secretion. Results: (a) In all experiments, a single broad peak of bile flow and bile acid secretion occurred at 35-55% of the MMC migration time. At this time the MMC had migrated to a point 70-85% of the distance along the small intestine. (b) During bile acid pool washout the peak of bile flow and bile acid secretion occurred with the distal migration of the first MMC and then bile flow and bile acid secretion rates decreased to a minimum and stabilized. (c) In bile acid pool washout experiments the radiolabeled bile acids instilled into the duodenum prior to duodenal phase III were secreted and peaked with peak endogenous bile acid secretion. The secretion of radiolabeled bile acids instilled into the duodenum after duodenal phase III was delayed until the subsequent cycle of the MMC. 88% of the bile acid pool collected over 6 h was secreted during the distal migration of the first MMC (2.4 +/- 0.4 h). (d) After bile acid pool washout and during continuous duodenal infusion of taurocholic acid, hepatic bile flow and bile acid secretion continued to fluctuate with the same pattern observed with the EHC intact. Conclusions: (a) In the fasting state, the transport of intestinal bile acids to the liver is pulsatile rather than continuous and is determined by the MMC. Maximum hepatic secretion occurs when phase III of the MMC propels the intraluminal bile acid pool to its site of absorption in the distal small bowel. (b) The "housekeeping" action of the MMC is very efficient and clears 88% of the 6-h washout bile acid pool in one pass.
R B Scott, S M Strasberg, T Y El-Sharkawy, N E Diamant
Plasma low density lipoprotein (LDL) transport kinetics were determined from the disappearance of 125I-LDL injected into age- and weight-matched groups of 13 normal subjects, 20 mild diabetics, and 8 moderately severe diabetic patients (fasting plasma glucose less than 150 and greater than 150 mg/100 ml, respectively). In mild diabetics, LDL apo-lipoprotein-B (apo-B) synthetic rate (SR) was significantly greater than normal. The fractional catabolic rate (FCR), however, was also increased so that plasma LDL concentration remained normal. In moderately severe diabetics, LDL SR was normal but FCR was reduced resulting in increased plasma LDL cholesterol and apo-B concentrations. In normal subjects, moderate obesity was associated with increased LDL secretion. In diabetic subjects, however, changes in LDL turnover were of equal magnitude in obese and nonobese patients. In normolipemic and hyperlipemic mild diabetic subjects with equal degrees of glucose intolerance, both LDL apo-B SR and FCR were greater than normal. The magnitude of these increases, however, was lower in the hyperlipemic individuals. Stepwise regression analysis revealed that both LDL SR and FCR correlated positively and linearly with insulin response to glucose loading, but negatively and curvilinearly with fasting plasma glucose and glucose response. We propose that in noninsulin-dependent diabetes, mild hyperglycemia is accompanied by increased LDL turnover, despite normal plasma LDL levels, whereas moderately severe hyperglycemia is associated with decreased LDL catabolism, resulting in increased plasma LDL levels. These changes cannot be attributed to the presence of obesity or hypertriglyceridemia, and may relate to varying degrees of insulin resistance and decreased insulin secretion affecting plasma very low density lipoprotein (VLDL) secretion, VLDL conversion to LDL, and LDL catabolism. Both increased LDL turnover in mild diabetes and delayed removal of LDL in moderately severe diabetes could increase cholesterol ester availability to peripheral tissues, and may result in an increased risk of atherosclerosis.
A H Kissebah, S Alfarsi, D J Evans, P W Adams
The effect of penicillin treatment of Streptococcus sanguis in vitro, on subsequent bacterial density in the bloodstream and on cardiac valves in the rabbit model of endocarditis was studied. As experimental tools for this study, isogenic pairs of S. sanguis differing in resistance to streptomycin or rifampin were prepared by genetic transformation. Rabbits with traumatized heart valves received an intravenous inoculation of penicillin treated (1 μg/ml) and untreated S. sanguis, each marked by resistance to either streptomycin or rifampin. The number of penicillin-treated and untreated bacteria attached to the valvular surfaces was determined by differential counting on streptomycin or rifampin containing media. Penicillin pretreatment reduced cardiac valve colonization 5 min after inoculation (“adherence ratio” × 108 was 4.11 for the control and 3.66 for the penicillin-treated bacteria, P < 0.001). The results were not due to differences in serum killing or bacterial densities in the bloodstream. There was no difference in valvular bacterial densities 24 h after bacterial inoculation (adherence ratio × 108, 7.26 untreated vs. 6.34 penicillin-pretreated, P > 0.10).
Franklin D. Lowy, Daniel S. Chang, Ellen G. Neuhaus, Diane S. Horne, Alexander Tomasz, Neal H. Steigbigel
To assess the pharmacologic effects of aspirin on endogenous prostacyclin and thromboxane biosynthesis, 2,3-dinor-6-keto PGF1 alpha (PGI-M) and 2,3-dinor-thromboxane B2 (Tx-M) were measured in urine by mass spectrometry during continuing administration of aspirin. To define the relationship of aspirin intake to endogenous prostacyclin biosynthesis, sequential urines were initially collected in individuals prior to, during, and subsequent to administration of aspirin. Despite inter- and intra-individual variations, PGI-M excretion was significantly reduced by aspirin. However, full mass spectral identification confirmed continuing prostacyclin biosynthesis during aspirin therapy. Recovery of prostacyclin biosynthesis was incomplete 5 d after drug administration was discontinued. To relate aspirin intake to indices of thromboxane biosynthesis and platelet function, volunteers received 20 mg aspirin daily followed by 2,600 mg aspirin daily, each dose for 7 d in sequential weeks. Increasing aspirin dosage inhibited Tx-M excretion from 70 to 98% of pretreatment control values; platelet TxB2 formation from 4.9 to 0.5% and further inhibited platelet function. An extended study was performed to relate aspirin intake to both thromboxane and prostacyclin generation over a wide range of doses. Aspirin, in the range of 20 to 325 mg/d, resulted in a dose-dependent decline in both Tx-M and PGI-M excretion. At doses of 325-2,600 mg/d Tx-M excretion ranged from 5 to 3% of control values while PGI-M remained at 37-23% of control. 3 d after the last dose of aspirin (2,600 mg/d) mean Tx-M excretion had returned to 85% of control, whereas mean PGI-M remained at 40% of predosing values. Although the platelet aggregation response (Tmax) to ADP ex vivo was inhibited during administration of the lower doses of aspirin the aggregation response returned to control values during the final two weeks of aspirin administration (1,300 and 2,600 mg aspirin/d) despite continued inhibition of thromboxane biosynthesis. These results suggest that although chronic administration of aspirin results in inhibition of endogenous thromboxane and prostacyclin biosynthesis over a wide dose range, inhibition of thromboxane biosynthesis is more selective at 20 than at 2,600 mg aspirin/d. However, despite this, inhibition of platelet function is not maximal at the lower aspirin dosage. Doses of aspirin in excess of 80 mg/d resulted in substantial inhibition of endogenous prostacyclin biosynthesis. Thus, it is unlikely that any dose of aspirin can maximally inhibit thromboxane generation without also reducing endogenous prostacyclin biosynthesis. These results also indicate that recovery of endogenous prostacyclin biosynthesis is delayed following aspirin administration and that the usual effects of aspirin on platelet function ex vivo may be obscured during chronic aspirin administration in man.
G A FitzGerald, J A Oates, J Hawiger, R L Maas, L J Roberts 2nd, J A Lawson, A R Brash
Dermal fibroblasts in culture from a woman with a mild to moderate form of osteogenesis imperfecta synthesize two species of the pro alpha 2-chain of type I procollagen. One chain is normal. The abnormal chain has a slightly faster mobility than normal during electrophoresis in sodium dodecyl sulfate polyacrylamide gels. Analysis of cyanogen bromide peptides of the pro alpha-chain, the alpha-chain, and of the mammalian collagenase cleavage products of the pro alpha- and alpha-chains indicates that the abnormality is confined to the alpha 2(I)CB4 fragment and is consistent with loss of a short triple-helical segment. Type I collagen production was decreased, perhaps because the molecules that contained the abnormal chain were unstable, with a resultant alteration in the ratio of type III to type I collagen secreted into culture medium. Collagen fibrils in bone and skin had a normal periodicity but their diameters were 50% of control; the bone matrix was undermineralized. The structural abnormality in the alpha 2(I)-chain in this patient may affect molecular stability, intermolecular interactions, and collagen-mineral relationships that act to decrease the collagen content of tissues and affect the mineralization of bone.
P H Byers, J R Shapiro, D W Rowe, K E David, K A Holbrook
We have developed a model in the rat that leads to a predictable degree of severe uremia to study the role of the liver in the insulin-resistant state of uremia. The uremic animals were euglycemic and had increased serum immunoreactive insulin when compared with their pair-fed controls. Insulin action, binding, internalization, and degradation were characterized in freshly isolated hepatocytes from uremic animals, sham-operated pair-fed, and ad lib.-fed controls. The basal rate of aminoisobutyric acid (AIB) uptake was increased in hepatocytes from both uremic and pair-fed control rats. However, while hepatocytes from uremic animals were refractory to insulin with regard to AIB uptake, there was no significant difference in the absolute increment above basal AIB uptake by hepatocytes from pair-fed and fed ad lib. animals at any insulin concentration studied. 125I-Insulin binding at 24 degrees C was higher in hepatocytes from uremic rats at every insulin concentration studied when compared with fed ad lib. controls. The time course of 125I-insulin binding to the cell and to the fractions that were membrane bound or internalized were studied at 37 degrees C. An increase in membrane-bound 125I-insulin at 37 degrees C was present also in hepatocytes from uremic animals. The same fraction of membrane-bound 125I-insulin was internalized in hepatocytes from all groups of animals. Extracellular and receptor-mediated 125I-insulin degradation at the plasma membrane and after internalization was studied at 37 degrees C by gel chromatography. There was a delayed and decreased rate of 125I-insulin degradation in hepatocytes from uremic rats in the three compartments. We conclude: (a) In chronic uremia the liver is refractory to insulin with regard to AIB uptake. (b) Insulin resistance in uremic rat liver is not due to defects in insulin binding or internalization. (c) Despite the high level of circulating immunoreactive insulin, hepatocytes from uremic rats did not show the expected "down regulation" of their insulin receptors or an increased rate of insulin degradation. These studies further emphasize the primary role of postbinding events in the regulation of insulin binding and degradation. The mechanism as to how the coordinated steps of insulin metabolism in the liver are disrupted in a pathological state is presently unknown.
J M Kauffman, J F Caro
The effect of glucose ingestion on insulin action was investigated in isolated human fat cells. Subcutaneous adipose tissue was obtained from eight normal adult volunteers before and 1 h after oral intake of 100 g of glucose. Lipolysis (glycerol release) and specific insulin receptor binding were determined. Insulin binding increased significantly by 20-30% after glucose ingestion. This was due to an increase in insulin binding affinity, without any change in the receptor number. The concentration of insulin producing half-maximum inhibition (ED50) of basal lipolysis was 50 μU/ml before and 0.25 μU/ml after glucose ingestion (P < 0.01), which represented a 200-fold difference. As regards isoprenaline-induced lipolysis, the ED50 for insulin inhibition was 30 μU/ml before and 2.5 μU/ml after oral glucose (P < 0.01), which was a 12-fold difference. The maximum insulin-induced inhibition of basal and isoprenaline-induced lipolysis were not altered after oral glucose. It is concluded that glucose ingestion is accompanied by a marked increase in insulin sensitivity of human fat cells and this may be an important modulating factor in the overall scheme of insulin action.
Peter Arner, Jan Bolinder, Jan Östman
We compared serum gastrin concentrations and gastric acid secretion basally and in response to a mixed meal in age-matched women and men. Women had significantly higher basal serum gastrin concentrations (P < 0.01) and two- to threefold higher food-stimulated serum gastrin concentrations (P < 0.001) than men. Basal and food-stimulated serum gastrin concentrations in women did not fluctuate significantly during the menstrual cycle. Sex-related differences in food-stimulated serum gastrin concentrations were not due to differences in antral pH because pH after the meal in women and men had been kept constant at 5.0 by in vivo intragastric titration with sodium bicarbonate.
M. Feldman, C. T. Richardson, J. H. Walsh
The effect of glucagon at various infusion rates on plasma levels of somatostatin-like immunoreactivity (SLI) was examined in conscious normal and chronic alloxan diabetic dogs. In normal dogs glucagon infused at 6, 36, and 120 ng/kg per min did not affect the peripheral venous plasma SLI levels. In diabetic dogs, however, peripheral venous plasma SLI levels in inferior vena cava rose significantly from a mean base-line value of 181±9 pg/ml to a peak value of 279±38 pg/ml during the infusion of 120 μg/kg per min of glucagon, which raised plasma immunoreactive glucagon to >5,000 pg/ml. This glucagon-mediated increase was completely abolished by coinfusion of 7 mU/kg per min of insulin, a rate that maintained the ratio of insulin to glucagon at approximately the baseline value.
Koichi Kawai, Roger H. Unger
Micropuncture studies have shown that glomerular filtration rate (GFR) falls in response to a rise in Na+ or Cl- concentrations in the loop of Henle, whereas studies in isolated kidneys have shown that GFR falls in response to osmotic diuresis. To define the separate effects of an acute increase in plasma sodium (PNa), chloride (PCl) or osmolality (Posmol), changes in renal blood flow (RBF) and GFR were measured during intrarenal infusions of hypertonic NaCl, NaHCO3, Na acetate, dextrose, NH4Cl or NH4acetate to denervated kidneys. The infusions raised Posmol at the experimental kidney by 30-45 mosmol. RBF increased abruptly by 10-30% with all hypertonic infusions indicating that an acute increase in plasma tonicity causes renal vasodilatation. Renal vasodilatation persisted or increased further during infusion of dextrose, NaHCO3 and Na acetate, but GFR was unchanged. In contrast, during infusion of the two Cl-containing solutions, vasodilatation was reversed after 1-5 min and RBF and GFR decreased (P < 0.01) below preinfusion levels. Prior salt depletion doubled the vasoconstriction seen with hypertonic NaCl infusions. Overall, changes in RBF were unrelated to changes in PNa or fractional Na or fluid reabsorption but correlated with changes in PCl (r = -0.91) and fractional Cl- reabsorption (r = 0.94). The intrafemoral arterial infusion of the two Cl-containing solutions did not increase femoral vascular resistance. In conclusion, hyperchloremia produces a progressive renal vasoconstriction and fall in GFR that is independent of the renal nerves, is potentiated by prior salt depletion and is related to tubular Cl- reabsorption. Chloride-induced vasoconstriction appears specific for the renal vessels.
Christopher S. Wilcox
The effects of systemic bicarbonate concentration and extracellular fluid volume status on proximal tubular bicarbonate absorption, independent of changes in luminal composition and flow rate, were examined with in vivo luminal microperfusion of rat superficial proximal convoluted tubules. Net bicarbonate absorption and bicarbonate permeability were measured using microcalorimetry. From these data, net bicarbonate absorption was divided into two parallel components: proton secretion and passive bicarbonate diffusion. The rate of net bicarbonate absorption was similar in hydropenic and volume-expanded rats when tubules were perfused with 24 mM bicarbonate, but was inhibited in volume-expanded rats when tubules were perfused with 5 mM bicarbonate. Volume expansion caused a 50% increase in bicarbonate permeability, which totally accounted for the above inhibition. The rate of proton secretion was unaffected by volume expansion in both studies. The rate of net bicarbonate absorption was markedly inhibited in alkalotic expansion as compared with isohydric expansion. Bicarbonate permeabilities were not different in these two conditions, and the calculated rates of proton secretion were decreased by greater than 50% in alkalosis. Net bicarbonate absorption was stimulated in acidotic rats compared to hydropenic rats. This stimulation was attributable to a 25% increase in the rate of proton secretion. We conclude that (a) proton secretion is stimulated in acidosis, inhibited in alkalosis, and is not altered by volume status; (b) bicarbonate permeability is increased by volume expansion but is not altered by increases in plasma bicarbonate concentration; (c) when luminal bicarbonate concentrations are similar to those of plasma, net bicarbonate absorption is dominated by proton secretion and is thus sensitive to peritubular bicarbonate concentrations, and insensitive to extracellular fluid volume; (d) when luminal bicarbonate concentrations are low and proton secretion is slowed, bicarbonate permeability and thus extracellular fluid volume have a greater influence on net bicarbonate absorption.
R J Alpern, M G Cogan, F C Rector Jr
The mechanism of the intestinal esterification of retinol has been obscure. Recently, an acyl-Coenzyme A (CoA):retinol acyltransferase (ARAT) was found in rat intestinal microsomes, and experiments were therefore conducted to determine whether a corresponding enzyme exists in human small intestine. When microsomes were incubated with [3H]retinol and palmitoyl-CoA, or retinol and [1-14C]palmitoyl-CoA, radioactive retinyl palmitate was formed as identified by alumina column chromatography and reverse-phase high-pressure liquid chromatography. Heating the microsomes for 30 min at 60 degrees C resulted in loss of activity. The esterification was negligible without exogenous acyl-CoA and markedly stimulated by palmitoyl-, oleoyl-, and stearoyl-CoA in concentrations up to 20 microM. The acyl-CoA was successfully replaced by an acyl-CoA generating system, but not by unactivated palmitate (2.5-200 microM). The assay was dependent on the presence of albumin with optimum activity at 2-10 mg/ml. The optimal retinol concentration was 20-30 microM and pH approximately 7.4. The esterifying activity was completely inhibited by 8 mM of taurocholate and to 90% by 1 mM of 5,5'-dithiobis(2-nitrobenzoic acid). Activity was found throughout the small intestine. In jejunum the rate of retinol esterification was: 3.44 +/- 2.24 nmol [3H]retinyl ester formed . mg microsomal protein-1 . min-1 (mean +/- SD, n = 12). The corresponding activity in whole homogenates of biopsies were 1.17 +/- 0.28 (n = 8). It is concluded that human small intestine contains a microsomal acyl-CoA:retinol acyltransferase. Due to its high activity in vitro this enzyme is likely to be responsible for the intestinal esterification of retinol.
P Helgerud, L B Petersen, K R Norum
Evidence is presented indicating that oxidants are generated in lungs of patients with the adult respiratory distress syndrome (ARDS). The evidence was derived from observations that alpha-1-PI, recovered in bronchoalveolar lavage (BAL) fluid, had been inactivated by oxidation, presumably oxidation of the methionyl residue in the reaction site of the molecule. This was indicated by findings that activity of the alpha-1-PI could be restored by exposure to the reducing agent, dithiothreitol in the presence of methionyl sulfoxide peptide reductase. The amount of activity restored was proportional to the amount of inactive alpha-1-PI present at 52,000 D. Oxidation of the 52,000-D alpha-1-PI was also revealed by the finding that the inactive molecule was subject to proteolytic cleavage to 47,000 D when exposed to porcine pancreatic elastase, a characteristic of alpha-1-PI with oxidized methionyl residues in the reactive site. Inactivation of the alpha-1-PI in vivo also resulted from complexing to an active enzyme, shown previously to be neutrophil elastase, and from proteolytic cleavage in vivo, that produced a fragment of 47,000 mol wt. In contrast to that in BAL fluids, the alpha-1-PI in plasma of patients with respiratory distress syndrome was found to be greater than 90% active in 14 of 22 cases and 50-90% active in 8 cases. This suggested that for the most part, alpha-1-PI was inactivated after leaving the vessels and entering the lung. The circulating alpha-1-PI in patients with the respiratory distress syndrome was found to be equally susceptible to oxidative inactivation as alpha-1-PI from normal individuals. It seems improbable therefore that patients develop ARDS because of labile alpha-1-PI inhibitor.
C G Cochrane, R Spragg, S D Revak
A qualitative platelet abnormality and a bleeding tendency are frequently associated with renal failure and uremia. We demonstrated previously that uremic patients display an abnormal platelet aggregation to arachidonic acid and reduced malondialdehyde production in response to thrombin and arachidonic acid. The objectives of this investigation were: (a) to compare platelet prostaglandin (PG) and thromboxane (TX) production in whole blood and in platelet-rich plasma (PRP) of 21 uremic patients and 22 healthy subjects; (b) to evaluate the concentration and activity of platelet PG- and TX-forming enzymes; (c) to assess the functional responsiveness of the platelet TXA2/PGH2 receptor; (d) to explore the hemostatic consequences of partially reduced TXA2 production.
G. Remuzzi, A. Benigni, P. Dodesini, A. Schieppati, M. Livio, G. De Gaetano, J. S. Day, W. L. Smith, E. Pinca, P. Patrignani, C. Patrono
When grown in nude mice, cultured renal carcinoma cells from a hypercalcemic patient produced marked hypercalcemia that was reversed by resection of tumor. Conditioned medium from this cell line contained a protein with activity in a renal adenylate cyclase bioassay for parathyroid hormone (PTH) which was blocked by the competitive PTH antagonist [8norleucyl, 18norleucyl, 34tyrosinyl]bPTH (3-34)amide. However, the biologically active protein was eluted from gel filtration columns as a larger molecular size component that PTH and was not recognized by any of four region-specific PTH antisera. The properties of this factor resemble those of the postulated PTH-like substance(s) in humoral hypercalcemia of malignancy.
G J Strewler, R D Williams, R A Nissenson
The most common form of beta-thalassemia among Mediterraneans results from a single nucleotide substitution within the first intervening sequence (IVS-1) of the beta-globin gene. This particular mutation is not detectable in uncloned DNA by restriction enzyme analysis. Using synthetic DNA of 19-nucleotides in length corresponding to the normal and mutant IVS-1 sequences as probes, we have developed a direct assay for this gene defect. Under carefully controlled experimental conditions these synthetic probes detect only their homologous sequences in restriction digests of both cloned and uncloned DNA samples. The method is sufficiently sensitive to establish the genotype of individuals with respect to this defect using approximately 20 micrograms total DNA. This assay provides an alternative to fetal blood and DNA linkage analysis for the prenatal diagnosis of this variety of beta-thalassemia, particularly among Greek families where it is especially common.
S H Orkin, A F Markham, H H Kazazian Jr
Erythrocytes from humans with Melanesian elliptocytosis are resistant to invasion by Plasmodium falciparum in vitro and epidemiological evidence suggests they may be resistant to P. vivax and P. malariae. We have examined the ability of P. knowlesi merozoites to invade Melanesian elliptocytes in vitro as a definitive means of examining these cells for resistance to invasion by malarial species with different receptor requirements. The Melanesian elliptocytes were highly resistant to invasion by P. knowlesi merozoites showing that the resistance associated with this erythrocyte variant lies at a level common to the invasion pathway(s) of P. falciparum and P. knowlesi. This makes Melanesian elliptocytosis unique as no other human erythrocyte variant has been shown to be resistant to invasion by both species.
T Hadley, A Saul, G Lamont, D E Hudson, L H Miller, C Kidson