CORRECTION

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Chronic myelogenous leukemia: in vitro studies of hematopoietic regulation in a patient undergoing intensive chemotherapy.

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A patient heterozygous for the X-linked enzyme glucose-6-phosphate dehydrogenase and with Philadelphia chromosome-positive chronic myelogenous leukemia (CML) was treated with combination chemotherapy and had a partial loss of Philadelphia chromosome accompanied by partial restoration of nonclonal hematopoiesis as determined by glucose-6-phosphate dehydrogenase. Studies of in vitro hematopoiesis were performed after chemotherapy to evaluate the influences of neoplastic stem cells on normal cells and to determine whether there were physical and cell kinetic differences between leukemic stem cells and their normal counterparts. The data revealed the following: (a) The frequencies of normal committed granulocytic stem cells (CFU-C) and erythroid stem cells (BFU-E) in blood did not differ from the frequencies in marrow. (b) Normal late erythroid progenitors (CFU-E) were found at a significantly lower frequency that the more primitive BFU-E. Calculations indicated that not only was there a decrease in CFU-E production by normal BFU-E, but there was also abnormal clonal expansion of CML BFU-E (CFU-E:BFU-E ratio for normal progenitors was 1.1, whereas for the CML clone it was 11.5). (c) No increase in frequency of normal CFU-C was found after marrow cells were exposed to high specific activity tritiated thymidine. (d) Normal CFU-C and those from the CML clone were not separable on the basis of density. (e) The frequency of normal BFU-E was consistently greater than that of CFU-C, suggesting that regulatory differences influence the commitment of normal progenitors to the two pathways.

Authors

J W Singer, J W Adamson, Z A Arlin, S J Kempin, B D Clarkson, P J Fialkow

Jejunal and ileal adaptation to alterations in dietary calcium: changes in calcium and magnesium absorption and pathogenetic role of parathyroid hormone and 1,25-dihydroxyvitamin D.

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Previous balance studies have shown that fractional calcium absorption is increased by a low and reduced by a high calcium diet. The present studies were done to determine which segment of the small intestine is most sensitive to alterations in dietary calcium, and to see if dietary calcium intake has an effect on the intestinal absorption of another divalent cation, magnesium. Absorption was measured during constant perfusion of 30-cm segments of jejunum and ileum of normal subjects after 4 or 8 wk of a high (1,900 mg/d) or a low (20 mg/d) calcium diet. We found that calcium absorption rate was higher when subjects had been on a low than when they had been on a high calcium diet; the ileum responded more rapidly and more completely than the jejunum. Similar results were obtained with magnesium, but only the difference in the ileum was statistically significant. Sodium and xylose absorption were not influenced by dietary calcium intake. The serum concentrations of parathyroid hormone and 1,25-dihydroxyvitamin D were higher on the low than on the high calcium diet. We conclude that the ileum is more sensitive than the jejunum to changes in dietary calcium intake, and that ileal adaptation probably plays a major role in protecting the body against a deficiency or excess of body calcium that otherwise would occur when dietary calcium is abnormally low or high. Calcium intake influences ileal magnesium absorption in a similar fashion; it is not known whether or not this serves a protective function. Our data are compatible with the concept that adaptation to dietary calcium intake is mediated by changes in the serum concentrations of parathyroid hormone and 1,25-dihydroxyvitamin D.

Authors

D A Norman, J S Fordtran, L J Brinkley, J E Zerwekh, M J Nicar, S M Strowig, C Y Pak

Hyperosmolar Triggering of Histamine Release from Human Basophils

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Idiopathic reactions occurring during the infusion of hyperosmolar solutions, such as radiocontrast dyes, cause a significant number of deaths each year. These reactions are similar to those which follow mediator release during allergen-induced anaphylaxis. In attempting to explain these nonimmunologic reactions, we examined the direct effect of hyperosmolarity on normal human basophils with emphasis on release induced by mannitol.

Authors

Steven R. Findlay, Ann M. Dvorak, Anne Kagey-Sobotka, Lawrence M. Lichtenstein

Plasma Lipoprotein Induction and Suppression of the Generation of Cellular Procoagulant Activity in Vitro: REQUIREMENTS FOR CELLULAR COLLABORATION

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Isolated human plasma very low density, intermediate density, and high density lipo-proteins at physiologic concentrations have been demonstrated in the preceding report to induce significant increases in the procoagulant activity of human peripheral blood mononuclear cells in vitro, whereas low density lipoprotein did not. The monocyte was identified in this study by cellular fractionation and by direct cytologic assays as the source of this inducible activity, thus identifying the procoagulant activity as a monokine. The generation of these lipoprotein-induced procoagulant monokines was entirely dependent upon the presence of lymphocytes. Isolated lymphocytes that had been exposed to the stimulatory lipoproteins could induce monocytes to produce the procoagulant activity, whereas neither the culture medium from lipoprotein-stimulated lymphocytes, homogenates of lymphocytes, nor other cells such as platelets could substitute for this requirement. The interaction of the stimulatory lipoproteins with lymphocytes was rapid, reaching completion within 30 min, and was equally effective at either 4° or 37°C. Low density lipoprotein did not stimulate lymphocytes to induce monocyte procoagulant activity, but did actively suppress the production of the procoagulant monokines induced by each of the stimulatory lipoproteins, as well as bacterial lipopolysaccharide. The monocyte was identified as the cell sensitive to low density lipoprotein suppression, and no suppression of lymphocyte triggering was observed. These observations on the interaction of plasma lipoproteins with lymphocytes and monocytes in vitro introduce two new regulatory events by which plasma lipoproteins influence the function of cells, and define a regulatory network by which certain lipoprotein classes trigger lymphocytes, which can in turn induce monocytes to express procoagulant activity. Only this latter phase is subject to lipoprotein suppression by physiologic concentrations of low density lipoprotein.

Authors

Gary A. Levy, Bradford S. Schwartz, Linda K. Curtiss, Thomas S. Edgington

Identification of Spermine as an Inhibitor of Erythropoiesis in Patients with Chronic Renal Failure

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Fetal mouse liver and normal human bone marrow cell cultures were used for studies on the inhibition of erythroid colony formation (CFU-E) by sera from anemic patients with end-stage renal failure and the polyamine spermine. Sera from each of eight predialysis uremic anemic patients with end-stage renal failure produced a significant (P < 0.001) inhibition of erythroid colony formation in the fetal mouse liver cell cultures when compared to sera from normal human volunteers. In vivo or in vitro dialysis of the uremic sera with a 3,500-dalton exclusion limit membrane removed the inhibitor from uremic sera. The uremic serum dialysate provided by the membrane fractionation was significantly inhibitory in the erythroid cell cultures. When this dialysate was applied to gel filtration chromatography (Bio-Gel P-2) the inhibitor was found to be in the same molecular weight range as [14C]spermine. The polyamine spermine produced a dose-related inhibition of erythroid colony formation (CFU-E) in fetal mouse liver and normal human bone marrow cultures. Thus, the following evidence is provided that the in vitro inhibitor of erythropoiesis found in chronic renal failure patients' sera is identical with the polyamine spermine: (a) the inhibitor and radiolabeled spermine appeared in identical Bio-Gel P-2 effluent fractions; (b) when spermine was added to normal human sera at concentrations reported in sera of uremic patients, and studied in both the fetal mouse liver cell culture and normal human bone marrow cultures, a dose-related inhibition of erythroid colony (CFU-E) formation was noted; and (c) the inhibitory effects of crude uremic serum, uremic serum dialysate, and fractions of uremic serum dialysate from a Bio-Gel column, on erythroid colony formation were completely abolished by the addition of a specific rabbit antiserum to spermine.

Authors

Heinz W. Radtke, Arvind B. Rege, Max B. Lamarche, Dagmar Bartos, Frantisek Bartos, Robert A. Campbell, James W. Fisher

Effects of age and diabetes mellitus on the solubility and nonenzymatic glucosylation of human skin collagen.

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Collagen from human skin was fractionated into neutral salt-soluble, acid-soluble, pepsin-released, and insoluble fractions. No age-related changes were observed in the proportion of collagen extracted by neutral salt. A significant age-related decrease in the proportion of acid-soluble collagen was found. A highly significant (P less than 0.001) age-related decrease in the amount of collagen released by pepsin digestion was observed, with a concomitant age-related increase in the fraction of insoluble collagen. The amount of ketoamine-linked glucose bound to this insoluble collagen also increased significantly with age. Skin collagen from three juvenile onset diabetics (JOD) and one young maturity onset diabetic (MOD) appeared to have undergone accelerated aging. JOD and the young MOD had significantly less collagen released by pepsin digestion and significantly more insoluble collagen than would be predicted by their ages. The collagen released by pepsin digestion of the diabetic samples had more high molecular weight components than similar fractions obtained from age-matched nondiabetic controls. There was also more ketoamine-linked glucose bound to the insoluble collagen of JOD than to that fraction from comparably aged control subjects. The apparent acceleration of collagen aging in diabetes mellitus may play a role in complications of diabetes that occur in collagen-rich tissues.

Authors

S L Schnider, R R Kohn

Role of insulin in the intermediary metabolism of the activated thymic-derived lymphocyte.

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The hypothesis that a role for insulin in the metabolism of T cells would be evident after cell activation when receptors appear was tested to validate the T cell model and to analyze the mechanism by which insulin may function in immunoregulation. Measuring the flux rates of 3-O-[methyl-3H]-D-glucose and aminoisobutyric acid, alpha-[1-14C], lactate production and oxidation, and glucose oxidation from carbon 1- and carbon 6-labeled substrates, it was determined that (a) mitogens such as phytohemagglutinin enhance basal T lymphocyte intermediary metabolism, (b) physiologic concentrations of insulin have no impact on the metabolism of unstimulated, cultured, receptor-negative lymphocytes, and (c) insulin provided to receptor bearing lymphocytes augments intermediary metabolism above mitogen stimulated levels. The importance of the pentose phosphate shunt pathway for energy metabolism in the stimulated lymphocyte was confirmed. These studies demonstrate that insulin has a classical physiologic role to play in the activated lymphocyte further validating the use of this cell to examine potential receptor defects in disorders of carbohydrate metabolism. By enhancing energy metabolism of stimulated lymphocytes, insulin serves biologic economy and thus may perform its immunoregulatory role.

Authors

J H Helderman

Abnormalities in membrane phospholipid organization in sickled erythrocytes.

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In contrast to the wealth of information concerning membrane phospholipid asymmetry in normal human erythrocytes, very little is known about membrane phospholipid organization in pathologic erythrocytes. Since the spectrin-actin lattice, which has been suggested to play an important role in stabilizing membrane phospholipid asymmetry, is abnormal in sickled erythrocytes, we determined the effects of sickling on membrane phospholipid organization. We used two enzymatic probes: been venom phospholipase A2 and Staphylococcus aureus sphingomyelinase C, which do not penetrate the membrane and react only with phospholipids located in the outer leaflet of the bilayer. Our results suggest that the distribution of glycerophospholipids within the membrane of sickled cells is different from that in nonsickled cells. Compared with the normal erythrocyte, the outer membrane leaflet of the deoxygenated, reversibly sickled cells (RSC) and irreversibly sickled cells (ISC) was enriched in phosphatidyl ethanolamine in addition to containing phosphatidyl serine. These changes were compensated for by a decrease in phosphatidyl choline in that layer. The distribution of sphingomyelin over the two halves of the bilayer was unaffected by sickling. In contrast to ICS, where the organization of phospholipids was abnormal under both oxy and deoxy conditions, reoxygenation of RSC almost completely restored the organization of membrane phospholipids to normal. These results indicate that the process of sickling induces an abnormality in the organization of membrane phospholipids to normal. These results indicate that the process of sickling induces an abnormality in the organization of membrane lipids in RSC which become permanent in ISC.

Authors

B Lubin, D Chiu, J Bastacky, B Roelofsen, L L Van Deenen

Plasma Lipoprotein Induction and Suppression of the Generation of Cellular Procoagulant Activity In Vitro: TWO PROCOAGULANT ACTIVITIES ARE PRODUCED BY PERIPHERAL BLOOD MONONUCLEAR CELLS

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In the process of analyzing the effects of lipoproteins on functions of lymphoid cells, it was observed that physiological concentrations of isolated human plasma lipoproteins possess varying capacities to rapidly enhance the expression of procoagulant activity of human peripheral blood mononuclear cells in vitro. In a strict dose-dependent fashion, very low density lipoprotein, intermediate density lipoprotein, and high density lipoprotein enhanced both the surface expression by viable cells and the total cellular content of procoagulant activity during a 6-h incubation. Very low density lipoprotein induced a maximal 6.7-fold increase in the expression of a thromboplastin activity, which was consistent with tissue factor, in that it was dependent on Factors VII, X, and II. Both intermediate density lipoprotein and high density lipoprotein induced approximately a 12-fold increase of a different procoagulant activity which appears to be a direct prothrombin activator. This prothrombinase was calcium dependent and was inhibited by 2.5 mM diisopropylfluorophosphate, but was not neutralized by anti-Factor X antibodies or by inhibitors of Factor Xa. In contrast to the other lipoprotein density classes, low density lipoprotein did not stimulate procoagulant activity, but instead actively suppressed the generation of the two procoagulant activities induced by the stimulatory lipoproteins. Suppression by low density lipoprotein was clearly evident at molar ratios of low density lipoprotein to stimulatory lipoproteins of 1:3 or less. Reconstitution of all lipoproteins to physiological concentrations was not stimulatory as a consequence of the suppressive effects of low density lipoprotein. These data indicate that isolated plasma lipoproteins are capable of regulating the expression of two different procoagulant activities of peripheral blood mononuclear cells in vitro. The possibility that these interactions may be implicated in the association between certain types of hyperlipoproteinemias and thromboembolic disease merits study.

Authors

Bradford S. Schwartz, Gary A. Levy, Linda K. Curtiss, Daryl S. Fair, Thomas S. Edgington

Decreased rates of methionine synthesis by methylene tetrahydrofolate reductase-deficient fibroblasts and lymphoblasts.

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Methionine synthesis from homocysteine was measured in intact human fibroblasts and lymphoblasts using a [14C]formate label. Seven fibroblast lines and two lymphoblast lines derived from patients with 5,10-methylene tetrahydrofolate reductase deficiency had rates of methionine synthesis that were from 4 to 43% of normal. When the patients were divided by clinical status into mildly (two patients), moderately (two patients), and severely (three patients) affected, methionine biosynthesis expressed as a percent of control values was 43 and 33%, 11 and 10%, and 7, 6, and 4%, respectively, in fibroblasts. Similar data for the two lymphoblast lines were 36 and 26% for a mildly and moderately affected patient, respectively. These data are to be contrasted with the measurement of residual enzyme activity in cell extracts which agrees less precisely with the clinical status of the patients. In the presence of normal methionine synthetase activity, the rate of synthesis of methionine from homocysteine is a function of the activity of the enzyme 5,10-methylene tetrahydrofolate reductase, and measurement of the methionine biosynthetic capacity of cells deficient in this enzyme accurately reflects the clinical status of the patient from whom the cells were derived.

Authors

G R Boss, R W Erbe

A factor X-activating cysteine protease from malignant tissue.

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A proteolytic procoagulant has been identified in extracts of human and animal tumors and in cultured malignant cells. It directly activated Factor X but its similarity to other Factor S-activating serine proteases was not clear. This study describes work done to determine whether this enzyme, cancer procoagulant, is a serine or cysteine protease. Purified cancer procoagulant from rabbit V2 carcinoma was bound to a p-chloromercurialbenzoate-agarose affinity column and was eluted with dithiothreitol. The initiation of recalcified, citrated plasma coagulation activity by cancer procoagulant was inhibited by 5 mM diisopropylfluorophosphate, 1 mM phenylmethylsulfonylfluoride, 0.1 mM HgCl2, and 1 mM iodoacetamide. Activity was restored in the diisopropylfluorophosphate-, phenylmethylsulfonylfluoride-, and HgCl2-inhibited samples by 5 mM dithiothreitol; iodoacetamide inhibition was irreversible. Russell's viper venom, a control Factor X-activating serine protease, was not inhibited by either 0.1 mM HgCl2 or 1 mM iodoacetamide. The direct activation of Factor X by cancer procoagulant in a two-stage assay was inhibited by diisopropylfluorophosphate and iodoacetamide. Diisopropylfluorophosphate inhibits serine proteases, and an undefined impurity in most commercial preparations inhibits cysteine proteases. Hydrolysis of diisopropylfluorophosphate with CuSO4 and imidazole virtually eliminated inhibition of thrombin, but cancer procoagulant inhibition remained complete, suggesting that cancer procoagulant was inhibited by the undefined impurity. These results suggest that cancer procoagulant is a cysteine endopeptidase, which distinguishes it from other coagulation factors including tissue factor. This and other data suggest that neoplastic cells produce this unique cysteine protease which may initiate blood coagulation.

Authors

S G Gordon, B A Cross

Turnover and splanchnic metabolism of free fatty acids in hyperthyroid patients.

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The arterial concentration and turnover rate and the splanchnic exchange of FFA were examined after an overnight fast in a group of 11 female patients with clinical and laboratory evidence of hyperthyroidism. [14C]oleic acid was infused intravenously and the hepatic venous catheter technique was used. As compared with healthy control individuals, the arterial concentrations of FFA and oleic acid were elevated by 30--40% in the hyperthyroid group. Both the turnover rate and the fractional turnover of oleic acid were significantly increased. The turnover rate correlated directly with arterial concentration of oleic acid in both the control and the patient group but the slope was steeper in the patients. The splanchnic uptake of oleic acid was three times higher than in the control group. The augmented uptake was a consequence of elevated arterial concentrations and increased hepatic plasma flow, whereas fractional splanchnic uptake remained unchanged. Ketone body production was four- to fivefold greater in the patients and could be largely accounted for by increased splanchnic FFA uptake. In six patients studied after treatment resulting in a return to normal thyroid function, a significant reduction was observed in arterial FFA, estimated hepatic blood flow, oleic acid turnover, and ketone body production. It is concluded that hyperthyroidism is characterized by increased turnover and splanchnic uptake of FFA and augmented ketogenesis. These findings can be explained on the basis of elevated arterial FFA concentrations and increased blood flow, particularly to the splanchnic bed.

Authors

L Hagenfeldt, A Wennlung, P Felig, J Wahren

Splanchnic Metabolism of Plasma Apolipoprotein B: STUDIES OF ARTERY-HEPATIC VEIN DIFFERENCES OF MASS AND RADIOLABEL IN FASTED HUMAN SUBJECTS

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The metabolism of apoprotein B-containing plasma lipoproteins by human splanchnic tissues has been studied in 29 men undergoing coronary angiography. Before catheterization autologous radio-iodinated lipoproteins were infused into a peripheral vein: 10 subjects received 125I-labeled Sf 12-60 lipoproteins; 12 received 125I-labeled Sf 12-60 plus 131I-labeled Sf 100-400 lipoproteins; and 7 received 125I-labeled Sf 12-60 plus 131I-labeled Sf 0-12 lipoproteins. Paired arterial and hepatic vein blood samples were subsequently collected for replicate measurements of apoprotein B (apo B) mass, radioactivity and specific activity in each lipoprotein class. Splanchnic plasma flow was measured with indocyanine green. All studies were conducted after a 14-h overnight fast.

Authors

P. R. Turner, N. E. Miller, C. Cortese, W. Hazzard, J. Coltart, B. Lewis

Effect of vasoactive intestinal polypeptide on active and passive transport in the human jejunum.

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The effect of intravenous vasoactive intestinal polypeptide (VIP) on normal transport mechanisms in the human jejunum in vivo was examined with the triple-lumen, steady-state perfusion technique. By using special test solutions that revealed different aspects of jejunal transport, we were able to evaluate the effect of VIP on specific transport processes, such as active bicarbonate absorption, active chloride secretion, and passive absorption or secretion of sodium chloride. At an infusion rate of 200 pmol/kg per h, VIP inhibited active bicarbonate absorption by approximately 42%, stimulated active chloride secretion to a slight extent, and slightly reduced passive sodium chloride absorption. A larger dose of VIP, 400 pmol/kg per h, had essentially the same effect on active bicarbonate absorption and active chloride secretion, but it markedly depressed passive sodium chloride absorption and also inhibited passive secretion induced by mannitol. VIP reduced the lumen-to-plasma unidirectional sodium and chloride flux rates, while the plasma-to-lumen flux rates were decreased to a lesser extent or remained unchanged. The potential difference became more lumen-negative with VIP, but the sodium diffusion and glucose-stimulated potential were not affected. We conclude that the major effect of VIP in the human jejunum is to decrease the normal absorption of water and electrolytes--not only active bicarbonate-mediated absorption, but also the passive absorption in response to osmotic forces generated by active or facilitated absorptive processes. Although an increase in chloride secretion does occur, this does not appear to be of major importance.

Authors

G R Davis, C A Santa Ana, S G Morawski, J S Fordtran

Effects of Arachidonic Acid, Monohydroxyeicosatetraenoic Acid and Prostaglandins on the Release of Mucous Glycoproteins from Human Airways In Vitro

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Human lung explants maintained in culture for 7 d incorporate [3H]glucosamine into mucous glycoproteins. Ethanol-precipitable, glucosamine-labeled mucous secretion was measured, and the effects of different pharmacologic agents upon this secretion were investigated. Anaphylaxed human lung generates prostaglandin (PG) synthesis and increased mucous release. Arachidonic acid (AA), PGA2, PGD2, and PGF2α significantly increased mucous glycoprotein release, whereas PGE2 significantly reduced release. Evidence which suggests that lipoxygenase products of AA augment mucous release includes the following: (a) Nonsteroidal anti-inflammatory drugs (NSAID: acetylsalicylic acid and indomethacin) increase mucous release while preventing prostaglandin formation. (b) The increase in mucous release induced by AA or NSAID is additive once the agents are combined. (c) Several nonspecific lipoxygenase inhibitors (eicosa-5,8,11,14-tetraynoic acid; vitamin E; nordihydroguaiaretic acid; and α-naphthol) inhibit mucous release. Three additional lines of evidence directly indicate that monohydroxyeicosatetraenoic acid (HETE) causes increased mucous release: (a) the addition of a mixture of synthetic HETE (24-600 nM) increases mucous release; (b) pure 12-HETE (1-100 nM) also increases mucous release; (c) mucous release is increased synergistically by the combination of HETE and NSIAD.

Authors

Zvi Marom, James H. Shelhamer, Michael Kaliner

Selectivity of Dobutamine for Adrenergic Receptor Subtypes: IN VITRO ANALYSIS BY RADIOLIGAND BINDING

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The cardiovascular responses elicited by dobutamine are distinctly different from those produced by other adrenergic or dopaminergic agonists. To test the hypothesis that dobutamine could have differential affinities for adrenergic receptor subtypes, and that such subtype selectivity could be related to its relatively unique pharmacologic properties, we assessed the ability of dobutamine to displace adrenergic radioligands from membrane receptors in a number of tissues of previously characterized adrenergic receptor subtype. For beta adrenergic receptors identified by (−) [3H]dihydroalprenolol (DHA), dobutamine had significantly greater affinity for the β1 subtype (KD = 2.5 μM in rat heart and 2.6 μM in turkey erythrocyte) than for the β2 subtype (KD = 14.8 μM in frog heart and 25.4 μM in rat lung) (P < 0.001). For alpha adrenergic receptors, dobutamine had markedly greater affinity for the α1-subtype identified by [3H]prazosin (KD = 0.09 μM in rat heart and 0.14 μM in rabbit uterus) than for the α2-subtype identified by [3H]dihydroergocryptine (DHE) (KD = 9.3 μM in human platelet) or by [3H]yohimbine (KD = 5.7 μM in rabbit uterus) (P < 0.001).

Authors

R. Sanders Williams, Timothy Bishop

Role of Gallbladder Mucus Hypersecretion in the Evolution of Cholesterol Gallstones: STUDIES IN THE PRAIRIE DOG

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Because mucin glycoproteins may be important in the pathophysiology of gallstones, we studied the relationship among biliary lipids, gallbladder mucin secretion, and gallstone formation in cholesterol-fed prairie dogs. Organ culture studies of gallbladder explants revealed that the incorporation of [3H]glucosamine into tissue and secretory gallbladder glycoproteins was significantly increased at 3, 5, 8, and 14 d of feeding. Peak secretion of labeled mucin occurred at 5 d, when total tissue and secreted glycoprotein production was fivefold greater than control. Gel filtration of the secreted glycoprotein on Sepharose 4B indicated that the majority of radioactivity was present in a macromolecule of > 1 million molecular weight. The increased secretion of gallbladder mucin was organ specific, in that [3H]glucosamine incorporation into glycoproteins of stomach and colon was unaffected by cholesterol feeding. Similarly, the incorporation of [3H]mannose into gallbladder membrane glycoproteins was not altered by cholesterol feeding. The rate of glycoprotein synthesis and secretion returned to normal upon withdrawal of the cholesterol diet, and ligation of the cystic duct before cholesterol feeding prevented gallbladder mucin hypersecretion. Both results indicate that the stimulus to mucin secretion was a constituent of bile. Gallbladder bile after 5 d contained cholesterol in micelles, liquid crystals, and crystals, whereas hepatic bile remained a single micellar phase throughout cholesterol feeding. For this reason the cholesterol-saturation indices of gallbladder bile were compared in both homogenized and centrifuged samples. The micellar phase of gallbladder bile was appreciably less saturated than homogenized bile at 5 and 8 d, which reflects the continuous nucleation of cholesterol in the gallbladder. Purified human gallbladder mucin gels were shown to induce nucleation of lecithin-cholesterol liquid crystals from supersaturated hepatic bile. These in turn gave rise to cholesterol monohydrate crystals within 18 h. Control supersaturated hepatic bile could not be nucleated by the addition of other proteins, and was stable for days upon standing. These results suggest that the increase in cholesterol content of bile in cholesterolfed prairie dogs stimulates gallbladder mucus hypersecretion, and that gallbladder mucus gel is a nucleating agent for biliary cholesterol.

Authors

Sum P. Lee, J. Thomas Lamont, Martin C. Carey

Arrangement of the respiratory burst oxidase in the plasma membrane of the neutrophil.

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Evidence was obtained regarding the way the O-2-forming NADPH oxidase of human neutrophils is arranged within the plasma membrane. O-2 production by particles from zymosan-activated human neutrophils rose two- to threefold when the particles were assayed in the presence of appropriate concentrations of Triton X-100. The portion of activity revealed by the detergent was not affected by treating the particles with trypsin or with p-chloromercuribenzene sulfonate, a nonpenetrating sulfhydryl reagent, but the activity detectable in the absence of detergent was abolished by these treatments. O-2 production by phagocytic vesicles was not augmented by detergent, and was almost entirely eliminated by tryptic digestion of the vesicles regardless of whether or not detergent was present during the assay. These results suggest that the O-2-forming oxidase is embedded in the plasma membrane with a portion extending into the cytoplasm and the rest buried in the lipid bilayer. It is proposed that the pyridine nucleotide-binding site is located on the cytoplasmic extension and the oxygen binding site is on the intramembranous portion of the enzyme.

Authors

G L Babior, R E Rosin, B J McMurrich, W A Peters, B M Babior

Epinephrine plasma thresholds for lipolytic effects in man: measurements of fatty acid transport with [l-13C]palmitic acid.

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To determine the plasma epinephrine thresholds for its lipolytic effect, 60-min epinephrine infusions at nominal rates of 0.1, 0.5, 1.0, 2.5, and 5.0 micrograms/min were performed in each of four normal young adult men while they also received a simultaneous infusion of [1-13C]palmitic acid to estimate inflow transport of plasma free fatty acids. These 20 infusions resulted in steady-state plasma epinephrine concentrations ranging from 12 to 870 pg/ml. Plasma epinephrine thresholds for changes in blood glucose, lactate, and beta-hydroxybutyrate were in the 150--200-pg/ml range reported by us previously (Clutter, W. E., D. M. Bier, S. D. Shah, and P. E. Cryer. 1980. J. Clin. Invest. 66: 94--101.). Increments in plasma glycerol and free fatty acids and in the inflow and outflow transport of palmitate, however, occurred at lower plasma epinephrine thresholds in the range of 75 to 125 pg/ml. Palmitate clearance was unaffected at any steady-state epinephrine level produced. These data indicate that (a) the lipolytic effects of epinephrine occur at plasma levels approximately threefold basal values and (b) lipolysis is more sensitive than glycogenolysis to increments in plasma epinephrine.

Authors

A D Galster, W E Clutter, P E Cryer, J A Collins, D M Bier

Thyroid Hormone-Carbohydrate Interaction in the Rat: CORRELATION BETWEEN AGE-RELATED REDUCTIONS IN THE INDUCIBILITY OF HEPATIC MALIC ENZYME BY TRIIODO-l-THYRONINE AND A HIGH CARBOHYDRATE, FAT-FREE DIET

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Previous studies from this laboratory have demonstrated an age-related decrease in hepatic malic enzyme (ME) levels and in the response of ME to triiodo-l-thyronine (T3). Moreover, we have recently shown a synergistic interaction of T3 and a high carbohydrate diet in the induction of this enzyme. Studies were therefore undertaken to assess the response of aging rats to a high carbohydrate diet and to test the effect of such dietary manipulations on the responsiveness of ME to T3. For this purpose, a new radio-immunoassay for ME was developed that, because of a 10-fold higher sensitivity, was particularly suited to the measurement of the low concentrations of hepatic enzyme in older animals. The level of ME per milligram of DNA fell ∼70% between 1 and 6 mo with only minor further changes demonstrated between 6 and 18 mo. In contrast, the level of ME per milligram DNA in brain was slightly increased in the older animals. Although the absolute increment of hepatic ME resulting from seven daily injections of T3 (15 μg/100 g body wt) fell with age, the ratio of the ME content per milligram DNA to that observed in control animals maintained on a regular chow diet remained relatively constant with an average value of 11.1. The responsivity of hepatic ME to a high carbohydrate, fat-free diet also decreased with age and could not be attributed exclusively to a reduction in food consumption. The age-related reduction in ME responsivity to dietary stimuli appeared to be due to a reduction in the formation of the specific messenger, (m)RNA for ME as determined in an in vitro translational assay. Our data are consistent with the following hypothesis. There is an age-related decreased hepatic responsivity to a high carbohydrate dietary stimulus. Thyroid hormone administration, as previously postulated by us, interacts with a product or an intermediate of carbohydrate metabolism in a multiplicative fashion. As a consequence, the absolute increment of ME induced by T3 administration also declines with age.

Authors

Mary Ann Forciea, Harold L. Schwartz, Howard C. Towle, Cary N. Mariash, F. E. Kaiser, J. H. Oppenheimer

Hydrolysis of human milk fat globules by pancreatic lipase: role of colipase, phospholipase A2, and bile salts.

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Human milk fat globules were used to explore how dietary triglycerides are hydrolyzed by pancreatic lipase. These triglycerides were hydrolyzed very slowly by lipase alone as if the surface layer of proteins and phospholipids impeded the action of the enzyme. The inhibition of lipase activity could be overcome by addition either of colipase or of pancreatic phospholipase A2. Colipase enhanced triglyceride hydrolysis in a dose-dependent manner whether bile salts were present or not. Bile salts had no effect on the activity of pancreatic lipase alone but further enhanced the activity at all concentrations of colipase tested. Bile salts were a prerequisite to relieve inhibition of lipase activity by phospholipase A2. Human milk fat globules exposed to phospholipase A2 should be representative of a physiological substrate for pancreatic lipase. A major new observation was that bile salts, even at high concentrations, stimulated triglyceride hydrolysis of such phospholipase-treated globules by pancreatic lipase also in the absence of colipase.

Authors

L Bläckberg, O Hernell, T Olivecrona

Reversal of Secondary Hyperparathyroidism by Cimetidine in Chronically Uremic Dogs

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Abstract

Chronic cimetidine therapy has been shown to suppress circulating concentrations of immunoreactive parathyroid hormone (iPTH) in hemodialysis patients. To evaluate the long-term metabolic effects of cimetidine treatment, we studied seven chronically uremic dogs for 20 wk. The dogs were studied under metabolic conditions before, during, and after cimetidine therapy. iPTH fell progressively in the five treated dogs from 536±70 μleq/ml (mean±SE) (nl < 100 μleq/ml) before treatment to 291±25 μleq/ml at 12 wk (P < 0.001) and 157±32 μleq/ml at 20 wk (P < 0.001). The control dogs showed no consistent change in iPTH. The fall in iPTH was not associated with a change in serum ionized calcium. However, serum phosphorus decreased from 5.7±0.9 mg/dl to 3.4±0.2 mg/dl by the 20th wk (P < 0.05). By contrast, the serum concentration of 1,25-dihydroxycholecalciferol increased in all treated dogs from 33.4±4.3 pg/ml to 51.8±2.4 pg/ml during treatment (P < 0.01). Calcium balance was negative in all seven dogs before cimetidine (−347±84 mg/72 h) and remained so in the control dogs; it became positive in the five treated dogs after 12 wk (1,141±409 mg/72 h) (P < 0.05). Phosphorus balance, 24-h fractional phosphate excretion, and creatinine clearance remained unchanged. Pooled samples of serum obtained during the control and 20th wk of therapy were fractionated by gel filtration and the eluates assayed for immunoreactivity. The decrease in iPTH was associated with a decrease in all the immunoreactive species, indicating suppression of parathyroid gland secretion.

Authors

Allan I. Jacob, Janet M. Canterbury, George Gavellas, Phillip W. Lambert, Jacques J. Bourgoignie

Comparison of the respiratory responses to external resistive loading and bronchoconstriction.

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Abstract

The effects of resistive loads applied at the mouth were compared to the effects of bronchospasm on ventilation, respiratory muscle force (occlusion pressure), and respiratory sensations in 6 normal and 11 asthmatic subjects breathing 100% O2. External resistive loads ranging from 0.65 to 13.33 cm H2O/liter per s were applied during both inspiration and expiration. Bronchospasm was induced by inhalation of aerosolized methacholine. Bronchospasm increased ventilation, inspiratory airflow, respiratory rate, and lowered PACO2. External resistive loading, on the other hand, reduced respiratory rate and inspiratory flow, but left ventilation and PACO2 unaltered. FRC increased to a greater extent with bronchospasm than external flow resistive loads. With both bronchospasm and external loading, occlusion pressure increased in proportion to the rise in resistance to airflow. However, the change in occlusion pressure produced by a given change in resistance and the absolute level of occlusion pressure at comparable levels of airway resistance were greater during bronchospasm than during external loading. These differences in occlusion pressure responses to the two forms of obstruction were not explained by differences in chemical drive or respiratory muscle mechanical advantage. Although the subjects' perception of the effort involved in breathing was heightened during both forms of obstruction to airflow, at any given level of resistance the sense of effort was greater with bronchospasm than external loading. Inputs from mechanoreceptors in the lungs (e.g., irritant receptors) and/or greater stimulation of chest wall mechanoreceptors as a result of increases in lung elastance may explain the differing responses elicited by the two forms of resistive loading.

Authors

S G Kelsen, T F Prestel, N S Cherniack, E H Chester, E C Deal Jr

Thyrotropin-releasing Hormone Stimulation of Prolactin Release from Clonal Rat Pituitary Cells: EVIDENCE FOR ACTION INDEPENDENT OF EXTRACELLULAR CALCIUM

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Abstract

Thyrotropin-releasing hormone (TRH) stimulates prolactin release and 45Ca2+ efflux from GH3 cells, a clonal strain of rat pituitary cells. Elevation of extracellular K+ also induces prolactin release and increases 45Ca2+ efflux from these cells. In this report, we distinguish between TRH and high K+ as secretagogues and show that TRH-induced release of prolactin and 45Ca2+ is independent of the extracellular Ca2+ concentration, but the effect of high K+ on prolactin release and 45Ca2+ efflux is dependent on the concentration of Ca2+ in the medium. The increment in 45Ca2+ efflux induced by 50 mM K+ during perifusion was reduced in a concentration-dependent manner by lowering extracellular Ca2+ from 1,500 to 0.02 μM (by adding EGTA), whereas 1 μM TRH enhanced 45Ca2+ efflux similarly over the entire range of extracellular Ca2+ concentrations. Although 50 mM K+ caused release of 150 ng prolactin from 40 × 106 GH3 cells exposed to 1,500 μM Ca2+ (control), reduction of extracellular Ca2+ to 2.8 μM decreased prolactin release caused by high K+ to <3% of controls and no prolactin release was detected after exposure to 50 mM K+ in medium with 0.02 μM free Ca2+. In contrast, TRH caused release of 64 ng of prolactin from 40 × 106 GH3 cells exposed to medium with 1,500 μM Ca2+, and release caused by TRH was still 50 and 35% of control in medium with 2.8 and 0.02 μM Ca2+, respectively. Furthermore, TRH transiently increased by 10-fold the fractional efflux of 45Ca2+ from GH3 cells in static incubations with 1,500 or 3.5 μM Ca2+, hereby confirming that the enhanced 45Ca2+ efflux caused by TRH in both low and high Ca2+ medium was not an artifact of the perifusion system.

Authors

Marvin C. Gershengorn, Sylvia T. Hoffstein, Mario J. Rebecchi, Elizabeth Geras, Brian G. Rubin

Regulation of Human Leukocyte Beta Receptors by Endogenous Catecholamines: RELATIONSHIP OF LEUKOCYTE BETA RECEPTOR DENSITY TO THE CARDIAC SENSITIVITY TO ISOPROTERENOL

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Abstract

High levels of beta receptor agonist have previously been shown to down-regulate beta receptor density on circulating leukocytes in man; however, the factors controlling receptor density under physiological conditions have not previously been defined. To determine whether beta receptor density is normally down-regulated by circulating, physiological levels of catecholamines we have examined the relationship between receptor density and catecholamine levels. Urinary epinephrine and norepinephrine were significantly reciprocally correlated to lymphocyte receptor density. A similar relationship existed between beta receptor density and supine plasma epinephrine, norepinephrine, upright epinephrine, and norepinephrine levels. Change in sodium intake from 10 to 400 meq/d caused a 52% increase in lymphocyte and a 48% increase in polymorphonuclear beta receptor density. The changes in receptor density were accompanied by an increase in the sensitivity to isoproterenol measured as a fall in the dose of isoproterenol required to raise the heart rate by 25 beats per minute. Beta receptor density on both lymphocyte and polymorphonuclear cells was significantly correlated to the cardiac sensitivity to isoproterenol. Propranolol administration resulted in an increase in the density of beta receptors on lymphocyte and polymorphonuclear cells that correlated with the subject's pretreatment catecholamine levels.

Authors

J. Fraser, J. Nadeau, D. Robertson, A. J. J. Wood

Triiodothyronine-induced Thyrotoxicosis Increases Mononuclear Leukocyte β-Adrenergic Receptor Density in Man

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Abstract

β-Adrenergic receptors are increased in some tissues of experimentally thyrotoxic animals but are reported to be unchanged in mononuclear leukocytes of spontaneously thyrotoxic humans. We examined the effects of triiodothyronine (100 μg/d for 7 d) and placebo on high-affinity mononuclear leukocyte β-adrenergic receptors in 24 normal human subjects, using a double-blind design. β-Adrenergic receptors were assessed by specific binding of the antagonist (-)[3H]dihydroalprenolol. Triiodothyronine administration resulted in objective evidence of moderate thyrotoxicosis and an increase in mean (-)[3H]dihydroalprenolol binding from 25±3 to 57±9 fmol/mg protein (P < 0.001). The latter was attributable, by Scatchard analysis, to an increase in β-adrenergic receptor density (967 ± 134 to 2250 ± 387 sites per cell, P < 0.01); apparent dissociation constants did not change. Placebo administration had no effects. Marked inter- and intraindividual variation in mononuclear leukocyte β-adrenergic receptor density was also noted. Because this was approximately threefold greater than analytical variation, it is largely attributable to biologic variation. Thus, we conclude: (a) The finding of a triiodothyronine-induced increase in mononuclear leukocyte β-adrenergic receptor density in human mononuclear leukocytes, coupled with similar findings in tissues of experimentally thyrotoxic animals, provides support for the use of mononuclear leukocytes to assess receptor status in man. (b) There is considerable biologic variation in β-adrenergic receptor density in man. (c) The findings of thyroid hormone-induced increments in β-adrenergic receptor density provide a plausible mechanism for the putative enhanced responsiveness to endogenous catecholamines of patients with thyrotoxicosis.

Authors

Ann M. Ginsberg, William E. Clutter, Suresh D. Shah, Philip E. Cryer

Role of the endogenous kallikrein-kinin system in modulating vasopressin-stimulated water flow and urea permeability in the toad urinary bladder.

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Abstract

This study investigates the endogenous kallikrein-kinin system's role as a modulator of vasopressin action in the toad urinary bladder. Kalli-krein inhibition by aprotinin, which results in decreased kinin production, significantly increased both vasopressin and 8-Br-cyclic (c) AMP-stimulated water flow. Kinin potentiation by the kininase II inhibitor captopril (SQ 14225) significantly decreased vasopressin and 8-Br-cAMP-stimulated water flow. In contrast to water flow, vasopressin-stimulated urea permeability was decreased by aprotinin and increased by captopril. We conclude that the endogenous kallikrein-kinin system represents a significant modulator of vasopressin action and it permits separate control of vasopressin-stimulated water flow and solute transport.

Authors

C P Carvounis, G Carvounis, L A Arbeit

Immunoglobulin G heavy chain (Gm) allotypes in multiple sclerosis.

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Abstract

Serum samples from 70 Caucasian patients with multiple sclerosis were typed for nine Gm markers. Significant association was found with the Gm 1,17;21 phenotype, and the relative risk for individuals with this phenotype was calculated at 3.6. The data indicate that Caucasians positive for Gm 1,17;21 are almost four times more likely to develop multiple sclerosis than those without this phenotype.

Authors

J P Pandey, J M Goust, J P Salier, H H Fudenberg

Abnormal platelet response to thromboxane A2.

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Abstract

To determine the pathogenetic mechanism of a hereditary primary platelet release disorder, arachidonic acid metabolism via the cyclooxygenase pathway was investigated. The propositus' platelets exhibited defective release reaction and second-wave aggregation when stimulated by sodium arachidonate or U46619, a thromboxane A2 (TXA2) agonist. The lack of platelet response to U46619 suggested that the defect was beyond the thromboxane synthetase level. Furthermore, thromboxane B2 (TXB2) formation in the propositus' platelets (558.52 ng/10(8) platelets) was within the normal range (574.29 +/- SD 27.39 ng/10(8) platelets) and TXA2 formation appeared to be adequate for aggregating normal platelets. The results were indicative of an abnormal platelet response to TXA2. Failure of the propositus' platelets to aggregate in response to TXA2 formed in normal platelet-rich plasma induced by arachidonate confirmed this notion. To gain further insight, platelet cyclic (c) AMP content was determined. Prostacyclin induced a significant elevation of the propositus' platelet cAMP level comparable to normal values. U46619 suppressed prostaglandin I2-induced cAMP elevation in normal subjects but had no such effect in the patient. We conclude that the primary release disorder observed in this kindred is due to an abnormal platelet respnse to TXA2 possibly because of TXA2/PGH2 receptor abnormalities.

Authors

K K Wu, G C Le Breton, H H Tai, Y C Chen

Gyrate atrophy of the choroid and retina with hyperornithinemia: characterization of mutant liver L-ornithine:2-oxoacid aminotransferase kinetics.

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Abstract

Deficient activity of L-ornithine:2-oxoacid aminotransferase is associated with gyrate atrophy of the choroid and retina with hyperornithinemia, an autosomal recessive disease leading to blindness. Liver tissue from two patients contained trace activity of the enzyme. The Michaelis (Km) value of the mutant enzyme for ornithine was 200 mM, 50-fold higher than normal, but increasing the concentrations of alpha-oxoglutarate and pyridoxal 5'-phosphate to 10 times those giving maximal activity of the normal enzyme had no effect on the mutant enzyme. Substrate inhibition of the mutant could not be demonstrated at 1,000 mM ornithine concentration, whereas ornithine concentrations above 70 mM inhibited the normal enzyme. The data suggest that the abnormal L-ornithine:2-oxoacid aminotransferase in the two patients studied has an altered binding site for ornithine.

Authors

I Sipilä, O Simell, J J O'Donnell