J A Berzofsky
The functional interrelationship between biliary cholesterol secretion, sinusoidal lipoprotein cholesterol secretion and bile salt synthesis was studied in the rat. Diosgenin, fructose, and colestipol in the diet were used to, respectively, influence biliary cholesterol output, VLDL production and bile salt synthesis. In the acute bile fistula rat, biliary cholesterol output was 700% increased by diosgenin and 50% decreased by fructose. In the rats fed both diosgenin and fructose, biliary cholesterol secretion was increased only by approximately 200%, whereas biliary bile salts and phospholipid outputs were unchanged. In the isolated perfused liver, VLDL-cholesterol output was 50% reduced by diosgenin alone, but was unchanged following feeding of diosgenin plus fructose. However, the livers of rats fed diosgenin plus fructose exhibited a 700% increase in VLDL-triglyceride production and a 200% increase in VLDL-cholesterol output. A significant reciprocal relationship between VLDL-cholesterol secretion and the coupling ratio of cholesterol to bile salts in bile was observed. Colestipol added to the diet maintained both sinusoidal and biliary cholesterol outputs within the normal range. In the chronic bile fistula rat, colestipol increased bile salt synthesis by 100% while diosgenin and fructose diets had no effect. Similarly, the addition of fructose to the colestipol diet did not decrease bile salt synthesis. These data suggest a reciprocal relationship between biliary cholesterol secretion and hepatic secretion of cholesterol as VLDL particles. The free cholesterol pool used for bile salt synthesis seems functionally unrelated to the pool from which VLDL-cholesterol and biliary cholesterol originate. These findings support the idea that metabolic compartmentalization of hepatic cholesterol is a major determinant of the quantity of cholesterol available for recruitment by the bile salt-dependent biliary cholesterol secretory mechanism.
F Nervi, I Marinović, A Rigotti, N Ulloa
Arene oxide metabolites of aromatic anticonvulsants (phenytoin, phenobarbital, and carbamazepine) may be involved in the pathogenesis of hypersensitivity reactions. We investigated 53 patients with clinical sensitivity to anticonvulsants by exposing their lymphocytes in vitro to drug metabolites generated by a murine hepatic microsomal system. The diagnosis of a hypersensitivity reaction was corroborated by in vitro rechallenge for each drug (phenytoin, n = 34; phenobarbital, n = 22; carbamazepine, n = 25) when cytotoxicity (% dead cells) exceeded 3 SD above the mean result for controls. Cross-reactivity among the drugs was noted. 7 out of 10 patients who had received all three anticonvulsants had adverse reactions to each. 40 out of 50 patients tested to all three drugs in vitro were positive to each. Adverse reactions were indistinguishable among anti-convulsants. Skin rash (87%), fever (94%), hepatitis (51%), and hematologic abnormalities (51%) were common clinical features of each drug. 62% of reactions involved more than two organs. Cells from patients' parents exhibited in vitro toxicity that was intermediate between values for controls and patients. In vitro testing can help diagnose hypersensitivity to anticonvulsants. Cells from patients may also be used for prospective individualization of therapy to decrease risk of adverse reaction. Cross-reactivity among the major anticonvulsants is common and should be considered before deciding on alternative therapy.
N H Shear, S P Spielberg
The 7 alpha-hydroxylation of two cholesterol analogues, sitosterol and cholestanol, and their effect on the 7 alpha-hydroxylation of cholesterol were measured in rat and human hepatic microsomes. In untreated rat liver microsomes, the 7 alpha-hydroxylation of cholesterol was higher than that of cholestanol (1.4-fold) and sitosterol (30-fold). After removal of endogenous sterols from the microsomes by acetone treatment, the 7 alpha-hydroxylation of cholesterol was similar to that of cholestanol and only fourfold higher than that of sitosterol. Cholestanol and sitosterol competitively inhibited cholesterol 7 alpha-hydroxylase in both rat and human liver microsomes, with cholestanol the more potent inhibitor. Patients with sitosterolemia with xanthomatosis, who have elevated microsomal cholestanol and sitosterol, showed reduced cholesterol 7 alpha-hydroxylase activity relative to the activity in control subjects (13.9 and 14.7 vs. 20.3 +/- 0.9 pmol/nmol P-450 per min, P less than 0.01). Enzyme activity in these patients was 40% higher when measured in microsomes from which competing sterols had been removed. Ileal bypass surgery in one sitosterolemic patient decreased plasma cholestanol and sitosterol concentrations and resulted in a 30% increase in hepatic microsomal cholesterol 7 alpha-hydroxylase activity. Cholesterol 7 alpha-hydroxylase appears to have a specific apolar binding site for the side chain of cholesterol and is affected by the presence of cholestanol and sitosterol in the microsomal substrate pool. Reduced bile acid synthesis in sitosterolemia with xanthomatosis may be related to the inhibition of cholesterol 7 alpha-hydroxylase activity by endogenous cholesterol analogues.
S Shefer, G Salen, L Nguyen, A K Batta, V Packin, G S Tint, S Hauser
Pulmonary edema after ascent to altitude is well recognized but its pathogenesis is poorly understood. To determine whether altitude exposure increases lung vascular permeability, we exposed rats to a simulated altitude of approximately 14,500 feet (barometric pressure [Pb] 450 Torr) and measured the pulmonary transvascular escape of radiolabeled 125I-albumin corrected for lung blood content with 51Cr-tagged red blood cells (protein leak index = PLI). Exposures of 24 and 48 h caused significant increases in PLI (2.30 +/- 0.08 and 2.40 +/- 0.06) compared with normoxic controls (1.76 +/- 0.06), but brief hypoxic exposures of 1-13 h produced no increase in PLI, despite comparable increases in pulmonary artery pressure. There were associated increases in gravimetric estimates of lung water in the altitude-exposed groups and perivascular edema cuffs on histologic examination. Normobaric hypoxia (48 h; fractional inspired oxygen concentration [FIO2] = 15%) also increased lung transvascular protein escape and lung water. Dexamethasone has been used to prevent and treat altitude-induced illnesses, but its mechanism of action is unclear. Dexamethasone (0.5 or 0.05 mg/kg per 12 h) started 12 h before and continued during 48 h of altitude exposure prevented the hypoxia-induced increases in transvascular protein escape and lung water. Hemodynamic measurements (mean pulmonary artery pressure and cardiac output) were unaffected by dexamethasone, suggesting that its effect was not due to a reduction in pulmonary artery pressure or flow. The role of endogenous glucocorticoid activity was assessed in adrenalectomized rats that showed augmented hypoxia-induced increases in transvascular protein escape, which were prevented by exogenous glucocorticoid replacement. In summary, subacute hypoxic exposures increased pulmonary transvascular protein escape and lung water in rats. Dexamethasone prevented these changes independent of reductions of mean pulmonary artery pressure or flow, whereas adrenalectomy increased pulmonary vascular permeability and edema at altitude. Increases in vascular permeability in hypoxia could contribute to the development of high-altitude pulmonary edema and endogenous glucocorticoids may have an important influence on pulmonary vascular permeability in hypoxia.
T J Stelzner, R F O'Brien, K Sato, J V Weil
Insulin- and glyburide-stimulated changes in cytosolic free calcium concentrations [( Ca2+]i) were studied in gluteal adipocytes obtained from six obese women (139 +/- 3% ideal body wt) and six healthy, normal weight age- and sex-matched controls. Biopsies were performed after an overnight fast and twice (at 3 and 6 h) during an insulin infusion (40 mU/m2 per min) (euglycemic clamp). In adipocytes obtained from normal subjects before insulin infusion, insulin (10 ng/ml) increased [Ca2+]i from 146 +/- 26 nM to 391 +/- 66 nM. Similar increases were evoked by 2 microM glyburide (329 +/- 41 nM). After 3 h of insulin infusion, basal [Ca2+]i rose to 234 +/- 21 nM, but the responses to insulin and glyburide were completely abolished. In vitro insulin-stimulated 2-deoxyglucose uptake was reduced by insulin and glucose infusion (25% stimulation before infusion, 5.4% at 3 h, and 0.85% at 6 h of infusion). In obese patients, basal adipocyte [Ca2+]i was increased (203 +/- 14 nM, P less than 0.05 vs. normals). The [Ca2+]i response demonstrated resistance to insulin (230 +/- 23 nM) and glyburide (249 +/- 19 nM) stimulation. Continuous insulin infusion increased basal [Ca2+]i (244 +/- 24 nM) and there was no response to either insulin or glyburide at 3 and 6 h of study. Rat adipocytes were preincubated with 1-10 mM glucose and 10 ng/ml insulin for 24 h. Measurements of 2-deoxyglucose uptake demonstrated insulin resistance in these cells. Under these experimental conditions, increased levels of [Ca2+]i that were no longer responsive to insulin were demonstrated. Verapamil in the preincubation medium prevented the development of insulin resistance.
B Draznin, K E Sussman, R H Eckel, M Kao, T Yost, N A Sherman
Human aortic endothelial cells (EC) and smooth muscle cells (SMC) were isolated and used to form a multilayer of EC-SMC separated by a layer of collagen. SMC and/or collagen layers exerted minimal effects on Na+ transport but impeded the transport of LDL. The presence of an endothelial monolayer markedly reduced the transport of Na+ and LDL. When monocytes were presented to the complete coculture, in the absence of added chemoattractant, one monocyte entered the subendothelial space for every one to three EC present. In contrast, neither collagen nor SMC plus collagen nor EC plus collagen induced comparable monocyte migration. Despite massive migration of monocytes into the coculture, no significant alteration in Na+ transport was observed. LDL transport into the preparation during massive monocyte migration increased modestly, but this was far less than the amount of LDL transported in the absence of an endothelial monolayer. We conclude that (a) the endothelial monolayer was the principal permeability barrier, (b) a substantial migration of monocytes occurred in the absence of added chemoattractant when both EC and SMC were present in the coculture, (c) endothelial barrier function was largely maintained after monocyte migration; and (d) these experiments indicate the need to study all three cell types (monocytes, EC, and SMC) together to understand the complex interactions that occur between these cells.
M Navab, G P Hough, L W Stevenson, D C Drinkwater, H Laks, A M Fogelman
Autoantibodies from bullous pemphigoid (BP) patients define a 230-kD protein found in the basement membrane of stratified squamous epithelia. The purpose of this study was to isolate and characterize a cDNA clone with coding sequences for BP antigen. Poly(A+) RNA derived from total RNA of cultured keratinocytes was used, with oligo-dT priming, to construct a cDNA library in the lambda gt11 expression vector, which was screened by the immunoperoxidase method with one BP serum. One darkly stained clone, called here the BP clone, was further characterized. 9 of 9 BP sera, but none of 6 normal and 11 pemphigus sera, bound the plaques of this BP clone. Furthermore, BP IgG affinity purified on plaques of this clone, but not unrelated clones, bound the epidermal basement membrane by immunofluorescence and immunoprecipitated the 230-kD BP antigen from extracts of cultured keratinocytes. Eco RI digestion of the BP clone's cDNA insert demonstrated a 680- and 1,500-bp fragment. Northern blots of total keratinocyte RNA showed that complementary riboprobes transcribed from both fragments hybridized to a 9-kb RNA. Dideoxy DNA sequencing from the 5' end of the BP cDNA demonstrated a 1,992-bp open reading frame, encoding a peptide of 76 kD. This BP cDNA clone will be valuable for understanding the protein structure, expression, and gene organization of BP antigen.
J R Stanley, T Tanaka, S Mueller, V Klaus-Kovtun, D Roop
The effect of growth hormone (GH) on binding of epidermal growth factor (EGF) to liver membrane preparations was investigated in hypophysectomized mice and partially GH-deficient, genetic mutant "little" (lit/lit) mice. The EGF binding of normal male mice and testosterone-treated females was higher than in normal females. Due to diminished receptor concentration, hepatic EGF binding was decreased in male and female lit/lit mice to a level that was unaffected by gender or androgen treatment. GH replacement therapy by intermittent injections and continuous infusion restored the EGF binding of hypophysectomized mice to normal male and female levels, respectively, suggesting a role for the more pulsatile GH secretion in normal males. In lit/lit mice, however, both continuous and intermittent GH resulted in EGF binding levels comparable to those in normal females. In normal males continuous GH suppressed EGF binding. In conclusion, endogenous GH secretion induces EGF receptors in mice and this effect may be modulated by sex differences in GH secretion.
J O Jansson, S Ekberg, S B Hoath, W G Beamer, L A Frohman
We studied the effects of natural and recombinant human IL-2 (rIL-2) on secretion of prostacyclin (PGI2), vWf, and tissue-type plasminogen activator (tPA). IL-2 elicited a steady increase in PGI2 synthesis by cultured human umbilical vein endothelial cells (HUVECS) and bovine aortic endothelial cells but had no effect on vWf or tPA. Both purified natural IL-2 (nIL-2) and rIL-2 induced significant PGI2 synthesis. Substitution of the cysteine residue at position 125 of rIL-2 with serine or alanine led to loss of PGI2-stimulatory activity in HUVECS without affecting thymidine incorporation in lymphocytes. HPLC analysis of arachidonate metabolites detected predominantly 6 keto-PGF1 alpha (6KPGF1 alpha) peak. Treatment of cultured endothelial cells with cycloheximide and actinomycin D resulted in inhibition of 6KPGF1 alpha synthesis. The Western blot using a polyclonal antibody against PGH synthase revealed an increment in the 70-kD subunit of PGH synthase by nIL-2 and rIL-2, but not by alanine-substituted rIL-2. We conclude that IL-2 stimulated sustained PGI2 production by a mechanism that includes the de novo synthesis of PGH synthase. This mechanism for regulating AA metabolism probably has important physiologic implications.
K Frasier-Scott, H Hatzakis, D Seong, C M Jones, K K Wu
The chronic and acute effects of different types of dietary fat on postprandial lipoprotein metabolism were studied in eight normolipidemic subjects. Each person was placed for 25 d on each of three isocaloric diets: a saturated fat (SFA), a w-6 polyunsaturated fat (w-6 PUFA) and a w-3 polyunsaturated fat (w-3 PUFA) diet. Two vitamin A-fat loading tests were done on each diet. The concentrations in total plasma and chylomicron (Sf greater than 1,000) and nonchylomicron (Sf less than 1,000) fractions of retinyl palmitate (RP) were measured for 12 h postprandially. Compared with the SFA diet, the w-6 PUFA diet reduced chylomicron and nonchylomicron RP levels 56 and 38%, respectively, and the w-3 PUFA diet reduced these levels 67 and 53%, respectively. On further analysis, the main determinant of postprandial lipoprotein levels was the type of fat that was chronically fed, which appeared to mediate its effect by changing the concentration of the endogenous competitor for the system that catabolizes triglyeride-rich lipoproteins. However, there was a significant effect of the acute dietary fat load, which appeared to be due to a differential susceptibility to lipolysis of chylomicrons produced by SFA as opposed to PUFA fat loads. The levels of postprandial lipoproteins are determined by the interaction of these chronic and acute effects.
M S Weintraub, R Zechner, A Brown, S Eisenberg, J L Breslow
To investigate which parts of the acetylcholine receptor are involved in the initiation and development of myasthenia gravis (MG), peptides representing different sequences of the human acetylcholine receptor alpha-subunit were synthesized. These peptides were tested for their ability to stimulate T cells of myasthenic patients and healthy control patients in proliferation assays and to bind to sera antibodies. Three of eight peptides discriminated significantly between the two groups in the proliferation assay, as well as in their ability to bind to serum antibodies. HLA-DR3 and DR5 were associated with proliferative responses to specific AChR peptides in the group of myasthenics. Acetylcholine receptor epitopes that might play a specific role in myasthenia gravis thus were demonstrated.
S Brocke, C Brautbar, L Steinman, O Abramsky, J Rothbard, D Neumann, S Fuchs, E Mozes
In vitro studies were undertaken to determine whether the level of anti-DNA antibody can be modulated in humans with systemic lupus erythematosus (SLE). DNA fragments of different sizes, i.e., oligonucleotide (N20-30) or oligonucleotide (N10-100), were covalently linked either to human gammaglobulin (HGG) and used as tolerogens or to keyhole limpet hemocyanin and used as immunogens. Experiments were done to determine whether such tolerogens specifically diminish antibodies to denatured DNA, native DNA, or both. PBL were obtained from 87 patients with SLE, 55 of whom spontaneously produced anti-DNA antibodies in vitro. Furthermore, of these 55 test subjects 23 made anti-DNA antibodies in response to antigen challenge in vitro. Exposure of PBL to tolerogenic oligonucleotide-HGG reduced spontaneous antibody formation in 34 of the 55 patients' PBL and abrogated the in vitro-induced response in all instances. The suppression was tolerogen specific. In some SLE patients lymphoid cells were suppressed by both (N10-100)-HGG and (N20-30)-HGG, while in others lymphoid cells were suppressed by only one. Longitudinal studies of spontaneous antibody production showed that the same tolerogens consistently reduced anti-DNA antibody formation in lymphoid cells of 12 patients on several occasions over a 2-yr interval, but in 8 others the results were either variable or inconsistent. In contrast, tolerogens consistently abrogated the antigen-induced response in all 23 patients' PBL. These results obtained in humans in vitro suggest that the principle of carrier-determined tolerance could be applied as a specific therapy for SLE in vivo.
Y Borel, H Borel
To investigate the effect of persistent HIV infection on the immune system, we studied leukocyte functions in 14 asymptomatic homosexual men (CDC group II/III) who were at least two years seropositive, but who still had normal numbers of circulating CD4+ T cells. Compared with age-matched heterosexual men and HIV-negative homosexual men, the CD4+ and CD8+ T cells from seropositive men showed decreased proliferation to anti-CD3 monoclonal antibody and decreased CD4+ T-helper activity on PWM-driven differentiation of normal donor B cells. Monocytes of HIV-infected homosexual men showed decreased accessory function on normal T cell proliferation induced by CD3 monoclonal antibody. The most striking defect in leukocyte functional activities was observed in the B cells of HIV-infected men. B cells of 13 out of 14 seropositive men failed to produce Ig in response to PWM in the presence of adequate allogeneic T-helper activity. These findings suggest that HIV induces severe immunological abnormalities in T cells, B cells, and antigen-presenting cells early in infection before CD4+ T cell numbers start to decline. Impaired immunological function in subclinically HIV-infected patients may have clinical implications for vaccination strategies, in particular the use of live vaccines in groups with a high prevalence of HIV seropositivity.
F Miedema, A J Petit, F G Terpstra, J K Schattenkerk, F de Wolf, B J Al, M Roos, J M Lange, S A Danner, J Goudsmit
Nodular sclerosing Hodgkin's disease is characterized by dense collagen fibrosis. Although transforming growth factor-beta (TGF-beta) is an important bifunctional growth factor for fibroblasts and is stored and released by many cells, it requires acidification to pH 2.0-3.0 before it becomes a biologically active growth factor. We show here that the L-428 Hodgkin's cell releases a high molecular weight TGF that competes for the TGF-beta cell membrane receptor but not the TGF-alpha receptor. This growth factor is most active at physiologic pH and is 97% inactivated by acidification. Hodgkin's TGF is also inactivated by proteases and can be preserved by protease inhibitors. The Hodgkin's TGF can be separated from an autocrine growth factor using either column chromatography or electroelution from gels and is shown to have a molecular weight of approximately 350,000. Incubation of the Hodgkin's TGF in SDS releases a 25,000-D protein with reduced biological activity but which cross-reacts with anti-TGF-beta IgG. We propose that L-428 nodular sclerosing Hodgkin's disease fibrosis is mediated by a potent high molecular weight TGF-beta which, unlike TGF-beta characterized to date, is secreted in a form most active at physiologic pH.
S R Newcom, M E Kadin, A A Ansari, V Diehl
Rat hepatocytes contain several types of Ca2+-linked receptors, all of which stimulate glycogen breakdown by increasing cytosolic free Ca2+ concentration [( Ca2+]c). In vivo desensitization of this Ca2+ messenger system was studied in hepatocytes isolated from either pheochromocytoma (PHEO)-harboring and chronically norepinephrine (NE)-infused rats. Homologous desensitization for alpha 1-adrenergic receptor-mediated phosphorylase activation developed in the early stage of PHEO rats (3-4 wk after implantation), whereas, in the later stage of tumor development or in the NE-infused rats, phosphorylase responses to all Ca2+-mobilizing stimulations were subsensitive (heterologous desensitization). In the homologous desensitization, the [Ca2+]c response to alpha 1-adrenergic stimulation was selectively reduced. We found, using the phenoxybenzamine inactivation method, that there was a linear relationship between alpha 1 receptor density and the [Ca2+]c response; consequently, the blunted [Ca2+]c response to alpha 1-adrenergic stimulation could not be explained by the 34% downregulation of alpha 1 receptors seen in these rats. These results indicated that uncoupling at a step proximal to alpha 1 receptor-stimulated [Ca2+]c increase is also of primary importance in homologous desensitization of phosphorylase activation. On the other hand, heterologous desensitization also involved alteration(s) at steps distal to the rise in [Ca2+]c. Our data demonstrate that prolonged exposure to catecholamines results in desensitization of the [Ca2+]c mobilization pathway and may involve multiple mechanisms.
G Tsujimoto, A Tsujimoto, K Kato, K Hashimoto
To determine whether the hypoferremic response to inflammation requires neutrophils, we administered human recombinant IL-1 to mice made neutropenic with cyclophosphamide. With single intraperitoneal injections of IL-1 the plasma iron concentrations decreased significantly in mice with either normal neutrophil counts or neutropenia. After single injections transferrin concentrations were not significantly changed, but the decrease in serum iron lowered mean transferrin saturations from a baseline of 45 to 24-30% in nonneutropenic mice, and from 99 to 70-77% in neutropenic mice. Similar changes were observed after intraperitoneal injections of Escherichia coli. 4-d continuous infusions of IL-1 also led to reductions in serum iron concentrations, but transferrin concentrations doubled. The combination of a decrease in serum iron and an increase in transferrin concentration after chronic infusion in neutropenic mice led to a greater decline in mean transferrin saturations, from a baseline of 110 to 25%. In mice not given cyclophosphamide, chronic IL-1 infusion was associated with a reduction in mean hemoglobin concentrations from 14.7 to 13.5 g/dl, consistent with restricted availability of iron for erythropoiesis associated with low saturation of transferrin. We conclude that IL-1 can decrease the serum iron despite profound peripheral neutropenia and that transferrin in a positive acute phase reactant in the mouse.
V R Gordeuk, P Prithviraj, T Dolinar, G M Brittenham
Previous studies have shown that pemphigus vulgaris (PV) IgG will fix early complement components (C1q, C4, and C3) to cultured murine epidermal cell surfaces and that PV IgG and complement alter epidermal cell membrane integrity. The present study was undertaken to determine if assembly of terminal complement components (C5, C6, C7, C8, and C9) and expression of C5b-9 neoantigens occur when PV IgG interacts with human keratinocyte (HuK) cell surface antigens in the presence of a source of complement. Monoclonal antibodies specific for C5, C6, C7, C8, C9, and C5b-9 neoantigens were screened for reactivity to the individual complement components in an assembled complex of human C5b-9 on rabbit red blood cell ghosts. Monoclonal antibodies (tissue culture supernatants) that bound to antigenic determinants accessible in the C5b-9 complex were selected for this study using immunofluorescence methods. HuK treated with PV IgG fixed C5, C6, C7, C8, C9, and C5b-9 neoantigens in a characteristic speckled pattern, while normal IgG did not. Heat inactivation or EDTA treatment of the complement source, or substitution of C2-depleted serum abolished C5, C6, C7, C8, C9, and C5b-9 neoantigen staining. PV IgG and complement also resulted in significant cytotoxicity to cell membranes as assessed using an ethidium bromide-fluorescein diacetate assay. These results suggest that PV IgG will activate the membrane attack complex of the complement system on HuK cell surfaces, resulting in cytotoxicity to cell membranes, further implicating complement in the pathogenesis of pemphigus.
P Xia, R E Jordon, W D Geoghegan
The ability of mononuclear phagocytes to assemble and activate components of the fibrinolytic system on their surfaces may be crucial in effecting an efficient inflammatory response. Lys-plasminogen, the plasmin modified form of this zymogen, was found to bind specifically and with high affinity to murine peritoneal macrophages and to cells of the human monocytoid line U937. This modified plasminogen has been shown to be a more efficient substrate for plasminogen activators than native Glu-plasminogen. Binding was lysine binding site dependent, rapid and reversible. In contrast, although native Glu-plasminogen bound specifically to these cells, affinity was low. Lys-plasminogen inhibited the binding of Glu-plasminogen but the opposite was not true. Molecular analysis of the bound ligands indicated that Glu-plasminogen was converted to Lys-plasminogen and Lys-plasminogen to plasmin on the cell surface but not in the supernatant. Peritoneal macrophages from patients with indwelling catheters and tissue macrophages in chronic inflammatory lesions were shown to express immunologically identified Lys-plasminogen on their surfaces. Therefore binding and surface activation of kinetically favored Lys-plasminogen may provide an important physiological mechanism for localizing proteolytic activity on the surface of inflammatory cells.
R L Silverstein, R J Friedlander Jr, R L Nicholas, R L Nachman
Two plasmin-resistant mutant forms of pro-urokinase (pro-UK) constructed by site-directed mutagenesis of Lys158 to Val158 and Met158 were used to evaluate the intrinsic enzymatic and fibrinolytic properties of pro-UK as distinct from those of its two-chain UK (TC-UK) derivative. Both mutants, while resistant to plasmin activation, were as sensitive as pro-UK to degradation by thrombin. Since thrombin cleaves a peptide bond only two residues from the activation site, the integrity of this loop was maintained in the two mutants. The amidolytic and plasminogen-activating activities of the mutants averaged 0.14 and 0.12% that of TC-UK, respectively. The fibrin plate activities were 2,400 IU/ml and 700 IU/mg for the Met158 and Val158 mutants or about 1.5% that of TC-UK. These findings attest to a discrete but low intrinsic activity for pro-UK and suggest that the higher values reported in the literature may be related to UK contaminants or plasmin-induced TC-UK generation during the assay. Clot lysis by the mutants required doses greater than 100-fold higher than those of pro-UK to induce a comparable effect. From this it appears that pro-UK activation is a major determinant of the rate of clot lysis occurring with pro-UK. Clot lysis by the mutants was potentiated by plasmin pretreatment of the fibrin and by the addition of small amounts of TC-UK or tissue plasminogen activator (t-PA). Combinations of t-PA and the mutants were synergistic in their fibrinolytic effects. These findings mirror those previously obtained with pro-UK. We concluded that the previously described potentiation of pro-UK-induced clot lysis by UK or t-PA is mediated primarily by pro-UK itself rather than by a promotion of its activation.
V Gurewich, R Pannell, R J Broeze, J Mao
Studies were designed to explore the possibility that human polymorphonuclear leukocyte granule constituents in addition to elastase (HLE) had the potential to cause emphysema. A two-step purification of three serine proteinases was developed. Granule extract proteins were initially separated by dye-ligand affinity chromatography. Fractions eluted were divided into four pools. Hamsters were given a single intratracheal instillation of saline +/- 0.1 mg protein of each pool. While pool 2 contained HLE and cathepsin G, the most dramatic bullous emphysema developed in animals treated with pool 4. The esterase from pool 4, designated proteinase 3 (PR-3) was purified, characterized in vitro, and tested for its ability to cause emphysema. PR-3 is a neutral serine proteinase with isoenzyme forms. Its ability to degrade elastin at pH 6.5 is slightly greater than that of HLE, but it is less active than HLE at pH 7.4 or 8.9. PR-3 has weak activity against azocasein. Its ability to degrade hemoglobin is intermediate to that of HLE and cathepsin G at pH 7.4. PR-3 has no activity against chromogenic substrates specific for HLE or cathepsin G. Its pI is substantially less than HLE or cathepsin G. It is also immunologically distinct from HLE. It induces emphysema in hamsters commensurate with that of HLE. We conclude that PR-3 may be important in the pathogenesis of human emphysema.
R C Kao, N G Wehner, K M Skubitz, B H Gray, J R Hoidal
Using a newly isolated cDNA clone encoding the TCR-delta gene and genomic probes, we have analyzed T cell receptor (TCR) delta gene rearrangement in 19 patients with T cell acute lymphoblastic leukemia (T-ALL) and 29 patients with B-precursor ALL. Five out of seven CD3- T-ALL and 4 of 12 CD3+ T-ALL showed bi-allelic rearrangements of the TCR-delta gene. In three CD3+ patients, a single allelic TCR-delta gene rearrangement was observed with rearrangement of the TCR-alpha gene on the other allele. In five CD3+ patients with bi-allelic rearrangements of the TCR-alpha gene, the TCR-delta gene locus was deleted. Transcription of the TCR-delta gene was also analyzed in six T-ALL. Five patients expressed TCR-delta transcripts. Only one T-ALL, presumably derived from the most immature T lineage cells, did not have TCR-delta transcripts, but expressed TCR-gamma and 1.0-kb truncated TCR-beta transcripts. In B-precursor ALL, 20 patients (69%) showed rearrangements of the TCR-delta gene. The frequency of TCR-delta gene rearrangement was higher than TCR-alpha (59%), gamma (52%), or beta (31%) genes. These findings suggest that TCR-alpha gene rearrangements may take place after rearrangements of the TCR-delta gene with concomitant deletion of rearranged TCR-delta genes in T cell differentiation. Among leukemic cells of B lineage, the TCR-delta gene is the earliest rearranging TCR gene, followed by TCR-gamma and beta gene rearrangements.
J Hara, S H Benedict, E Champagne, Y Takihara, T W Mak, M Minden, E W Gelfand
Platelet-activating factor (PAF) acetylhydrolase has been recognized as an enzyme that inactivates PAF. We developed a convenient and reproducible method for determining human serum PAF acetylhydrolase activity. The assay was based on measurement of [14C]acetate produced from 1-O-alkyl-2-[14C]-acetyl-sn-glycero-3-phosphocholine upon precipitation of the complex of radioactive substrate and albumin with TCA. The apparent Km value of PAF acetylhydrolase (near the physiological concentration of serum protein) was 1.5 X 10(-4) M PAF. 32 subjects with serum PAF acetylhydrolase deficiency were found among 816 healthy Japanese adults. The low PAF acetylhydrolase activity in the deficient serum might not be due to the presence of enzyme inhibitor. Both the sensitivity to PAF and the metabolism of PAF in platelets from PAF acetylhydrolase-deficient subjects were almost the same as those of normal subjects. Deficiency in serum PAF acetylhydrolase appeared to be transmitted by autosomal recessive heredity among five Japanese families. Among healthy adults, healthy children, and asthmatic children, who were grouped into five classes on the basis of respiratory symptoms (remission, wheezy, mild, moderate, and severe groups), the probability of PAF acetylhydrolase deficiency was significantly higher in groups with severe symptoms (moderate and severe) (P less than 0.01). These results suggest that deficiency of serum PAF acetylhydrolase might be one of the factors leading to severe respiratory symptoms in asthmatic children.
M Miwa, T Miyake, T Yamanaka, J Sugatani, Y Suzuki, S Sakata, Y Araki, M Matsumoto
Mesangial cells in culture change shape and become less adhesive in response to cAMP elevation (e.g., treatment with isoproterenol plus isobutylmethylxanthine (IM). Inhibitors of serine proteases inhibit cellular shape change in response to IM. To further examine the role of cell surface proteases in shape change, adhesion plaque proteins (i.e., preparations of ventral membranes and extracellular matrix) were separated in SDS-polyacrylamide gels containing gelatin with and without plasminogen. Four discrete zones of lysis were evident in plasminogen gels (indicative of activation of plasminogen) from control adhesion plaques: one inconspicuous zone with a Mr approximately 150 kD, another at approximately 115 kD, and a doublet at approximately 35-32 kD. Another diffuse zone of lysis centered around Mr approximately 70 kD and contained a defined band of approximately 56 kD. Adhesion plaques contained most of the plasminogen activators (PA). 5 min after IM treatment, the Mr approximately 150- and approximately 115-kD PA were increased in activity. Vasopressin (VP), which prevented shape change and adhesion loss when added along with IM, inhibited the increase in these PA. Preincubation with monoclonal or polyclonal antibodies to urokinase-type plasminogen activator (uPA) totally inhibited the IM-inducible shape change and adhesion loss. Activation of plasminogen throughout the gels revealed multiple protease resistant bands that markedly increased with IM treatment (maximal at 45 min). These may represent focal control mechanisms. uPA thus may mediate focal proteolysis, which results in shape change and decreased adhesion.
W F Glass 2nd, R A Radnik, J A Garoni, J I Kreisberg
The previous identification of a membrane fatty acid binding protein (MFABP) in brush border plasma membranes of the jejunum suggested that mucosal cell uptake of fatty acids might represent a carrier-mediated transport system. For evaluation of this hypothesis cellular influx kinetics (V0) of [3H]-oleate were examined in isolated rat jejunal mucosal cells. With increasing unbound oleate concentration in the medium V0 was saturable (Km = 93 nM; Vmax = 2.1 nmol X min-1 per 10(6) cells) and temperature dependent with an optimum at 37 degrees C. Pretreatment of the cells with a monospecific antibody to MFABP significantly inhibited V0 of oleate, other long-chain fatty acids, and D-monopalmitin, but not of L-alanine. Moreover, in the in vivo system of isolated perfused jejunal segments the physiologic significance of MFABP in the directed overall intestinal absorption process of fatty acids was documented. In the presence of the anti-MFABP oleate absorption was markedly reduced, whereas uptake of L-alanine remained unaltered. By antibody inhibition studies it was suggested that this membrane carrier also reveals transport competence for various other long-chain fatty acids, D-monopalmitin, L-lysophosphatidylcholine, and cholesterol. These data support the hypothesis that absorption of fatty acids is mediated by a fatty acid binding membrane protein.
Rotaviruses are major causes of infectious gastroenteritis in humans and other animals. We found that a variety of protease inhibitors suppressed the replication of the SA-11 strain of rotavirus in MA-104 cell cultures. Three of these compounds, leupeptin, pentamidine, and bis (5-amidino-2-benzimidazolyl) methane (BABIM) also restricted the intestinal replication of the murine strain of rotavirus when protease inhibitor and virus were administered simultaneously to suckling mice. Repeated administration of BABIM resulted in significantly reduced levels of intestinal rotaviral antigen even if administration of the compound was begun as late as 48 h after viral inoculation. Additionally, BABIM-treated animals had significantly less intestinal replication of rotavirus than did placebo-treated controls when placed in a heavily rotavirus-contaminated environment. The use of protease inhibitors represents a novel approach to the control of this important gastrointestinal pathogen and is a potential modality for the prevention and treatment of diseases caused by other enteric viruses, for which proteolytic cleavage is necessary for efficient replication.
S L Vonderfecht, R L Miskuff, S B Wee, S Sato, R R Tidwell, J D Geratz, R H Yolken
The purpose of this study was to investigate myocardial substrate utilization during moderate intensity exercise in humans. Coronary sinus and arterial catheters were inserted in nine healthy trained male subjects (mean age, 25 +/- 6 (SD) years). Dual carbon-labeled isotopes were infused, and substrate oxidation was quantitated by measuring myocardial production of 14CO2. Supine cycle ergometer exercise was performed at 40% of the subject's maximal O2 uptake. With exercise there was a significant increase in the arterial lactate level (P less than 0.05). A highly significant positive correlation was observed between the lactate level and the isotopic lactate extraction (r = 0.93; P less than 0.001). The myocardial isotopic lactate uptake increased from 34.9 +/- 6.5 mumol/min at rest to 120.4 +/- 36.5 mumol/min at 5 min of exercise (P less than 0.005). The 14CO2 data demonstrated that 100.4 +/- 3.5% of the lactate extracted as determined by isotopic analysis underwent oxidative decarboxylation. Myocardial glucose uptake also increased significantly with exercise (P less than 0.04). The [14C]glucose data showed that only 26.0 +/- 8.5% of the glucose extracted underwent immediate oxidation at rest, and during exercise the percentage being oxidized increased to 52.6 +/- 7.3% (P less than 0.01). This study demonstrates for the first time in humans an increase in myocardial oxidation of exogenous glucose and lactate during moderate intensity exercise.
E W Gertz, J A Wisneski, W C Stanley, R A Neese
In inflammatory diseases such as rheumatoid arthritis, functions of chondrocytes including synthesis of matrix proteins and proteinases are altered through interactions with cells of the infiltrating pannus. One of the major secreted products of mononuclear inflammatory cells is IL-1. In this study we found that recombinant human IL-1 beta suppressed synthesis of cartilage-specific type II collagen by cultured human costal chondrocytes associated with decreased steady state levels of alpha 1 (II) and alpha 1(IX) procollagen mRNAs. In contrast, IL-1 increased synthesis of types I and III collagens and levels of alpha 1(I), alpha 2(I), and alpha 1(III) procollagen mRNAs, as we described previously using human articular chondrocytes and synovial fibroblasts. This stimulatory effect of IL-1 was observed only when IL-1-stimulated PGE2 synthesis was blocked by the cyclooxygenase inhibitor indomethacin. The suppression of type II collagen mRNA levels by IL-1 alone was not due to IL-1-stimulated PGE2, since addition of indomethacin did not reverse, but actually potentiated, this inhibition. Continuous exposure of freshly isolated chondrocytes from day 2 of culture to approximately half-maximal concentrations of IL-1 (2.5 pM) completely suppressed levels of type II collagen mRNA and increased levels of types I and III collagen mRNAs, thereby reversing the ratio of alpha 1(II)/alpha 1(I) procollagen mRNAs from greater than 6.0 to less than 1.0 by day 7. IL-1, therefore, can modify, at a pretranslational level, the relative amounts of the different types of collagen synthesized in cartilage and thereby could be responsible for the inappropriate repair of cartilage matrix in inflammatory conditions.
M B Goldring, J Birkhead, L J Sandell, T Kimura, S M Krane
In animals injected with a bolus of isoproterenol, beta-adrenergic receptors in both mononuclear leukocytes (MNL) and heart were sequestered away from the cell surface, and the time course (0-120 min) and dose-response patterns were similar in the two tissues. In guinea pigs given a constant infusion of isoproterenol, 0.15 mg/(kg.h), down-regulation of total receptor number occurred more quickly and to a greater extent in the MNL than in the heart. We also compared receptor sequestration after aortic constriction-induced acute heart failure. Negligible sequestration (9%) of beta-adrenergic receptors occurred in the MNL of animals treated in this manner, whereas the number of receptors in the sarcolemmal fraction decreased 61%. This selective sequestration of cardiac receptors may result from the action of high concentrations of norepinephrine (which is selective for beta 1 over beta 2 receptors) present at sympathetic nerve-cardiac cell synapses. We conclude that although receptor redistribution occurs similarly in MNL and heart in response to a circulating nonselective agonist, beta-adrenergic receptor redistribution may occur selectively in the heart in response to such stimuli as aortic constriction-induced acute heart failure that activate the sympathetic nervous system.
A S Maisel, M G Ziegler, S Carter, P A Insel, H J Motulsky
Oxidant-induced damage to the intima of pulmonary and systemic vessels is thought to be an important mechanism of injury in a variety of syndromes of vascular damage. Hydrogen peroxide (H2O2) is an active oxygen metabolite that may induce intimal injury by cytolytic attack or by inducing biochemical and functional alterations in the endothelial cells (EC); however, mechanisms involved in noncytolytic perturbation of EC are largely unknown. We found that H2O2 stimulated the synthesis of platelet-activating factor (PAF) by primary cultures of bovine pulmonary artery endothelium (BPAEC) and by human umbilical vein endothelium (HUVEC). In each cell type the incorporation of [3H]acetate into [3H-acetyl]PAF was concentration- and time-dependent and was temporally dissociated from severe plasma membrane disruption and cytolytic cell injury; the newly synthesized PAF remained associated with the EC. H2O2 caused permeabilization of EC to 45Ca2+ and an increase in intracellular Ca2+, suggesting that a transmembrane Ca2+ flux is the signal that initiates PAF synthesis. H2O2 also induced the endothelial cell-dependent adhesion of neutrophils to HUVEC monolayers. This response was rapid, with an onset within minutes and a subsequent time course that paralleled the time course of PAF accumulation, and was dependent on extracellular Ca2+ but not on de novo protein synthesis. These studies demonstrate that H2O2 can induce two rapid activation responses of endothelium, PAF synthesis and EC-dependent neutrophil adhesion, events that may be important in physiologic and pathologic inflammation.
M S Lewis, R E Whatley, P Cain, T M McIntyre, S M Prescott, G A Zimmerman
Human mononuclear cells (MNC) secrete histamine-releasing factor(s) (HRF) when cultured in vitro. HRF induces the release of histamine and other mediators from basophils and mast cells. We have shown that MNC also produced a histamine release inhibitory factor (HRIF), and that the synthesis is augmented by culture with physiologic concentrations of histamine (10(-10) to 10(-6) M) and by the mitogen concanavalin A (Con A). HRIF does not affect release initiated by other secretagogues such as allergen, anti-IgE, C5a, Con A, and phorbol myristate acetate. HRIF requires a preincubation with the cells for 5-10 min for maximal inhibition, and this effect is not abolished by washing the cells after the preincubation. The biological activity of HRIF is protease-sensitive, neuraminidase-resistant, and relatively heat-stable. HRIF can be distinguished from HRF by a lower apparent molecular mass (8,000-10,000 D).
R Alam, J A Grant, M A Lett-Brown
To study the roles of substance P and endogenous neutral endopeptidase in mediating cough, we measured cough responses in awake guinea pigs in response to exogenous substance P and capsaicin aerosols in the presence and absence of the neutral endopeptidase inhibitors leucine-thiorphan and phosphoramidon. Substance P stimulated cough in very low concentrations (10(-17)-10(-16) M). In a second study where the investigator did not know whether substance P or diluent alone was aerosolized, substance P (10(-16) M) caused cough. Leucine-thiorphan (10(-5) M) and phosphoramidon (10(-5) M) potentiated substance P-induced cough; NEP inhibitors also potentiated capsaicin-induced cough significantly. These findings suggest that substance P is a potent stimulator of cough responses, that capsaicin-induced cough is mediated by substance P or another similar neuropeptide, and that cough responses are modulated by endogenous neutral endopeptidase.
H Kohrogi, P D Graf, K Sekizawa, D B Borson, J A Nadel
We investigated the determinants of hepatic clearance functions in a rat model of liver cirrhosis induced by phenobarbital/CCl4. Aminopyrine N-demethylation (ABT), galactose elimination (GBT), and serum bile acids (SBA) were determined in vivo. The livers were then characterized hemodynamically: intrahepatic shunting (IHS) was determined by microspheres and sinusoidal capillarization by measuring the extravascular albumin space (EVA) by a multiple indicator dilution technique. The intrinsic clearance was determined by assaying the activity of the rate-limiting enzymes in vitro. Hepatocellular volume (HCV) was measured by morphometry. ABT and SBA, but not GBT, differentiated cirrhotic from normal liver. IHS ranged from normal to 10%; all cirrhotic livers showed evidence of sinusoidal capillarization (reduced EVA). The cirrhotic livers showed a bimodal distribution of HCV, HCV being decreased in 50% of the cirrhotic livers. Multivariate analysis showed EVA and portal flow to be the main determinants of microsomal (ABT) and cytosolic (GBT) clearance function; SBA, by contrast, were determined solely by IHS. We conclude that sinusoidal capillarization is the main determinant of hepatic clearance, while serum bile acids reflect intrahepatic shunting. These findings emphasize the importance of alterations of hepatic nutritional flow to explain reduced clearance function in cirrhosis of the liver.
J Reichen, B Egger, N Ohara, T B Zeltner, T Zysset, A Zimmermann
Whereas the greatest relative increase in body mass occurs during the third trimester of fetal life, the source of the cholesterol that supports this growth is uncertain. These studies used [3H]water and 125I-cellobiose-labeled low density lipoproteins to quantitate absolute rates of cholesterol acquisition in vivo by the fetus of the rat. Preliminary studies demonstrated that [3H]water administered intravenously to the mother rapidly equilibrated with the body pool of water in the fetus and that 22-microgram atoms of H from the water pool were incorporated into each micromole of newly synthesized cholesterol. After administration of [3H]water to pregnant rats, the rates of sterol synthesis per 100 g of whole body weight were severalfold higher in the fetus than in the dams. Individual organs of the dam such as the liver, however, had much higher synthetic rates than those in the fetus. When maternal hepatic cholesterol synthesis was suppressed by cholesterol feeding, newly synthesized cholesterol disappeared from the maternal blood yet there was essentially no change in the rate of appearance of newly synthesized sterol in the fetus, placenta, and fetal membranes. The placenta did take up low density lipoproteins at rates equal to about one-third of that seen in the maternal liver, but none of the apolipoprotein or cholesterol was transferred to the fetus. These studies indicate that the rat fetus receives little or no cholesterol from the mother but, rather, satisfies its need for cholesterol during fetal development through local synthesis. Furthermore, the fetal membranes appear to be an important site for sterol synthesis in the fetal compartment.
W M Belknap, J M Dietschy
To investigate the pathogenetic mechanisms of tubule nephrotoxicity of low molecular weight proteins (LMWP), proximal tubules (PT) of rats were perfused in vivo with artificial tubule fluid (ATF) containing one of five LMWPs: three human Bence Jones proteins (BJP), beta-lactoglobulin (BLG), and rabbit myoglobin (MYG). Volume (JV), chloride (JCl) and glucose (JG) fluxes in these perfused PTs were compared with those determined using ATF alone. In separate experiments, perfused nephrons were examined with electron and immunoelectron microscopy. After exposure to BJP1 or BLG, JV, JCl, and JG were less (P less than 0.05) than corresponding control fluxes. Cell damage of these perfused PTs, along with cellular debris in the distal tubules, was prominent. The PT lysosomes often appeared atypical and contained crystals. In contrast, perfusion with BJP2, BJP3, or MYG did not alter JV, JCl, or JG. These findings were corroborated by the normal ultrastructure of these PTs despite immunohistochemical evidence of endocytosis of the BJPs. Isoelectric point, molecular form, and isotype were not factors associated with PT damage. In addition, proteins with pI less than 7.4 precipitated in the distal nephron, forming acellular casts. Thus, certain nephrotoxic LMWPs damaged the PT, while others precipitated in the distal tubule, obstructing the nephron. These two pathogenetic mechanisms may independently be responsible for tubulointerstitial nephropathy of LMWPs in humans.
P W Sanders, G A Herrera, A Chen, B B Booker, J H Galla
Infection of monocyte-macrophages with human immunodeficiency virus may be central to the pathogenesis of the acquired immunodeficiency syndrome. The ability of infected macrophages to prime T cells through IL-1 production was investigated in vitro. Purified human monocytes maintained in suspension culture were infected with strain HIV-DV. Intracellular expression of virus p24 antigen increased from undetectable levels immediately after infection to 13-59% of cells by 10-14 d; infected macrophages remained viable for up to 60 d. Supernatants collected between 14 and 20 d after infection were examined in the murine thymocyte co-mitogenesis assay and demonstrated to contain a potent IL-1 inhibitor, designated contra-IL-1. Contra-IL-1 activity was present in all supernatants examined after 4 d of infection, and peaked coincident with peak p24 antigen expression. Inhibitory activity was not present in uninfected cells. Contra-IL-1 activity eluted after gel filtration with an approximate molecular weight of 9 kD. Inhibitory activity was removed by exposure to heat or acid pH, or by incubation with chymotrypsin or staphylococcal V8 protease. Contra-IL-1 did not inhibit IL-2- or IL-4-dependent proliferation of murine T cell lines. Despite its ability to inhibit IL-1 activity, contra-IL-1 did not interfere with the binding of recombinant IL-1 beta to a fibroblast cell line. Contra-IL-1 inhibited the proliferation of normal peripheral blood mononuclear cells to both concanavalin A and tetanus toxoid; inhibition could be attenuated by the addition of exogenous IL-1. Messenger RNA extracted from infected macrophages was examined by Northern analysis for the presence of message to IL-1 beta. No message was apparent, suggesting that the presence of contra-IL-1 was not obscuring the concomitant release of IL-1. Infected macrophages stimulated with endotoxin generated readily detectable message for IL-1 beta. Spleen macrophages purified from two patients with AIDS complicated by immune thrombocytopenia spontaneously expressed p24 antigen in vitro and released contra-IL-1 activity into the media. Contra-IL-1 may contribute to the immune dysfunction of AIDS.
R M Locksley, S Crowe, M D Sadick, F P Heinzel, K D Gardner Jr, M S McGrath, J Mills
The metabolism of radioiodinated apo B-100 in large VLDL from normal and Watanabe heritable hyperlipidemic (WHHL) rabbits, with diameters exceeding 450 A, was studied in corresponding recipient rabbits. In both cases approximately 87% of the particles contained apolipoprotein (apo) E (B,E particles). In normal rabbits, apo B in these B,E particles was removed from blood plasma much more rapidly than apo B in B,E particles in smaller VLDL and few of the large B,E particles were converted to lipoproteins of higher density. In WHHL rabbits, approximately 60% of the apo B in B,E particles in large VLDL was removed at a comparably rapid rate, but an appreciable fraction of the remainder, which was removed slowly, was converted to particles of higher density, as are the B,E particles in smaller VLDL. From kinetic analysis of these and other data, an hypothesis was formulated from which it is estimated that apo B in large VLDL accounts for 18 and 41% of apo B transport in normal and WHHL rabbits, respectively, despite the fact that these lipoproteins contain less than 5% of the apo B in total VLDL. Failure to account for the contribution of large VLDL to VLDL turnover may lead to serious underestimation of total apo B transport in the blood.
N Yamada, D M Shames, K Takahashi, R J Havel
The distribution of vacuolar H+ATPase in rat kidney was examined by immunocytochemistry using affinity-purified antibodies against the 31-, 56-, and 70-kD subunits of the bovine kidney proton pump. Proximal convoluted tubules were labeled over apical plasma membrane invaginations, and in the initial part of the thin descending limb, apical and basolateral plasma membranes were moderately stained. Thick ascending limbs and distal convoluted tubules were apically stained although the intensity was greater in the distal convoluted tubule. Collecting duct principal cells were virtually unlabeled, but intercalated cells had intense staining with an apical, basolateral or diffuse pattern in the cortex, and exclusively apical staining in the medulla. These results (a) show the presence of an H+ATPase in the apical plasma membrane of the proximal tubule that may contribute to H+ transport in this segment; (b) provide direct evidence that the intercalated cell contains most of the H+ATPase detectable in the collecting duct, supporting its proposed role in H+ transport; (c) demonstrate that subpopulations of cortical intercalated cells have opposite polarities of an H+ATPase, consistent with the presence of both proton- and bicarbonate-secreting cells; and (d) suggest a role for the H+ATPase in acid/base regulation or H+ transport in segments other than the collecting duct and the proximal tubule.
D Brown, S Hirsch, S Gluck
The effects of adenosine on the human His-Purkinje system (HPS) were studied in nine patients with complete atrioventricular (AV) block. Adenosine had minimal effect on the control HPS cycle length, but in the presence of isoproterenol increased it from 906 +/- 183 to 1,449 +/- 350 ms, P less than 0.001. Aminophylline, a competitive adenosine antagonist, completely abolished this antiadrenergic effect of adenosine. In isolated guinea pig hearts with surgically induced AV block, isoproterenol decreased the HPS rate by 36%, whereas in the presence of 1,3-dipropyl-8-phenyl-xanthine, a potent adenosine antagonist, the HPS rate decreased by 48% and was associated with an increased release of adenosine. Therefore, by blocking the effects of adenosine at the receptor level, the physiologic negative feedback mechanism by which adenosine antagonizes the effects of catecholamines was uncoupled. The results of this study indicate that adenosine's effects on the human HPS are primarily antiadrenergic and are thus consistent with the concept of accentuated antagonism. These effects of adenosine may serve as a counterregulatory metabolic response that improves the O2 supply-demand ratio perturbed by enhanced sympathetic tone. Some catecholamine-mediated ventricular arrhythmias that occur during ischemia or enhanced adrenergic stress may be due to an imbalance in this negative feedback system.
B B Lerman, R C Wesley Jr, J P DiMarco, D E Haines, L Belardinelli
Cultured porcine aortic endothelial cells were conditioned through two passages to mimic euglycemic and hyperglycemic conditions (5.2 mM, normal glucose; 15.6 mM, elevated glucose). After incubation with 1 microM [14C]arachidonic acid for 24 h, the cells were stimulated with 1 microM A23187 for times up to 30 min. Uptake of [14C]arachidonic acid and its distribution among cell lipids were unaffected by the increased glucose concentration. The release of eicosanoids from labeled cells and unlabeled cells was measured by reverse-phase HPLC and by RIA, respectively. Compared with cells stimulated in the presence of normal glucose concentrations, cells stimulated in the presence of elevated glucose released 62.6% less free [14C]arachidonic acid, but released 129% more 14C-labeled 15-hydroxyeicosatetraenoic acid (HETE). Increased release of 15-HETE in the presence of elevated glucose in response to A23187, bradykinin, and thrombin was confirmed by RIA. A similar increase in 5-HETE release was observed by RIA after A23187 treatment. The release of both radiolabeled and unlabeled prostanoids was equal at both glucose concentrations. The data indicate that glucose may play an important role in the regulation of release and metabolism of arachidonic acid after agonist stimulation. In the presence of elevated glucose concentrations, such as those associated with diabetes mellitus, the extent and pattern of eicosanoid release from endothelial cells is markedly altered.
M L Brown, J A Jakubowski, L L Leventis, D Deykin
Protein S is a vitamin K-dependent protein cofactor to the anticoagulant, activated protein C (APC). This study examines the inhibition of human protein S anticoagulant activity by prothrombin. In the absence of protein S, the anticoagulant activity of APC measured in a Factor Xa recalcification time, was comparable using normal or plasma adsorbed with Al(OH)3. Protein S was an effective cofactor to APC in Al(OH)3-adsorbed plasma, but was significantly less active in normal plasma. Analysis of the difference in the two plasmas revealed that normal plasma contained an inhibitor to the anticoagulant activity of protein S that was removed by Al(OH)3 adsorption. Purification of this inhibitory activity demonstrated that it was mediated by the vitamin K-dependent protein, prothrombin. Prothrombin purified by conventional techniques caused immediate, dose-dependent inhibition of the cofactor activity of protein S in the presence of phospholipids or platelets, but had no effect on the anticoagulant activity of APC. The inhibition was demonstrable using a Factor Xa recalcification time, and studies of the rates of inactivation of purified Factor Va. Increasing concentrations of protein S overcame the inhibition by prothrombin and kinetic analysis of the interaction demonstrated that prothrombin acted as a competitive inhibitor to protein S. Immunoabsorption of prothrombin from plasma using immobilized antiprothrombin antibodies was associated with the complete removal of the protein S inhibitory activity. We conclude that the anticoagulant activity of protein S is modulated by prothrombin and that this may represent another regulatory mechanism of the natural anticoagulant system.
C A Mitchell, S M Jane, H H Salem
A new inborn error in bile acid synthesis, manifest in identical infant twins as severe intrahepatic cholestasis, is described involving the delta 4-3-oxosteroid 5 beta-reductase catalyzed conversion of the key intermediates, 7 alpha-hydroxy-4-cholesten-3-one and 7 alpha,12 alpha-dihydroxy-4-cholesten-3-one for chenodeoxycholic and cholic acid synthesis, to the respective 3 alpha-hydroxy-5 beta (H) products. This defect was detected by fast atom bombardment ionization-mass spectrometry from an elevated excretion and predominance of taurine conjugated unsaturated hydroxy-oxo-bile acids. Gas chromatography-mass spectrometry confirmed these to be 7 alpha-hydroxy-3-oxo-4-cholenoic and 7 alpha,12 alpha-dihydroxy-3-oxo-4-cholenoic acids (75-92% of total). Fasting serum bile acid concentrations were greater than 37 mumol/liter; chenodeoxycholic acid was the major bile acid, but significant amounts of allo(5 alpha-H)-bile acids (approximately 30%) were present. Biliary bile acid concentration was less than 2 mumol/liter and consisted of chenodeoxycholic, allo-chenodeoxycholic, and allo-cholic acids. These biochemical findings, which were identical in both infants, indicate a defect in bile acid synthesis involving the conversion of the delta 4-3-oxo-C27 intermediates into the corresponding 3 alpha-hydroxy-5 beta(H)-structures, a reaction that is catalyzed by a delta 4-3-oxosteroid-5 beta reductase enzyme. This defect resulted in markedly reduced primary bile acid synthesis and concomitant accumulation of delta 4-3-oxo-and allo-bile acids. These findings indicate a pathway in bile acid synthesis whereby side chain oxidation can occur despite incomplete alterations to the steroid nucleus, and lend support for an active delta 4-3-oxosteroid 5 alpha-reductase catalyzing the conversion of the delta 4-3-oxosteroid intermediates to the respective 3 alpha-hydroxy-5 alpha(H)-structures.
K D Setchell, F J Suchy, M B Welsh, L Zimmer-Nechemias, J Heubi, W F Balistreri
Present evidence suggests that in the small intestine, villus cells are primarily absorptive and crypt cells are primarily secretory. In order to further confirm that there are differences in transport properties between villus and crypt cells, we have separated villus from crypt cells, using calcium chelations techniques, and determined the distribution of Na:H and Cl:HCO3 exchange activity on brush border membrane and basolateral membrane preparations from these two cell populations. Separation of cells was determined utilizing alkaline phosphatase and maltase activity as a marker of villus cells and thymidine kinase activity as a marker of crypt cells. Utilizing these techniques, we were able to sequentially collect cells along the villus-crypt axis. Na-stimulated glucose and alanine uptake in brush border membrane vesicles diminished from the villus to the crypt region in the sequentially collected cells fractions, further suggesting separation of these cells. Brush border and basolateral membranes were then prepared from cells from the villus and crypt areas, utilizing a continuous sucrose gradient. In the villus cells, Na:H exchange activity was found associated with both the brush border and basolateral membrane, whereas, in crypt cells, Na:H exchange activity was only found on the basolateral membrane. Cl:HCO3 exchange activity was found only on the brush border membrane, in both villus and crypt cells. These studies suggest functional heterogeneity in ion transport between villus and crypt cells.
R G Knickelbein, P S Aronson, J W Dobbins
In past reports of hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency a marked degree of molecular heterogeneity has been noted. We have previously described two apparently unrelated subjects with partial HPRT deficiency, G.S. and D.B., who have a mutant form of HPRT with remarkably similar alterations in physical and kinetic properties. The mutation in G.S. is a serine to leucine substitution at amino acid 110 as determined by amino acid sequence analysis. This mutant enzyme has been designated HPRTLondon. We have examined HPRT cDNA from D.B. using two different methods to determine if the similar properties of mutant HPRT from these two subjects are the result of a common mutation. HPRT cDNA clones were obtained by routine cloning techniques and by polymerase chain reaction amplification of single-stranded cDNA reverse transcribed from mRNA derived from subject D.B. Dideoxynucleotide sequencing revealed a single mutation, a C to T transition at bp 329 in clones generated by both methods. This mutation in D.B. predicts the identical amino acid substitution described in HPRTLondon. A C to T nucleotide transition at 329 in D.B. creates an Hpa I site in exon 4 of the HPRT gene. Southern blot analysis of genomic DNA isolated from lymphoblasts derived from G.S. and D.B. revealed that both have this additional Hpa I site, indicating that the similarly altered protein sequence is due to the identical transition in the HPRT gene.
B L Davidson, S J Chin, J M Wilson, W N Kelley, T D Palella
We used a physiological stimulus, chronic dynamic exercise, in pigs to examine resultant changes in chronotropic responsiveness to catecholamine and biochemical features of cardiac beta-adrenergic receptors and the stimulatory guanine nucleotide-binding protein, GS. Long-term treadmill running resulted in a substantial (44%) down-regulation of right atrial beta-adrenergic receptors, but the dose of isoproterenol yielding a 50% maximal increase in heart rate was decreased by 57% (from 0.07 +/- 0.03 to 0.03 +/- 0.01 microgram/kg; P less than 0.02) despite this decrease in receptor number. This disparity between receptor number and physiological responsiveness suggested altered signal transduction. We therefore quantitated GS in myocardial membranes obtained before and after chronic exercise in a competitive ELISA based on an antipeptide antibody developed to the alpha S portion of GS. We found a 42% increase in the amounts of GS in right atrial membranes (from 11.4 +/- 0.8 to 16.2 +/- 2.0 pmol/mg; P less than 0.05) and a 76% increase in the amounts of GS in left ventricular membranes (from 15.6 +/- 2.6 to 27.4 +/- 5.2 pmol/mg; P = 0.02) after chronic running. These data suggest that in the heart physiological perturbations can result in changes in the levels of GS, that GS and beta-adrenergic receptor number are not coordinately regulated, and that GS may contribute to altered adrenergic responsiveness independently of changes in beta-adrenergic receptor number.
H K Hammond, L A Ransnas, P A Insel
Two T helper cell clones recognizing the gp 120 envelope protein of HIV were generated from the peripheral blood of a healthy seropositive individual. These cells were type specific as they proliferated and produced IL 2 when stimulated by an epitope in the amino-terminal half of gp 120 of HIVSF2, but not by a similar region of HIVZr6, a Zairian HIV-1 isolate. These two viruses differ by 26% in the deduced amino sequence of the gp 120 protein. Moreover, the antigenic site(s) recognized by the cloned T cells are distinct from those recognized by envelope-specific antibodies. These observations have important implications for the development and use of anti-HIV vaccines.
C M Walker, K S Steimer, K L Rosenthal, J A Levy
We have previously demonstrated that recombinant soluble CD4 protein (rsT4) blocks both HIV-1 infection of CD4 bearing lymphocytes and syncytium formation in vitro. (Recombinant soluble CD4 is designated by rsT4). Hence, we suggested the use of rsT4 in therapy for AIDS or the prevention of HIV-1 infection in individuals with a known risk of exposure. However, concerns arose that rsT4 might be immunosuppressive because of its implicated role in the enhancement of certain lymphocyte activation events through its engagement of MHC class II molecules on target cells. We therefore assessed the effect of recombinant soluble CD4 upon a number of functional and activation parameters of lymphocytes, including cellular proliferation, IL-2 secretion, and cytolytic capability, after antigenic or mitogenic stimulation. We report here that rsT4, at 60-fold over the concentration needed to block acute HIV-1 infection in vitro, does not significantly inhibit the activation of human peripheral blood lymphocytes by either PHA, tetanus toxoid or allogeneic cells. These results indicate that rsT4 will potentially exert minimal immunosuppressive effects in vivo, thus supporting the feasibility of clinical trials of rsT4 in the treatment or prevention of AIDS. In addition, the implications of these results for the interactions between CD4 and MHC class II molecules during lymphocyte activation are discussed.
M A Liu, T Liu