Pharmacology and Inotropic Potential of Forskolin in Human Heart

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Human T lymphocyte subsets. Functional heterogeneity and surface recognition structures.

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P L Romain, S F Schlossman

Human neutrophils increase expression of C3bi as well as C3b receptors upon activation.

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We used monoclonal antibodies and flow cytometry to study the expression of the receptors for the complement fragments C3bi (CR3) and C3b (CR1) on human polymorphonuclear neutrophil leukocytes (PMN). Expression of both receptors was minimal on cells stained in anticoagulated whole blood incubated at 0 degree or 37 degrees C. PMN isolated with Percoll density gradients and held at 0 degree C also had only minimal expression of both receptors. With the isolated cells, however, a spontaneous increase in expression of both receptors occurred upon warming to 37 degrees C. This did not represent complete expression of either receptor since additional increments in surface expression could be induced upon stimulation with N-formyl-methionyl-leucyl-phenylalanine or Raji cell supernatant. The increases in complement receptor (CR) expression appeared to be specific since there were no changes in expression of the Fc gamma receptor or beta-2-microglobulin under any of these conditions. The increased CR expression seems to involve translocation from an intracellular pool since it is complete within minutes and is not blocked by puromycin or cycloheximide. These results demonstrate that both CR3 and CR1 expression increase rapidly upon activation of PMN and that isolated cells can be used to study this phenomenon, which may be a critical part of neutrophil function in vivo.

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M Berger, J O'Shea, A S Cross, T M Folks, T M Chused, E J Brown, M M Frank

Thermic effect of glucose in man. Obligatory and facultative thermogenesis.

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The contribution of the sympathetic nervous system to the thermic effect of intravenously infused glucose and insulin was studied in 10 healthy young men before and after beta-adrenergic receptor blockade with propranolol during conditions of normoglycemia (90 mg/dl) at two levels of hyperinsulinemia (approximately 90 microU/ml and approximately 620 microU/ml). During steady state conditions of glucose uptake (0.515 +/- 0.046 and 0.754 +/- 0.056 g/min), significant increases were observed in energy expenditure (0.10 +/- 0.02 kcal/min, P less than 0.001, and 0.21 +/- 0.02 kcal/min, P less than 0.01, respectively). Similarly, glucose oxidation increased from 0.100 +/- 0.015 to 0.266 +/- 0.022 g/min (P less than 0.001) at approximately microU/ml insulin and from 0.082 +/- 0.013 to 0.295 +/- 0.018 g/min (P less than 0.001) at approximately 620 microU/ml insulin. Concomitantly, the rate of nonoxidative glucose disposal or "glucose storage" was 0.249 +/- 0.033 and 0.459 +/- 0.048 g/min, respectively. At this time the thermic effect of infused glucose/insulin was 5.3 +/- 0.9 and 7.5 +/- 0.7%, and the energy cost of "glucose storage" was 0.50 +/- 0.16 kcal/g and 0.47 +/- 0.04 kcal/g at the two different levels of glucose uptake. After beta-adrenergic receptor blockade with propranolol, glucose uptake, oxidation, and "storage" were unchanged in both studies, but significant decreases in energy expenditure were observed (1.41 +/- 0.06-1.36 +/- 0.05 kcal/min, P less than 0.01 at approximately 90 microU/ml insulin, and 1.52 +/- 0.07-1.43 +/- 0.05 kcal/min, P less than 0.005 at approximately 620 microU/ml insulin) causing significant falls in both the estimated thermic effect of infused glucose/insulin and the energy cost of "glucose storage". Regression analysis of the results from both studies indicated a mean energy cost for "glucose storage" of 0.36 kcal/g (r = 0.74, P less than 0.001), which fell significantly (P less than 0.005) to 0.21 kcal/g (r = 0.49, P less than 0.05) during beta-adrenergic receptor blockade with propranolol. The latter is in close agreement with that calculated on theoretical grounds for the metabolic cost of glucose storage as glycogen, i.e., obligatory thermogenesis. It is concluded that beta-adrenergically mediated sympathetic nervous activity is responsible for almost the entire rise in energy expenditure in excess of the obligatory requirements for processing and storing glucose during conditions of normoglycemia and hyperinsulinemia in healthy man, and that the energy cost of "glucose storage" is not different at normal (approximately 90 microU/ml) and supraphysiological (approximately 620 microU/ml) plasma insulin concentrations.

Authors

K J Acheson, E Ravussin, J Wahren, E Jéquier

Neutrophil-endothelial cell interactions. Modulation of neutrophil adhesiveness induced by complement fragments C5a and C5a des arg and formyl-methionyl-leucyl-phenylalanine in vitro.

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Neutrophil adherence to vascular endothelial cells is the initial event in the emigration of neutrophils through blood vessel walls to tissue sites of inflammation; this process is attributed to the generation of extravascular chemotactic factors. To investigate the effect of chemotactic factors on neutrophil adherence to endothelium, we developed a sensitive, reproducible in vitro microtiter adherence assay. Base-line nonstimulated adhesion of human neutrophils to cultured human umbilical vein endothelial cell monolayers was 35.2 +/- 0.9%, which is equivalent to three to four neutrophils per endothelial cell. Addition of either purified complement fragment C5a des arg, or formyl-methionyl-leucyl-phenylalanine (FMLP), in concentrations ranging from 10(-10) to 10(-6) M, increased neutrophil adherence to endothelium in a dose-dependent manner. Purified C5a and C5a des arg were essentially equal in their ability to enhance neutrophil adherence, in contrast to the previously described greater in vitro potency of C5a compared with C5a des arg in stimulating neutrophil chemotaxis and enzyme release. Nonstimulated neutrophils adhered preferentially to human endothelial cells compared with fibroblasts or smooth muscle cells, suggesting that endothelial cells may make a unique contribution to the base-line adhesive interaction. However, chemotactic factors appear to enhance neutrophil adherence to endothelium by exerting an effect primarily on the neutrophil. In the presence of chemotactic factor, neutrophils adhered equally well to different cell types or to protein-coated plastic. Pretreatment of endothelial cells with chemotactic factor for as long as 4 h failed to increase subsequent neutrophil adherence. In contrast, pretreatment of neutrophils with chemotactic factor increased adherence to endothelium. Chemotactic factor-stimulated neutrophil adherence to endothelium occurred rapidly (within 2 min), diminished upon removal of stimulus, but could be rapidly and maximally restimulated upon readdition of the original dose of chemotactic factor. Thus, adherence to endothelium stimulated by chemotactic factor would appear to be a dynamic neutrophil response capable of rapid modulation, possibly important to the ability of neutrophils to adhere to and then migrate through vessel walls to localize at sites of inflammation.

Authors

M G Tonnesen, L A Smedly, P M Henson

Mechanism of sodium modulation of glomerular angiotensin receptors in the rat.

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Specific binding of 125I-angiotensin to high affinity glomerular receptors varies directly with the level of dietary sodium. To investigate the mechanism of sodium regulation of glomerular angiotensin receptors, groups of Sprague-Dawley rats were maintained on one of three levels of sodium intake for at least 5 d: high sodium (7.39 meq/24 h), moderate sodium (0.88 meq/24 h), and low sodium diets (0.01 meq/24 h). An additional group was given low sodium diet with daily injections of furosemide (1 mg/kg i.p.). To dissociate the effects of dietary sodium from those of circulating angiotensin II levels on glomerular receptor regulation, a fifth group was placed on high sodium diet and given a continuous infusion of angiotensin via an implanted minipump (100 ng/min) for 21 d. There was a strong negative correlation (r = -0.98, P less than 0.01) between plasma angiotensin II and glomerular angiotensin receptor density. Dietary sodium, potassium, or water consumption did not correlate with angiotensin II receptor concentration. The affinity constant did not vary in any of the groups (2.33 +/- 0.30 X 10(8) M-1). The time course of sodium regulation of glomerular angiotensin II receptors was studied in rats switched from a moderate sodium to either a high sodium diet or a low sodium diet plus furosemide. Receptor density was unchanged at 24 h, varied directly with sodium intake for 1-5 d when induction was maximal, and remained constant for at least 21 d. The time course of receptor regulation closely paralleled changes in plasma angiotensin II. Additional studies were undertaken to demonstrate that glomerular angiotensin II receptors are down-regulated by circulating hormone. Rats maintained on moderate sodium intake were killed 2 min after the induction of anesthesia with pentobarbital (50 mg/kg i.p.) or by rapid decapitation. Despite a 50-fold elevation of plasma angiotensin II in anesthetized rats (424 +/- 154 vs. 8.6 +/- 1.0 pg/ml, P less than 0.001) angiotensin receptor density was unchanged (anesthetized, 1,016 +/- 126 vs. unanesthetized, 1,290 +/- 84 fmol/mg). The infusion of angiotensin II (100 mg/min) for 15 min or 2 h into anesthetized rats maintained on moderate sodium intake resulted in a 50% reduction in specific angiotensin binding that could not be reversed by the dissociation of endogenous angiotensin. These data are compatible with modulation of receptor density by circulating hormone and can not be accounted for by prior receptor occupancy.

Authors

A Bellucci, B M Wilkes

Familial apolipoprotein AI and apolipoprotein CIII deficiency. Subclass distribution, composition, and morphology of lipoproteins in a disorder associated with premature atherosclerosis.

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Lipoprotein classes isolated from the plasma of two patients with apolipoprotein AI (apo AI) and apolipoprotein CIII (apo CIII) deficiency were characterized and compared with those of healthy, age- and sex-matched controls. The plasma triglyceride values for patients 1 and 2 were 31 and 51 mg/dl, respectively, and their cholesterol values were 130 and 122 mg/dl, respectively; the patients, however, had no measurable high density lipoprotein (HDL)-cholesterol. Analytic ultracentrifugation showed that patients' S degrees f 0-20 lipoproteins possess a single peak with S degrees f rates of 7.4 and 7.6 for patients 1 and 2, respectively, which is similar to that of the controls. The concentration of low density lipoprotein (LDL) (S degrees f 0-12) particles, although within normal range (331 and 343 mg/dl for patients 1 and 2, respectively), was 35% greater than that of controls. Intermediate density lipoproteins (IDL) and very low density lipoproteins (VLDL) (S degrees f 20-400) were extremely low in the patients. HDL in the patients had a calculated mass of 15.4 and 11.8 mg/dl for patients 1 and 2, respectively. No HDL could be detected by analytic ultracentrifugation, but polyacrylamide gradient gel electrophoresis (gge) revealed that patients possessed two major HDL subclasses: (HDL2b)gge at 11.0 nm and (HDL3b)gge at 7.8 nm. The major peak in the controls, (HDL3a)gge, was lacking in the patients. Gradient gel analysis of LDL indicated that patients' LDL possessed two peaks: a major one at 27 nm and a minor one at 26 nm. The electron microscopic structure of patients' lipoprotein fractions was indistinguishable from controls. Patients' HDL were spherical and contained a cholesteryl ester core, which suggests that lecithin/cholesterol acyltransferase was functional in the absence of apo AI. The effects of postprandial lipemia (100-g fat meal) were studied in patient 1. The major changes were the appearance of a 33-nm particle in the LDL density region of 1.036-1.041 g/ml and the presence of discoidal particles (12% of total particles) in the HDL region. The latter suggests that transformation of discs to spheres may be delayed in the patient. The simultaneous deficiency of apo AI and apo CIII suggests a dual defect in lipoprotein metabolism: one in triglyceride-rich lipoproteins and the other in HDL. The absence of apo CIII may result in accelerated catabolism of triglyceride-rich particles and an increased rate of LDL formation. Additionally, absence of apo CIII would favor rapid uptake of apo E-containing remnants by liver and peripheral cells. Excess cellular cholesterol would not be removed by the reverse cholesterol transport mechanism since HDL levels are exceedingly low and thus premature atherosclerosis occurs.

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T M Forte, A V Nichols, R M Krauss, R A Norum

Bile salt stimulation of colonic epithelial proliferation. Evidence for involvement of lipoxygenase products.

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Prostaglandin E2 (PGE2) and several other prostaglandins synthesized by colon suppress the proliferative activity of colonic epithelium. However, bile salts stimulate colonic epithelial proliferation despite the actions of bile salts to enhance the release of arachidonate and consequent colonic synthesis of PGE2. The current study was conducted to assess whether bile salt-induced increases in colonic formation of arachidonate metabolites other than PGE2 were linked to the stimulation of the proliferative activity of colonic epithelium. Within 10 min of addition, deoxycholate markedly stimulated the in vitro release of [14C]arachidonate from prelabeled rat colon. When given in vivo by intracolonic instillation deoxycholate (10 mumol) increased colonic accumulation of immunoreactive prostaglandin E (PGE), thromboxane B2 (TXB2), and the lipoxygenase product 12-hydroxyeicosatetraenoic acid (12-HETE) by two to fourfold over control in 30 min. This effect of intracolonic deoxycholate was followed by a ninefold increase in mucosal ornithine decarboxylase activity (4 h), and a subsequent two to threefold increase in [3H]thymidine [( 3H]Thd) incorporation into DNA of either mucosal scrapings or isolated pools of proliferative colonic epithelial cells (24 h). Intracolonic instillation of indomethacin (50 mumol) suppressed to low or undetectable levels both basal colonic accumulation of PGE and TXB2 and the increases in each parameter induced by subsequent instillation of deoxycholate. By contrast, indomethacin enhanced accumulation of 12-HETE in both control colons and those subsequently exposed to deoxycholate. The increases in 12-HETE induced by indomethacin alone were correlated with stimulation of mucosal ornithine decarboxylase activity and [3H]Thd incorporation into mucosal DNA. Indomethacin also enhanced the increases in these parameters induced by deoxycholate. Intracolonic instillation of phenidone (25-100 mumol) suppressed accumulation of PGE, TXB2, and 12-HETE in control colons and the increases in these parameters induced by a subsequent instillation of deoxycholate. Phenidone alone did not alter mucosal ornithine decarboxylase activity or [3H]thymidine incorporation into mucosal DNA. However, phenidone suppressed or abolished increases in these parameters induced by a subsequent instillation of deoxycholate. 4-(2-[IH-imidazol-1-yl]ethoxy) benzoic acid hydrochloride UK 37,248, which selectively reduced colonic TXB2 to undetectable levels without altering PGE or 12-HETE, had no effect on control or deoxycholate-induced increases in mucosal ornithine decarboxylase activity or [3H]Thd incorporation into DNA. Neither indomethacin nor phenidone altered the increases in [(14)C]arachidonate release induced in vitro by deoxycholate. Chenodeoxycholate and cholate also stimulated [(14)C]arachidonate release from colon in vitro within 10 min, and increased colonic 12-HETE (30 min) and mucosal ornithine decarboxylase activity (4 h) upon intracolonic installation. Prior installation of phenidone inhibited the increases in both 12-HETE and ornithine decarboxylase activity induced by these bile salts. The results support a role for bile salt-induced increases in colonic accumulation of lipoxygenase products, as reflected by 12-HETE, in the subsequent stimulation of the proliferative activity of colonic epithelium.

Authors

F R DeRubertis, P A Craven, R Saito

Complex formation of platelet thrombospondin with plasminogen. Modulation of activation by tissue activator.

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Thrombospondin (TSP), a multifunctional alpha-granule glycoprotein of platelets, binds fibrinogen, fibronectin, heparin, and histidine-rich glycoprotein and thus may play an important role in regulating thrombotic influences at vessel surfaces. In this study we have demonstrated that purified human platelet TSP formed a complex with purified human plasminogen (Plg). Complex formation was detected by rocket immunoelectrophoresis of mixtures of the purified radiolabeled proteins. Significant complex formation of fluid-phase Plg with adsorbed TSP was also demonstrated by enzyme-linked immunosorbent assay (ELISA). The complex formation was specific, saturable, and inhibited by excess fluid-phase TSP, with an apparent KD of approximately 35 nM. In both ELISA and rocket immunoelectrophoresis systems, complex formation was inhibited by 10 mM epsilon-amino-n-caproic acid, implying that there is a role for the lysine binding sites of Plg in mediating the interaction. TSP also formed a complex with plasmin as detected by ELISA but did not directly inhibit plasmin activity measured with a synthetic fluorometric substrate or with a 125I-fibrin plate assay. TSP, when incubated with Plg before addition to 125I-fibrin plates significantly inhibited the generation of plasmin activity by tissue plasminogen activator (TPA) in a manner that was calcium dependent. A kinetic study of Plg activation by TPA in the presence of TSP demonstrated that Michaelis-Menten kinetics were followed and that TSP acted as a noncompetitive inhibitor. These studies support the hypothesis that TSP, acting as a multifunctional regulator in focal areas of active hemostasis, could serve as a prothrombotic influence, leading to increased deposition of fibrin.

Authors

R L Silverstein, L L Leung, P C Harpel, R L Nachman

Effect of acetazolamide on cerebral blood flow and cerebral metabolic rate for oxygen.

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The aim of this study was to evaluate the effect of acetazolamide on cerebral blood flow (CBF) and cerebral metabolic rate for oxygen (CMRO2). CBF, arterial and jugular venous partial O2 pressure, partial CO2 pressure, pH, and O2 saturation percentage were measured in six patients before and 3 and 20 minutes after intravenous administration of 1 g of acetazolamide. CBF was measured by the intracarotid 133xenon injection technique. In addition, changes in CBF were estimated from the arteriovenous oxygen content difference. CBF increased in all patients after acetazolamide, by approximately 55 and 70% after 3 and 20 min, respectively. The CBF changes were of the same order whether calculated from the 133Xe clearance or from the arteriovenous oxygen differences (A-V)O2. CMRO2, calculated from (A-V)O2 differences and CBF, remained constant. Except for an increase in the venous oxygen saturation, the blood gases remained constant. Acetazolamide, in a dose sufficient to inhibit the erythrocyte carbonic anhydrase (EC 4.2.1.1), thus induced a rapid and marked increase in CBF, leaving CMRO2 unchanged. This effect of acetazolamide on CBF is probably explained by a decrease in brain pH rather than by brain tissue hypoxia due to inhibition of oxygen unloading in the brain capillaries.

Authors

S Vorstrup, L Henriksen, O B Paulson

2'-deoxyguanosine toxicity for B and mature T lymphoid cell lines is mediated by guanine ribonucleotide accumulation.

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Inherited deficiency of the enzyme purine nucleoside phosphorylase (PNP) results in selective and severe T lymphocyte depletion which is mediated by its substrate, 2'-deoxyguanosine. This observation provides a rationale for the use of PNP inhibitors as selective T cell immunosuppressive agents. We have studied the relative effects of the PNP inhibitor 8-aminoguanosine on the metabolism and growth of lymphoid cell lines of T and B cell origin. We have found that 2'-deoxyguanosine toxicity for T lymphoblasts is markedly potentiated by 8-aminoguanosine and is mediated by the accumulation of deoxyguanosine triphosphate. In contrast, the growth of T4+ mature T cell lines and B lymphoblast cell lines is inhibited by somewhat higher concentrations of 2'-deoxyguanosine (ID50 20 and 18 microM, respectively) in the presence of 8-aminoguanosine without an increase in deoxyguanosine triphosphate levels. Cytotoxicity correlates instead with a three- to fivefold increase in guanosine triphosphate (GTP) levels after 24 h. Accumulation of GTP and growth inhibition also result from exposure to guanosine, but not to guanine at equimolar concentrations. B lymphoblasts which are deficient in the purine salvage enzyme hypoxanthine guanine phosphoribosyltransferase are completely resistant to 2'-deoxyguanosine or guanosine concentrations up to 800 microM and do not demonstrate an increase in GTP levels. Growth inhibition and GTP accumulation are prevented by hypoxanthine or adenine, but not by 2'-deoxycytidine. 8-Aminoguanosine appears to effectively inhibit extracellular PNP activity; thus, it prolongs the extracellular half-life of 2'-deoxyguanosine and guanosine, but does not completely inhibit intracellular PNP activity in these lymphoid cells. As a result, 2'-deoxyguanosine and guanosine are phosphorolyzed and actively salvaged within the cell, accounting for the accumulation of GTP. Partial inhibition of PNP activity in vivo, therefore, may lead to nonselective cellular toxicity by a mechanism independent of dGTP accumulation.

Authors

Y Sidi, B S Mitchell

Biosynthesis of the human C3b/C4b receptor during differentiation of the HL-60 cell line. Identification and characterization of a precursor molecule.

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The C3b receptor (C3bR) of the human promyelocytic leukemia cell line (HL-60) was induced by incubating these cells with dimethylsulfoxide (DMSO) or retinoic acid. A majority of differentiated (DMSO- or retinoic acid-treated) but not undifferentiated cells formed rosettes with C3b-coated erythrocytes and were morphologically mature granulocytes. HL-60 cells were surface- or biosynthetically labeled and then solubilized in 1% Nonidet P-40 in the presence of multiple protease inhibitors. The C3bR was isolated either by immunoprecipitation with anti-C3bR antibodies or by affinity chromatography with hemolytically inactive components in which the internal thioester bond within the alpha-chain was cleaved (iC3)- or iC4-Sepharose. Autoradiographs of NaDodSO4-polyacrylamide gels indicated that the surface-labeled C3bR on the differentiated cells had an Mr of 210,000 (nonreduced) or 240,000 (reducing conditions). The bulk (approximately 85%) of the radiolabeled material that was isolated from biosynthetically labeled cells co-migrated with the surface-labeled band. A small fraction (approximately 15%) of the biosynthetically labeled material that was isolated by affinity chromatography or immunoprecipitation had an Mr of 188,000, which did not correspond to any surface-labeled band. This putative precursor molecule was characterized by pulse-chase experiments and by analysis of its carbohydrate. In pulse-chase (15-min pulse) studies of differentiated cells, only the 188,000-mol wt molecule was detected at 0 h. By 2 h, greater than 80% of counts had chased from the 188,000 to the 210,000-mol wt molecule. Treatment of these two molecules with endoglycosidases indicated that the 188,000-mol wt molecule possessed high mannose oligosaccharides, while the mature C3bR had complex oligosaccharides. We conclude from these data that the 188,000-mol wt molecule is a precursor of the C3b receptor of HL-60 cells. Other experiments indicated that the half-maximal time for newly synthesized receptor to attain an Mr of 210,000 was 45 min, and that the t1/2 for the disappearance of the receptor on the surface of differentiated HL-60 cells in tissue culture was approximately 10 h. The ability to observe the induction of the C3b receptor as the HL-60 cell line differentiates is an instructive model system to study the biosynthesis of a human integral membrane receptor glycoprotein.

Authors

J P Atkinson, E A Jones

Pathogenesis of hepatic steatosis in the parenterally fed rat.

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Hepatic steatosis frequently complicates total parenteral nutrition (TPN). Some of the mechanisms responsible were examined in rats receiving calories as dextrose (CHO-TPN) or dextrose plus lipid emulsion (Lipid-TPN). Hepatic triglyceride content increased approximately threefold after CHO-TPN and twofold after Lipid-TPN (P less than 0.02). Hepatic triglyceride fatty acid composition reflected endogenous synthesis. Hepatic acetyl-Coenzyme A carboxylase specific activity increased fourfold after CHO-TPN and twofold after Lipid-TPN, and it correlated positively with hepatic lipid content (r = 0.82). The activities of the microsomal enzymes of complex lipid synthesis were unchanged in the TPN groups. Both TPN regimens suppressed hepatic triglyceride secretion, measured by the rise in plasma triglyceride and the incorporation of [14C]palmitic acid into plasma triglyceride after intravenous Triton. Hepatic triglyceride secretion correlated negatively with total hepatic lipid content (r = -0.89). CHO-TPN increased the uptake of a radiolabeled triglyceride emulsion and increased hepatic lipase activity, whereas Lipid-TPN decreased both. Both adipose and cardiac lipase were higher for Lipid-TPN animals than for CHO-TPN or control animals. Hepatic 14C-triglyceride content was increased in both TPN groups as compared with controls after the injection of 1-[14C]-palmitic acid. This increment was proportional to the decreased hepatic secretion. Triglyceride fatty acid oxidation was significantly suppressed by CHO-TPN, less so by Lipid-TPN. Free fatty acid oxidation was suppressed only by CHO-TPN. The results suggest that the steatosis induced by TPN in rats was due to enhanced hepatic synthesis of fatty acid and reduced triglyceride secretion. Reduced hepatic triglyceride uptake, enhanced fatty acid oxidation, and enhanced peripheral tissue plasma triglyceride lipolysis when CHO-TPN is supplemented with lipid may modulate the accumulation of hepatic triglyceride and, along with reduced synthesis of fatty acid, lead to a lower hepatic triglyceride content.

Authors

R I Hall, J P Grant, L H Ross, R A Coleman, M G Bozovic, S H Quarfordt

Proteinases of Pseudomonas aeruginosa evoke mucin release by tracheal epithelium.

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We have determined the potential of exoproducts from pathogenic bacteria to stimulate the release of high molecular weight mucins from goblet cells of airway epithelium in a rabbit tracheal explant system. Culture supernatants from proteolytic strains of Pseudomonas aeruginosa and Serratia marcescens, but not supernatants from a number of non-proteolytic strains, released mucins from goblet cells. Highly purified elastase and alkaline proteinase from P. aeruginosa stimulated goblet cell mucin release in a dose-dependent fashion. Lipopolysaccharide, exotoxin A, and alginate of P. aeruginosa did not possess mucin release properties. Proteolytic activity was required for mucin release by P. aeruginosa elastase, but such release in goblet cells was not mediated by cyclic AMP. Morphologic studies suggested rapid release of mucins from goblet cells was response to elastase by a process resembling apocrine secretion. Several nonbacterial proteinases mimicked the effect of Pseudomonas proteases. These studies provide support for the hypothesis that bacterial and other play a role in the pathogenesis of mucus hypersecretion in acute and chronic lung infections.

Authors

J D Klinger, B Tandler, C M Liedtke, T F Boat

Furosemide inhibits glucose transport in isolated rat adipocytes via direct inactivation of carrier proteins.

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Furosemide inhibits 3-O-methyl-D-glucose equilibrium flux in isolated adipocytes. The inhibition is saturable with an increasing concentration of furosemide and shows a noncompetitive type of kinetics. Both basal and insulin-stimulated fluxes are equally affected by the inhibition. Hydrochlorothiazide and piretanide also inhibit the flux with a similar potency, whereas bumetanide, a more potent diuretic, is much less potent. To understand the molecular basis of this inhibition, effects of furosemide on the glucose-sensitive cytochaslasin B binding activities of adipocytes were studied. Furosemide inhibits the glucose-sensitive cytochalasin B binding of both microsomal and plasma membrane preparations. For both preparations, the inhibition is time dependent and only slowly reversible, is saturable with an increasing concentration of furosemide, shows a noncompetitive type of kinetics with apparent Ki (the inhibitor concentration that gives the half-maximum effect) of 3.5 and 0.7 mM after 2 and 18 h incubation, respectively, and is essentially identical between the basal and insulin-stimulated adipocytes. The inhibition develops with a first-order rate constant of approximately 0.12/h at 4 degrees C. These results indicate that furosemide inhibits glucose transport in adipocytes by directly inactivating transport carriers of both plasma membranes and microsomal reserve pool. This inactivation of glucose carrier may play a part in the diuretic-induced glucose intolerance frequently observed during diuretic therapy.

Authors

D B Jacobs, B K Mookerjee, C Y Jung

Role of phorbol diesters in in vitro murine megakaryocyte colony formation.

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In vitro megakaryocyte differentiation is regulated by two activities: a megakaryocyte colony-stimulating activity (Mk-CSA), which is required for proliferation, and an auxiliary factor, megakaryocyte potentiating activity, which plays a role in later differentiation events. Tumor-promoting phorbol esters alter many cellular differentiation-related events. Thus, it was hypothesized that phorbol esters may bring about megakaryocyte differentiation in vitro. 4 beta-Phorbol 12-myristate 13-acetate (PMA), when co-cultured with a source of Mk-CSA, stimulated a threefold increase in colony numbers. Co-culture of PMA and megakaryocyte potentiator activity did not stimulate colony formation, thus eliminating any action of PMA as an Mk-CSA. The direct effect of PMA on the formation of megakaryocyte colonies was established by (a) the function of PMA as a megakaryocyte potentiator in serum-free experiments, (b) the ability of PMA to stimulate megakaryocyte colony formation using bone marrow cells depleted of populations known to produce potentiating activity, (c) the inability of bone marrow adherent cells previously treated with phorbol, 12,13-dibutyrate (PDBu) to augment megakaryocyte colony formation, and (d) the ability of PMA to induce the growth of immature megakaryocytes into large single megakaryocytes. Structure:activity experiments resulted in equivalent activities for PMA and PDBu, whereas the nontumor promoter phorbol 12,13-diacetate and phorbol itself lacked activity. The observations in this study indicate that phorbol esters can bring about megakaryocyte differentiation, and during colony formation, can induce effects identical to those brought about by biological sources of megakaryocyte potentiator activity.

Authors

M W Long, J E Smolen, P Szczepanski, L A Boxer

Comparison of live human neutrophil and alveolar macrophage elastolytic activity in vitro. Relative resistance of macrophage elastolytic activity to serum and alveolar proteinase inhibitors.

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Elastin is an extracellular matrix protein critical to the normal structure and function of human lung. Recently reported data indicate that live human alveolar macrophages can degrade purified elastin in vitro. In this study, we directly compared the elastolytic activity of alveolar macrophages with that of human neutrophils. In the absence of proteinase inhibitors, human neutrophils degrade much more elastin than do human alveolar macrophages. However, macrophages cultured in 10% human serum and in contact with purified 3H-elastin degraded 4.7 micrograms elastin/10(6) cells per 24 h, as compared to less than 1 microgram/10(6) cells/24 h for neutrophils. We observed a similar pattern when the two cells were cultured in human alveolar fluid. We determined that the relative resistance of macrophage elastolytic activity to serum or alveolar proteinase inhibitors was not simply due to phagocytosis of substrate by the larger macrophages. Live macrophages as well as neutrophils degrade 125I-elastin coupled to noningestible sepharose beads. Again in serum-free media, neutrophils degraded eight-fold more elastin than macrophages but only macrophages degraded sepharose-coupled elastin in the presence of 10% serum. Because of these findings, we compared the enzymatic mechanisms of elastin breakdown by macrophages with that of neutrophils. Macrophage elastolytic activity is largely (65-80%) due to a cysteine proteinase(s), at least part of which is Cathepsin B. Approximately half of the cysteine proteinase activity appeared to be expressed at or near the cell surface. These experiments defined two enzymatically distinct pathways of elastin breakdown by human inflammatory cells: the classic, neutrophil derived soluble elastase(s) that is sensitive to serum and alveolar proteinase inhibitors, and a macrophage-mediated pathway that is largely cell associated and relatively resistant to inhibitors. The function of the two pathways depends on the relative excess or deficiency of soluble inhibitors. At inflammatory sites rich in proteinase inhibitors, tissue macrophages may degrade more extracellular matrix elastin than neutrophils. In smokers without antiproteinase deficiency, pulmonary macrophages, which are known to be increased in number, may be the more important cause of elastin breakdown and emphysema.

Authors

H A Chapman Jr, O L Stone

Platelet membrane glycoprotein IIIa contains target antigens that bind anti-platelet antibodies in immune thrombocytopenias.

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The precise pathogenic mechanism of platelet destruction in immune thrombocytopenias is not known, although many investigators have found that platelet-associated IgG is increased in these diseases. We report here the differentiation between specific binding of anti-platelet antibody, associated with platelet destruction, and the ubiquitous presence of nonspecific, platelet-associated IgG. Using an electrophoretic separation and antibody overlay technique, we have identified a specific membrane protein that bears target platelet antigens in immune thrombocytopenias. When posttransfusion purpura serum was studied, antibody binding to the PlA1 antigen on glycoprotein IIIa was readily distinguished from the nonspecific binding of immunoglobulin to a protein of 200,000 mol wt. After reduction of disulfide bonds, the PlA1 antigenicity was not observed, and IgG bound nonspecifically to a protein band with an apparent molecular weight of 45,000. We have also identified anti-platelet antibodies in patients with idiopathic thrombocytopenic purpura and determined their antigenic specificity. Antibodies which bind to a 100,000-mol wt protein were found in nine of thirteen patients with chronic disease. The antigens in three of these cases were studied in detail by using both reduced and nonreduced control and Glanzmann's thrombasthenic platelets. Target antigens were localized to glycoprotein IIIa, but are different from PlA1. The immune thrombocytopenic purpura antigenic system is clearly distinguished from nonspecific platelet-associated IgG. Sera from eight children with acute idiopathic thrombocytopenic purpura were also studied. In all cases, the nonspecific IgG binding to the 200,000-mol wt protein was observed. However, we were unable to demonstrate antibody binding to glycoprotein IIIa, which suggested that the acute childhood form of this disease may have a different pathogenic mechanism than that of the autoimmune chronic cases.

Authors

D S Beardsley, J E Spiegel, M M Jacobs, R I Handin, S E Lux 4th

Response to immunization with Haemophilus influenzae type b polysaccharide-pertussis vaccine and risk of Haemophilus meningitis in children with the Km(1) immunoglobulin allotype.

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Abstract

In experimental animals, immune responses to certain antigens are regulated by immunoglobulin allotype-linked genes. In an effort to detect such genes in humans, we examined the antibody responses of 74 healthy children with different Km(1) or Gm(23) allotypes to a Haemophilus influenzae type b vaccine (type b polysaccharide capsule-pertussis vaccine). The anticapsular antibody responses of black or white children with the Km(1) allotype were 4.6- to 9.5-fold higher than those of children who lacked this determinant (P less than 0.004). No significant differences were found in antibody response with respect to the Gm(23) allotype. The frequencies of Km(1) and Gm(23) also were examined in 170 patients with Haemophilus meningitis, 71 patients with epiglottitis, and 173 control children. Km(1) was detected less frequently in black patients with meningitis (38%) than in those with epiglottitis (81%, P less than 0.002) or in controls (66%, P less than 0.0007). The relative risk of meningitis thus was 3.2-fold lower among black children with the Km(1) allotype than in those who lacked this allotype (odds ratio = 0.3, 95% confidence interval 0.2 to 0.6). However, the risk of meningitis was not decreased in white children with the Km(1) allotype (odds ratio = 1.0). There were no significant differences in the frequency of Gm(23) among the patient groups and controls. The Km(1) allotype but not the Gm(23) thus defines a subpopulation of children of both races who are high responders to this vaccine, and black children but not white children with the Km(1) allotype are at decreased risk of developing Haemophilus meningitis. These data indicate that in blacks, genes associated with Km(1) may affect immune response to a prototype type b Haemophilus vaccine, and perhaps interact with another factor related to race to affect susceptibility to Haemophilus meningitis.

Authors

D M Granoff, J P Pandey, E Boies, J Squires, R S Munson Jr, B Suarez

Host granulomatous response in schistosomiasis mansoni. Antibody and cell-mediated damage of parasite eggs in vitro.

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Abstract

In chronic schistosomiasis mansoni the major pathologic lesions are granulomas surrounding eggs deposited in host tissues. Parasite ova release antigenic material that sensitize the host, resulting in the development of delayed-type hypersensitivity granulomas. The objectives of the present study were to assess the ability of components of the host granulomatous response to induce biochemical and biologic alterations in eggs in vitro, and to correlate these with the capacity of ova to induce granulomas in vivo. An assay of egg tricarboxylic acid cycle activity was developed by use of 2-[14C]acetate as substrate and measurement of accumulation of released 14CO2. Addition of human granulocytes (96% neutrophils, 4% eosinophils) to eggs (cell/egg ratio 1,000:1) and heat-inactivated normal human serum reduced predicted egg 14CO2 generation by 15.6 +/- 3.0%. This effect was greater in the presence of sera of subjects with schistosomiasis (25.6 +/- 2.8% reduction) or when complement was present (24.4 +/- 4.0%). Autologous eosinophils and neutrophils were equally effective in decreasing egg 2-[14C]acetate metabolism (25.6 and 21.4% reductions, respectively). Since the biological role of schistosome eggs relates to their ability to hatch and produce miracidia, we evaluated the effect of granulocytes and sera on this function. The hatching rate of eggs incubated with normal serum was 52.8 +/- 3.3 miracidia/100 eggs; this value decreased to 37.0 +/- 2.6 when granulocytes were added (P less than 0.01). Granulocytes plus antibody- or complement-containing sera led to hatching rates of 23 and 20 miracidia/100 eggs. When ova were pre-incubated with granulocytes and various sera and injected into mice, the areas of egg-induced pulmonary granulomas measured 8 d later were reduced 32 to 45% as compared with lesions elicited by parasite eggs not exposed to granulocytes. Exposure of antigen-coated Sepharose beads to granulocytes and immune serum before injection into mice also led to a reduction in granuloma formation as compared with beads pre-incubated with serum alone. These data indicate that granulocytes in conjunction with antibodies and complement inflict biologically relevant toxic effects on eggs that are manifest in vivo by a decreased ability to elicit granulomas.

Authors

P A de Brito, J W Kazura, A A Mahmoud

Correction of hypokalemia corrects the abnormalities in erythrocyte sodium transport in Bartter's syndrome.

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Abstract

In Bartter's syndrome, the defective renal tubular transport has been postulated to be a manifestation of a more generalized membrane abnormality. To explore this possibility, sodium concentration, ouabain-sensitive (pump transport), ouabain-resistant but furosemide-sensitive (Na-K-Cl cotransport), and ouabain- and furosemide-resistant (passive transport) 22Na effluxes were measured in erythrocytes obtained from nine patients with Bartter's syndrome before and during correction of hypokalemia. Intracellular [Na+] in erythrocytes obtained from nine patients with Bartter's syndrome was significantly (P less than 0.001) higher than that in 30 normal controls (11.8 +/- 1.8 vs. 7.3 +/- 1.4 mmol/liter cells). Pump transport and Na-K-Cl cotransport 22Na effluxes were significantly (P less than 0.01) increased, whereas the rate constant for these effluxes as well as for passive 22Na efflux did not differ from normal. Correction of hypokalemia and maintenance of a normal serum potassium decreased intracellular [Na+] to 8.2 +/- 1.8 mmol/liter cells, a normal value, and corrected the ouabain-sensitive and furosemide-sensitive 22Na effluxes. The results indicate that exposure of erythrocytes to a low potassium environment is responsible for the high intracellular [Na+] and, in turn, the high sodium efflux in Bartter's syndrome. The normal sodium efflux observed during correction of hypokalemia and the consistently normal rate constants for all three efflux parameters measured suggest that intrinsic sodium transport processes in erythrocytes are normal in Bartter's syndrome.

Authors

J M Korff, A W Siebens, J R Gill Jr

Platelet interaction with rabbit subendothelium in von Willebrand's disease: altered thrombus formation distinct from defective platelet adhesion.

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Abstract

Blood interaction with the subendothelium of rabbit aorta was investigated in an annular perfusion chamber using patients with von Willebrand's disease, hemophilia, and afibrinogenemia. The vessels were exposed to nonanticoagulated blood for a range of flow conditions (wall shear rates of 650-3,300 s-1) and exposure times (1.5-10 min). The resultant platelet and fibrin interaction was quantified by the use of several morphometric techniques, one of which was developed to measure more precisely the dimensions (height and volume) of platelet thrombi attached to the subendothelium. A major finding was that under flow conditions in which little or no defect in platelet adhesion was observed in von Willebrand's disease, platelet thrombus height and volume in this disorder were significantly reduced as compared with normal controls or patients with hemophilia. Thus, Factor VIII/von Willebrand factor (VIII/VWF) may mediate not only the adhesion of platelets to subendothelium but also platelet-platelet attachments necessary for normal thrombus development. The level of Factor VIII:coagulant activity (VIII: C) was also observed to influence the resultant thrombus height and volume deposited on subendothelium, presumably through the generation of thrombin or some other procoagulant factor preceding fibrin formation, since normal values of thrombus dimensions were always observed in a patient with a fibrinogen deficiency. The influence of VIII:C became greater as shear rate was reduced, whereas as shear rate was increased, VIII/VWF was more dominant in determining the resultant platelet deposition on subendothelium. Thus, the deficiencies of VIII:C and VIII/VWF in hemophilia and von Willebrand's disease can lead to various abnormalities in platelet and fibrin association with subendothelium. The importance of a particular deficiency will depend strongly on the local blood flow conditions.

Authors

V T Turitto, H J Weiss, H R Baumgartner

Nonenzymatic glycation of human lens crystallin. Effect of aging and diabetes mellitus.

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Abstract

We have examined the nonenzymatic glycation of human lens crystallin, an extremely long-lived protein, from 16 normal human ocular lenses 0.2-99 yr of age, and from 11 diabetic lenses 52-82-yr-old. The glucitol-lysine (Glc-Lys) content of soluble and insoluble crystallin was determined after reduction with H-borohydride followed by acid hydrolysis, boronic acid affinity chromatography, and high pressure cation exchange chromatography. Normal lens crystallin, soluble and insoluble, had 0.028 +/- 0.011 nanomoles Glc-Lys per nanomole crystallin monomer. Soluble and insoluble crystallins had equivalent levels of glycation. The content of Glc-Lys in normal lens crystallin increased with age in a linear fashion. Thus, the nonenzymatic glycation of nondiabetic lens crystallin may be regarded as a biological clock. The diabetic lens crystallin samples (n = 11) had a higher content of Glc-Lys (0.070 +/- 0.034 nmol/nmol monomer). Over an age range comparable to that of the control samples, the diabetic crystallin samples contained about twice as much Glc-Lys. The Glc-Lys content of the diabetic lens crystallin samples did not increase with lens age.

Authors

R L Garlick, J S Mazer, L T Chylack Jr, W H Tung, H F Bunn

Inhibition of lipolysis by adenosine is potentiated with age.

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Abstract

The ability of a variety of hormones to activate cells declines with age. We have investigated the mechanism for the reduced ability of beta adrenergic stimulation to activate lipolysis in fat cells from older rats. Previously, we have found that these cells have an intact lipolytic response to a cAMP analogue but diminished cAMP accumulation after isoproterenol stimulation, suggesting that the blunted cAMP response is rate limiting. In the present study we have tested the hypothesis that enhanced inhibition of lipolysis by endogenously released adenosine accounts for the diminished lipolysis. Adenosine deaminase was added to media containing the adipocytes from older rats to remove endogenous adenosine. Under these conditions beta adrenergic stimulation of lipolysis is intact in fat cells from older rats. The adenosine analogue N6-phenylisopropyladenosine more effectively inhibited lipolysis in the older group (77 +/- 6%) than in the younger group (46 +/- 5%), suggesting that enhanced efficacy of endogenous adenosine may account for the reduced lipolytic response to catecholamines. When pertussis vaccine was used to functionally inactivate adenosine receptors in adipocytes from the younger and older rats, the ability of isoproterenol to activate lipolysis was restored in the older group. All the data are consistent with the hypothesis that enhanced inhibitory effects of adenosine explain the diminished ability of beta adrenergic agonists to activate lipolysis. It is possible that enhanced inhibitory pathways may be involved in blunting responses to stimulatory hormones in other tissues from older animals.

Authors

B B Hoffman, H Chang, Z Farahbakhsh, G Reaven

Different stages of B cell differentiation in non-T acute lymphoblastic leukemia.

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Abstract

Immunoglobulin heavy chain gene rearrangement was evaluated in 19 cases of acute lymphoblastic leukemia (ALL) and correlated with the immunological phenotypic expression on primary or phorbol diester (12-O-tetradecanoylphorbol-13-acetate [TPA])-induced cells. One case of common ALL (cALL), one case of T-ALL, and one undifferentiated acute leukemia that responded to anti-myeloid drugs after unsuccessful anti-lymphoid induction therapy, had germ line heavy chain genes. Rearranged immunoglobulin genes were instead found in 15 of the 16 cALL cases studied and in a case of non-T, non-B, non-common ("null") ALL, which suggested the B cell origin of the neoplastic cells. All cases bearing a heavy chain gene rearrangement were HLA-DR positive. However, the unique cALL case with a germ line configuration was also HLA-DR positive, which confirmed that both the cALL antigen and HLA-DR antigen were not per se expression of B cell commitment. On the other hand, a complete search for B cell-related markers (BA-1 and B1 monoclonal antibodies, as well as cytoplasmic immunoglobulins [CyIg]) in the cALL cases showed that at least one B cell marker could be detected either on primary or on TPA-induced cells in all cases in which a gene rearrangement had occurred. Incubation with TPA allowed the detection of one B cell marker in a case in which the primary cells were negative, and increased the expression of B cell markers in all but one of the cALLs tested. The only cALL case that was not rearranged expressed no B cell markers either on primary or on TPA-induced cells. The non-T, non-B, non-common ("null") case that was rearranged also showed no phenotypic evidence of B cell markers on primary and induced cells. These findings indicate that: (a) practically all cases of cALL appear to be of B cell origin as shown by gene rearrangement analysis; (b) DNA studies are relevant for a more precise characterization of individual cases of undifferentiated acute leukemia; (c) a complete survey for B cell markers may establish the B cell origin of the cALL blasts, as long as the analysis on primary cells is complemented by differentiation induction assessment; and (d) most cases of non-T ALL appear to be characterized by the expansion of neoplastic cells "frozen" at different levels along the B cell differentiation pathway, the first detectable marker being heavy chain gene rearrangement, followed by BA-1, B1, and CyIg expression.

Authors

R Foa, N Migone, M Saitta, M T Fierro, M C Giubellino, P Lusso, L Cordero di Montezemolo, R Miniero, F Lauria

Role of thrombospondin in platelet aggregation.

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Abstract

Thrombospondin (TSP), the major alpha-granule protein of human platelets, binds to the activated platelet surface upon platelet stimulation. TSP has hemagglutinating (lectin-like) activity and forms a specific complex with fibrinogen. Based on these observations, it was postulated that the interaction of TSP and fibrinogen on the activated platelet surface may be an important step in the platelet aggregation process. To test this hypothesis, monospecific, affinity-purified anti-TSP Fab fragments were prepared and their effects on platelet aggregation and platelet fibrinogen binding were studied. Anti-TSP Fab caused significant interference with thrombin- and collagen-induced platelet aggregation, as monitored by both turbidometric aggregometry and particle counting measuring the disappearance of single platelets. Phase-contrast microscopy revealed that anti-TSP Fab caused a marked decrease in platelet macroaggregates and an increase in microaggregates and nonaggregated single platelets. Anti-TSP Fab did not affect the initial phase of ADP-induced platelet aggregation but caused rapid platelet disaggregation with the abolition of the secondary phase of aggregation. The effect of anti-TSP Fab was not mediated by a direct inhibition of platelet secretion. The effect of anti-TSP Fab on specific binding of labeled fibrinogen to thrombin-stimulated platelets was also studied. Anti-TSP Fab caused a marked decrease in the affinity of fibrinogen binding to the receptors on the activated platelet surface. Kinetic analyses revealed significant displacement of labeled fibrinogen by unlabeled fibrinogen in the presence of anti-TSP Fab, suggesting that TSP serves to stabilize fibrinogen binding to the activated platelet surface and reinforces the strength of interplatelet interactions. It is proposed that platelet aggregation is a dynamic, multistep process, governed initially by the platelet membrane glycoprotein IIb/IIIa-fibrinogen interaction, with the TSP-fibrinogen interaction playing an important role in determining the size and reversibility of platelet aggregates.

Authors

L L Leung

Comparative effects of cholestanol and cholesterol on hepatic sterol and bile acid metabolism in the rat.

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Abstract

Large amounts of cholestanol, the 5 alpha-dihydro derivative of cholesterol are found in tissues of patients with the rare inherited sterol storage disease cerebrotendinous xanthomatosis. Although small amounts of cholestanol are present in virtually every tissue of normal man, little is known about its metabolism and effect on cholesterol and bile acid formation. The purpose of this study is to investigate the absorption and metabolism of cholestanol and its early effects on hepatic morphology and on the rate-limiting enzymes of cholesterol and bile acid biosynthesis. After 2 wk on a diet supplemented with 2% cholestanol, total liver sterol content increased by 48% (3.26 vs. 2.20 mg/g), and resulted in a significant rise in hepatic cholestanol concentration to 1.4 mg/g. However, cholestanol was less efficiently absorbed from the intestine than cholesterol and interfered with cholesterol absorption. Furthermore, hepatic hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase activity rose 2.6-fold (from 150.3 to 397.0 pmol/mg per min) during cholestanol feeding, and was associated with a marked proliferation of the smooth endoplasmic reticulum of the centrilobular areas. In addition, significant amounts of allocholic acid (16%) and allochenodeoxycholic acid (5%) were formed from cholestanol and excreted in the bile. These results show that cholestanol is absorbed from the intestine, interferes with cholesterol absorption, and is deposited in the liver. However, in contrast to cholesterol, cholestanol feeding was associated with a marked elevation of HMG-CoA reductase activity. Thus, despite structural similarity between cholesterol and its 5 alpha-saturated derivative, cholestanol does not exert feedback inhibition on hepatic cholesterol biosynthesis.

Authors

S Shefer, S Hauser, G Salen, F G Zaki, J Bullock, E Salgado, J Shevitz

Effects of platelet depletion on the unanesthetized sheep's pulmonary response to endotoxemia.

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Abstract

The effect of platelet depletion on the unanesthetized sheep's pulmonary response to endotoxemia was studied in eight unanesthetized sheep. Platelets were depleted with rabbit anti-sheep platelet antibodies (APA). Bolus injections of APA alone caused marked pulmonary hypertension (PPA increased from 21 +/- 2 to 62 +/- 5 cm H2O +/- SE) and alterations in lung mechanics (dynamic compliance of the lung [Cdyn] decreased to 38.5 +/- 4.6% and resistance to air flow across the lung [RL] increased to 705 +/- 162% +/- SE of control), which were attenuated by pretreatment with meclofenamate. It was possible to deplete platelets before endotoxemia through a slow continuous infusion of APA without altering base-line values of the measured variables. Platelet depletion did not significantly attenuate the alterations in pulmonary hemodynamics, lung mechanics, lung fluid and solute exchange, or the normal increase in lung lymph concentrations of thromboxane B2 or 6-keto-PGF1 alpha observed following endotoxemia in the sheep. We conclude that normal circulating platelet counts are not required for the full expression of the sheep's response to endotoxemia.

Authors

J R Snapper, J M Hinson Jr, A A Hutchison, P L Lefferts, M L Ogletree, K L Brigham

Differential acute effects of aldosterone, dexamethasone, and hyperkalemia on distal tubular potassium secretion in the rat kidney.

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Abstract

To determine the specific effects on renal potassium transport of acute elevations in plasma aldosterone, dexamethasone, and potassium concentrations, we studied adrenalectomized rats prepared such that each factor could be varied independently. Clearance data alone could not be used to deduce the underlying tubular transport effects, however, since infusion of each of these agents was associated with a marked change in urinary flow rate, which may itself have influenced potassium excretion. We therefore used a technique of continuous microperfusion, in vivo, of single superficial distal tubules to evaluate potassium secretion at constant luminal flow rate during each experimental maneuver. Acute aldosterone infusion was associated with a 90% stimulation of potassium secretion by microperfused tubules. However, total kidney sodium excretion and urinary flow rate were markedly reduced, and these factors opposed the direct tubular action of aldosterone, resulting in no net change in the amount of potassium excreted into the final urine. Conversely, dexamethasone had no direct effect on potassium secretion by single microperfused tubules, but it caused a sharp increase in urinary flow and sodium excretion, and secondarily enhanced urinary potassium excretion by 50%. Hyperkalemia per se stimulated renal potassium excretion both via a direct tubular effect and by increasing urinary flow rate. We conclude that urinary potassium excretion after infusion of each of these agents represents the net result of direct tubular effects and secondary flow-mediated changes.

Authors

M J Field, B A Stanton, G H Giebisch

Nonenzymatic addition of glucocorticoids to lens proteins in steroid-induced cataracts.

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Abstract

A frequent manifestation of long-term glucocorticoid administration is the occurrence of posterior subcapsular cataracts. The molecular basis for this effect has not yet been elucidated. The addition of prednisolone to the rat lens in culture results in a time- and concentration-dependent lens opacification that correlates with the formation of covalent prednisolone-lens protein adducts. Prednisolone adduct formation was analyzed by [3H]prednisolone incorporation and by immunoprecipitation with antiserum specific for proteins modified by the nonenzymatic addition of prednisolone. In the rat lens, these adducts were localized in both the water-soluble and urea-soluble lens protein fractions. Gel electrophoresis and fluorography revealed that the most extensively modified proteins were two crystallins subunits. Lens proteins from 33 normal and cataractous human lenses were fractionated and analyzed for the presence of prednisolone-protein adducts by competitive radioimmunoassay. Adducts were detected only in those samples derived from glucocorticoid-induced cataractous lenses. We conclude that elevated glucocorticoid levels lead to the formation of glucocorticoid-lens protein adducts both in vitro and in vivo. Lens protein modification by glucocorticoids may lead to sufficient biochemical or structural alterations so as to result in cataract formation. The ability of glucocorticoids to form adducts with proteins in vivo also may play a role in some of the other toxic manifestations of long-term glucocorticoid therapy.

Authors

S Manabe, R Bucala, A Cerami

Mechanism of alteration of sodium potassium pump of erythrocytes from patients with chronic renal failure.

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Abstract

We examined intracellular electrolytes, K influx, and [3H]ouabain-binding capacity of erythrocytes from 32 normal subjects and 45 patients with end-stage renal failure on dialysis, including 16 with high intracellular Na (mean 17.3 +/- 3.9 mmol/liter cell water). The [3H]ouabain-binding capacity of erythrocytes with high cell Na was markedly reduced as compared with that of erythrocytes from normal subjects (274 +/- 52 vs. 455 +/- 59 sites/cell, P less than 0.001). The mean serum creatinine was higher in the uremic group with high cell Na. There was a significant linear correlation between intracellular Na and [3H]ouabain-binding in both normal and uremic subjects. Cross-incubation of normal cells with uremic plasma for 24 h failed to reduce [3H]ouabain-binding capacity of normal cells. In spite of a substantial increase in cell Na, K pump influx was not higher in uremic erythrocytes with high cell Na. When intracellular Na was altered with nystatin (cell Na equal to 120 mmol/liter cell water in both groups), K pump influx was proportional to the number of Na-K pump sites so that the ion turnover rate per pump site was similar in the two groups. Uremic plasma failed to depress K pump influx of normal erythrocytes. The passive net influx of Na in uremic cells with high intracellular Na was not different from that observed in erythrocytes from normal subjects. When erythrocytes were separated by age on Percoll density gradients, the number of Na-K pump sites of the youngest uremic cells was significantly lower than that of the youngest normal cells, suggesting that decreased synthesis of Na-K pump sites, rather than accelerated loss of Na-K pump sites during aging, was responsible for the decrease in [3H]ouabain-binding capacity of erythrocytes from uremic subjects. Taken together, these findings suggest that a decrease in the number of Na-K pump sites plays a major role in the abnormality of Na-K pump of erythrocytes from patients with chronic renal failure.

Authors

J T Cheng, T Kahn, D M Kaji

C-peptide and insulin secretion. Relationship between peripheral concentrations of C-peptide and insulin and their secretion rates in the dog.

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Abstract

Estimation of the insulin secretory rate from peripheral C-peptide concentrations depends upon the following characteristics of C-peptide kinetics: (a) equimolar secretion of insulin and C-peptide by pancreatic beta cells; (b) negligible hepatic extraction of C-peptide; (c) constant metabolic clearance rate (MCR) of C-peptide over a physiological and pathophysiological range of plasma levels; and (d) proportional changes in the secretion rate of C-peptide and its peripheral concentrations under varying physiological conditions. In the present experiments, the relationship between a variable intraportal infusion of C-peptide and its concentration in the femoral artery was explored in 12 pancreatectomized dogs. As the infusion of C-peptide was rapidly increased, the magnitude of its peripheral concentration initially increased less than the infusion rate by 20-30%. After an equilibration period of approximately 30 min, however, further increases and decreases in the intraportal infusion were accompanied by nearly proportional changes in its peripheral concentration. Estimates of the amount of C-peptide infused during the experiment based on the steady state C-peptide MCR and its peripheral concentration were within 20% of the amount of C-peptide actually infused. These experiments demonstrate that the portal delivery rate of C-peptide can be calculated from its MCR and peripheral concentration in the dog. They also provide a basis for testing the validity of more complicated models of insulin secretion based on peripheral C-peptide concentrations in the dog as well as other species, including man. Finally, we have shown that the hepatic extraction of endogenously secreted C-peptide is negligible in the basal state (3.1 +/- 6.1%), and does not change after oral glucose ingestion. The MCR of exogenous dog C-peptide was similar whether measured by constant peripheral intravenous infusion (12.3 +/- 0.7 ml/kg per min), constant intraportal infusion (13.4 +/- 0.6 ml/kg per min), or analysis of the decay curve after a bolus injection (13.5 +/- 0.7 ml/kg per min).

Authors

K S Polonsky, W Pugh, J B Jaspan, D M Cohen, T Karrison, H S Tager, A H Rubenstein

Protective effect of intrarenal calcium membrane blockers before or after renal ischemia. Functional, morphological, and mitochondrial studies.

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Abstract

The present study examined whether a pre- or postischemic infusion of verapamil (V) or a postischemic infusion of nifedipine (N), drugs which block calcium (Ca++) influx across plasma membranes, provides protection against ischemic acute renal failure (ARF) in dogs. Renal hemodynamics and excretory function were examined 1 h (initiation phase) and 24 h (maintenance phase) after a 40-min intrarenal infusion of norepinephrine (NE). In each case, the uninfused contralateral kidney served as control. Four groups were studied: (a) dogs receiving NE alone; (b) dogs receiving an intrarenal infusion of V for 30 min before NE (V + NE); (c) dogs in which intrarenal V was infused for 2 h, beginning immediately after completion of NE infusion (NE + V); and (d) dogs in which intrarenal N was infused for 2 h, beginning immediately after completion of NE infusion (NE + N). Glomerular filtration rate (GFR) in the NE kidneys, as assessed by inulin clearance, at 1 and 24 h averaged 2.4 +/- 1.1 and 5.0 +/- 2.0 ml/min, respectively, as compared with control kidney GFRs of 28.0 +/- 3.5 and 43.8 +/- 5.0 ml/min, respectively (both at least P less than 0.01). In the V + NE group, GFR at 1 and 24 h averaged 15.0 +/- 5.5 and 31.0 +/- 4.5 ml/min, respectively, both at least P less than 0.05 as compared with values from NE kidneys. GFRs in the NE + V group averaged 15.0 +/- 2.4 and 16.3 +/- 3.6 ml/min at 1 and 24 h, both at least P less than 0.02 as compared with values from NE kidneys. GFR in the NE + N group averaged 18.6 +/- 6.0 ml/min at 24 h (P less than 0.05 as compared with GFRs in the NE kidneys). In addition, function of cortical mitochondria (Mito) was examined at the end of the 40-min NE infusion and after 1 and 24 h of reperfusion in the NE alone and NE + V groups. Mito respiration, assessed by acceptor control ratios, was reduced at each period in the NE alone kidneys. After 24 h, these Mito had accumulated Ca++ and exhibited reduced Ca++ uptake and increased Ca++ release rates. Mito from NE + V kidneys respired normally, did not accumulate Ca++, and exhibited no alterations in Ca++ uptake or release. Light and electron microscopy also demonstrated morphological protection of V against tubular necrosis and cell injury. Mito from the NE + N kidneys also respired normally and did not accumulate significant amounts of Ca++. The results of the present studies therefore demonstrated that chemically dissimilar calcium entry blockers exert substantial functional, cellular, and morphological protection against experimental ischemic ARF. These findings are compatible with the hypothesis that increased cytosolic Ca++ is critically important in the maintenance of renal vasoconstriction and the development of cellular necrosis with subsequent tubular obstruction in NE-induced ischemic ARF. V or N may provide protection against renal injury by retarding any increase in cytosolic Ca++ in renal vasculature and epithelium.

Authors

T J Burke, P E Arnold, J A Gordon, R E Bulger, D C Dobyan, R W Schrier

Degradation of sulfated proteoglycans in the subendothelial extracellular matrix by human platelet heparitinase.

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Abstract

Cultured vascular and corneal endothelial cells produce an underlying extracellular matrix (ECM) which induces platelet adherence, aggregation, and release reaction. Incubation of a metabolically (35S)O = 4-labeled ECM with platelet-rich plasma or washed platelets, but not with platelet-poor plasma, resulted in degradation of its heparan sulfate-containing proteoglycans into labeled fragments four to five times smaller than intact glycosaminoglycan side chains. These fragments were sensitive to deamination with nitrous acid and were not produced in the presence of heparin, indicating that heparan sulfate in the ECM is susceptible to cleavage by the platelet heparitinase. This degradation required adhesion of platelets to the ECM rather than aggregation since it was not inhibited by aspirin, which prevented platelet aggregation but not adherence. The enzyme was not released during aggregation of platelets on the ECM but was readily liberated upon their exposure to thrombin. This liberation was inhibited in the presence of prostacyclin (PGI2). Isolated high molecular weight proteoglycans first released from the ECM by incubation with platelet poor plasma served as a substrate for further degradation by the platelet heparitinase, suggesting a cascade mechanism for degradation of heparan sulfate in the ECM. Heparitinase, although to a lower level, was also active when washed platelets were added on top of a confluent endothelial cell monolayer covering the (35S)O = 4-labeled ECM. It is suggested that the platelet heparitinase may be involved in the impairment of the integrity of the vessel wall and thus facilitate the extravasation of blood-borne cells.

Authors

J Yahalom, A Eldor, Z Fuks, I Vlodavsky

Electrical charge of the antigen determines intraarticular antigen handling and chronicity of arthritis in mice.

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Abstract

We studied the influence of antigenic charge on the handling of intraarticular antigen by the joint and on the ability of the antigen to induce chronic arthritis. Three different antigens were used: anionic native bovine serum albumin (BSA), and charge modified BSA made cationic (pI 8.5) either by methylation (mBSA), or amidation (aBSA). 125I-labeled antigen was injected into the knee joints of nonimmune mice and of mice immunized with antigen in Freund's complete adjuvant. Intraarticular antigen retention of the cationic antigens mBSA and aBSA was significantly increased compared with native BSA, both in immune and non-immune mice. In vitro studies indicated the electrostatic character of the binding of the cationic antigens to joint tissues and confirmed the large difference in antigen retention of the antigens found in vivo. A 100-fold amount of cationic antigen could be bound to non-cartilaginous collagenous tissue of the joint compared with antibody-mediated trapping of native BSA, and for hyaline articular cartilage, this difference was even greater. In immunized mice, chronic arthritis only developed after intraarticular injection of the cationic antigens. This phenomenon was apparently related to increased retention of mBSA and aBSA compared with BSA, since delayed hypersensitivity and humoral immunity were comparable for the three antigens used. Our data indicate that antigenic charge is an important determinant of antigen handling by the joint and, in addition, support the concept that the development of chronic arthritis depends on the amount of antigen retained in the joint.

Authors

W B van den Berg, L B van de Putte, W A Zwarts, L A Joosten

Examination of transepithelial exchange of water and solute in the rat renal pelvis.

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Abstract

Severance of the ureter beyond the renal papilla causes a fall in urinary osmolality, which suggests that exchange of water or solute between urine and renal parenchyma normally occurs in the intact renal pelvis. We examined water and solute flux in the renal pelvis with micropuncture and microcatheterization techniques. Four groups of antidiuretic rats were studied. Group I (n = 17) underwent micropuncture through the intact contracting ureter. Urine samples were obtained at the papillary tip, and in the pelvis beside the base of the extrarenal papilla. Urinary osmolality at the base, 880 +/- 97 mosmol/kg H2O (mean +/- SE), was less than that at the tip, 1,425 +/- 104 mosmol/kg H2O (P less than 0.005). In group II (n = 24), samples were analyzed for inulin and osmolality. In 15 rats (group IIA), comparison was made between base and tip samples. In the other nine animals (group IIB), comparisons were made among base, tip, and bladder samples and urea was also measured. In group II (A and B combined) urine-to-plasma (U/P) osmolality was lower at the base, 4.31 +/- 0.27, than at the tip, 6.08 +/- 0.23 (P less than 0.001), and U/P inulin was lower at the base, 192 +/- 25, than at the tip, 306 +/- 16 (P less than 0.001). In group IIB, the bladder urine had a lower U/P osmolality, 5.27 +/- 0.25, than the tip, 6.01 +/- 0.31 (P less than 0.02). The U/P urea was 59 +/- 10.6 (base), 98 +/- 9.4 (tip) (base vs. tip, P less than 0.05), and 81 +/- 6.5 (bladder, P less than 0.005, compared with tip). In group III (n = 8), samples were obtained by microcatheter from the fornices, the deepest intrarenal extensions of the pelvis, and compared with samples at the tip. Urinary osmolality was lower in the fornix, 646 +/- 106 mosmol/kg H2O, than at the tip, 1,296 +/- 99 mosmol/kg H2O (P less than 0.001). Similarly, U/P inulin was lower in the fornix, 48 +/- 14, than at the tip, 128 +/- 12 (P less than 0.001). The lower U/P inulin in the pelvic urine is the result of either the addition of fluid to the pelvis, or the backleak of inulin across the epithelium lining the pelvis. To verify that the pelvic epithelium was impermeable to inulin, in group IVA (n = 4) the left renal pelvis was superfused with a solution of chemical inulin. Cumulative absorption of inulin from the left kidney was 0.15 +/- 0.08% of that superfused. Using [14C]inulin in group IVB (n= 3), similar results were obtained (0.05 +/- 0.02%). These findings indicate that in the renal pelvis, fluid is added to urine after it emerges from the collecting ducts. We suggest that reflux of hyperosmotic urine over the renal papilla creates a transepithelial gradient for the flux of water into the pelvis. A model that incorporates diffusive and convective forces for water and solute transport is proposed to account for these findings.

Authors

J Bargman, S L Leonard, E McNeely, C Robertson, R L Jamison

In vivo influx of free and esterified plasma cholesterol into human aortic tissue without atherosclerotic lesions.

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Abstract

In order to determine the in vivo influx of plasma cholesterol into human aortic intimamedia tissue, specimens of the ascending aortic wall without visible atherosclerosis were obtained from patients undergoing aortic valve replacement. Before the operation the patients were intravenously injected with autologous plasma in which the lipoproteins were labeled with radioactive cholesterol. The influence of the duration of the exposure time (0.3-114 h) and of the distribution of radioactivity between free and esterified cholesterol in plasma on the amount of radioactivity found in the arterial wall was studied by the simultaneous use of 3H- and 14C-cholesterol. It was shown that the influx of free and esterified cholesterol into the intima-media layer of the tissue could be calculated from a set of linear equations that relate the labeled sterols in the tissue to the average specific activities in plasma. In nine patients between 50 and 70 yr of age with 4.2-5.9 mM total cholesterol in plasma, the influx of free cholesterol and of esterified cholesterol was 1.2-8.8 and 1.0-12.5 nmol X cm-2 X d-1, respectively. Both hydrolysis and esterification of the sterol fractions in the aortic tissue and exchange of free cholesterol between the plasma lipoproteins and the tissue were demonstrated. The cholesterol content of the intima-media layer was 0.6-2.3 mumol X cm-2. This corresponds to the influx of esterified cholesterol during a period of only 0.1-3.5 yr, which is short compared with the lifespan of the patient. Our data thus suggest that removal of esterified cholesterol from aortic tissue without visible atherosclerosis represents a major importance for the cholesterol concentration in the tissue.

Authors

S Stender, E Hjelms

Detection of the common acute lymphoblastic leukemia antigen in the serum of leukemia patients.

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Abstract

The common acute lymphoblastic leukemia antigen (CALLA) has been detected in biological fluids using a radioimmunoassay based on the inhibition of binding of 125I-labeled monoclonal anti-CALLA antibody to glutaraldehyde-fixed NALM-1 cells. With this assay, we showed first that CALLA was released in culture fluids from NALM-1 and Daudi cell lines but was absent from culture fluids from CALLA negative cell lines. Then, we found that the sera of 34 out of 42 patients (81%) with untreated common acute lymphoblastic leukemia (c-ALL) contained higher CALLA levels than any of the 42 serum samples from healthy controls. The specificity of these results was further demonstrated by testing in parallel the sera from 48 patients with CALLA negative leukemias, including 26 acute myeloid leukemia (AML), 12 T-cell acute lymphoblastic leukemia (T-ALL), and 10 acute undifferentiated leukemia (AUL). All of these sera gave negative results, except for one patient with AUL, who had a significantly elevated circulating CALLA level, and one patient with AML, who had a borderline CALLA level, 3 SD over the mean of the normal sera. Preliminary results suggest that circulating CALLA is associated with membrane fragments or vesicles, since the total CALLA antigenic activity was recovered in the pellet of the serum samples centrifuged at 100,000 g. In addition, the CALLA-positive pellets contained an enzyme considered as a membrane marker, 5'-nucleotidase. Evaluation of the clinical importance of repeated serum CALLA determinations for the monitoring of c-ALL patients deserves further investigation.

Authors

S Carrel, F Buchegger, D Heumann, C Girardet, C Barras, G Losa, J P Mach, V von Fliedner

Effect of corticotropin-like intermediate lobe peptide on pancreatic exocrine function in isolated rat pancreatic lobules.

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Abstract

Naturally occurring derivatives of pro-opiomelanocortin (POMC) have been identified in various extra-pituitary sites, including the endocrine and exocrine pancreas. Corticotropin-like intermediate lobe peptide (CLIP = ACTH18-39), a naturally occurring derivative of POMC, has been suggested to be an insulin secretagogue. To determine whether CLIP might also affect the exocrine pancreas, we measured its effect on amylase secretion and protein synthesis and secretion in isolated rat pancreatic lobules. Lobules were dual-pulsed with trace amounts of 14C- and 3H-leucine, both in the presence and absence of CLIP (10(-9)-10(-6) M), using a technique that permitted the labeling of both the synthetic and secretory compartments. The effect of CLIP on protein synthesis was determined by comparing 3H-leucine incorporation into lobules with and without CLIP. The secretory effect of CLIP was determined by measuring (a) secreted 14C-labeled protein as a percent of total incorporated radiolabeled protein, and (b) amylase release into incubation medium. The effect of CLIP on amylase release was compared with that of secretin, cholecystokinin-octapeptide, and carbamylcholine. To localize the biologically active region of CLIP, we similarly studied synthetic ACTH25-39. We demonstrated that CLIP stimulates amylase and protein secretion in a dose-dependent manner and is of similar potency to secretin and carbamylcholine. This effect appears to require the ACTH18-24 region of CLIP and results from stimulus-secretion coupling rather than augmented protein synthesis. We also confirmed the presence of immunoreactive-adrenocorticotropic hormone (IR-ACTH) in rat pancreatic extract using a COOH-terminally directed antibody to ACTH1-39 and demonstrated that this IR-ACTH co-eluted with synthetic CLIP. These findings suggest that CLIP might be an endogenous modulator of pancreatic exocrine function.

Authors

J B Marshall, L P Kapcala, L D Manning, A J McCullough

Iron and copper promote modification of low density lipoprotein by human arterial smooth muscle cells in culture.

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Abstract

Modification of low density lipoproteins by human arterial smooth muscle cells was characterized by increased electrophoretic mobility and increased content of malondialdehyde-like oxidation products reactive with thiobarbituric acid. Lipoprotein modification was promoted by micromolar concentrations of iron or copper in the culture medium and was metal ion concentration- and time-dependent. The ability of diverse media to promote smooth muscle cell-mediated low density lipoprotein modification correlated with their iron concentration. Therefore, metal ion concentration of culture media contributes substantially to low density lipoprotein modification in vitro. Human monocyte-derived macrophages took up and esterified the cholesterol from modified low density lipoprotein more extensively than from native low density lipoprotein. Metal ion-mediated modification of low density lipoprotein may be a contributing factor to the pathogenesis of arteriosclerosis.

Authors

J W Heinecke, H Rosen, A Chait