Potentiation of cholecystokinin-induced exocrine secretion by both exogenous and endogenous insulin in isolated and perfused rat pancreata.

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Abstract

Using an isolated perfused rat pancreas preparation, the interrelationship between the endocrine and exocrine portions of the pancreas were studied. Addition of exogenous rat insulin (1-20 mU/ml) to the perfusing solution potentiated the action of cholecystokinin (CCK) (1 mU/ml) to increase both pancreatic juice flow and the release of the enzyme, amylase. Raising the glucose concentration in the perfusing solution from 2.5 to 17.5 mM both increased endogenous insulin release and potentiated the CCK-induced exocrine secretory response. Two lines of evidence indicated that this effect of glucose on the exocrine pancreas was mediated by endogenous insulin release. First, the addition of comparable amounts of xylose or galactose to the perfusion medium neither released insulin nor potentiated the CCK-induced response. Second, epinephrine blocked the effect of high glucose on both insulin release and potentiation of CCK action. Epinephrine alone did not affect the action of CCK. The magnitude of the exocrine response induced by high glucose was comparable to that of 2.5 mU/ml exogenous insulin. It seems possible that pancreatic acinar cells can be exposed to insulin levels of this magnitude in situ.

Authors

A Saito, J A Williams, T Kanno

Concentration of Myo-inositol in Skeletal Muscle of the Rat Occurs without Active Transport

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Abstract

The cellular uptake of nonphosphorylated myo-inositol (MI) and its incorporation into phosphoinositide in the rat epitrochlearis muscle was measured. Cellular uptake of [2-3H]MI was determined by the difference between total uptake and [2-3H]MI present in the extracellular fluid determined with [1-14C]mannitol. Cellular uptake was parabolic and directly proportional to medium MI concentrations between 25 and 3,200 μM. Saturation of a MI carrier was not evident. Moreover, uptake was not inhibited by 2 mM ouabain, 0.3 mM 2,4-dinitrophenol, or 22 mM glucose. Insulin, 100 mU/ml, was without effect on either cellular uptake of [2-3H]MI or its incorporation into phosphoinositides. In muscles that were preloaded with [2-3H]MI and then incubated in media that contained a constant amount of MI but no [2-3H]MI, 44.3% of the [2-3H]MI was released after 10 min increasing to 62.5% by 120 min. Cellular MI concentrations were 0.18 μmol/g wet tissue (four times plasma levels) in rapidly isolated and frozen epitrochlearis muscle. When muscle was incubated without MI, 48% of endogenous MI was lost rapidly. Restoration of cellular MI in 50 μM MI media occurred in two phases, a rapid uptake phase lasting 10 min and a subsequent slow phase of MI uptake.

Authors

Bruce A. Molitoris, Irene E. Karl, William H. Daughaday

Diminished synthesis of immunoglobulin by peripheral lymphocytes of patients with idiopathic membranous glomerulonephropathy.

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Abstract

Some studies of animal models of serum-sickness nephritis have shown that the lesions of membranous nephropathy develop in animals exhibiting a poor antibody response to the administered antigen (if given in constant amounts). It is postulated that patients with idiopathic membranous nephropathy may share a similar characteristic, namely, a diminished capacity to produce sufficient amounts of antibody. To test this hypothesis, we examined the ability of lymphocytes isolated from 11 patients with this disorder to produce immunoglobulin (Ig)G and IgM on stimulation with a polyclonal B-cell activator, pokeweed mitogen. The peripheral blood lymphocytes (2 x 10(6) cells) from 24 normal individuals had geometric mean production rates of 1,779 ng for IgG, and 2,940 ng for IgM after 7 d of culture in the presence of pokeweed mitogen. By contrast, under identical conditions, lymphocytes from the 11 patients with membranous nephropathy produced significantly lower quantities of both immunoglobulins, with geometric mean concentrations of 511 ng for IgG and 439 ng for IgM. When lymphocytes from patients with membranous nephropathy were co-cultured with normal lymphocytes, the production of immunoglobulin by normal lymphocytes was depressed by 22-82%, suggesting that a population of suppressor cells was responsible for this disturbance in B-cell function. By co-culturing normal lymphocytes with patient lymphocytes depleted of either T cells or monocytes, the suppressor cell was identified as a monocyte.

Authors

B S Ooi, Y M Ooi, A Hsu, P E Hurtubise

Adverse Effects of Cytomegalovirus Vaccination in Mice

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Abstract

Studies of live attenuated cytomegalovirus (CMV) vaccine have recently been initiated in man. The possibilities of latent infection and disease resulting from reactivation of vaccine virus are major concerns. Because markers for attenuation of tissue culture-passaged mouse CMV (MCMV) exist, studies of potential adverse effects of vaccination were initiated in mice. Plaque-purified MCMV was passed 12 times in cell culture (“vaccine virus”) and shown to be attenuated by virtue of loss of lethality and diminished replication in reticuloendothelial organs of normal mice. Although subcutaneous inoculation of 105 plaque-forming units of wild virus was lethal for mice immunosuppressed with antilymphocyte serum (18/18 died), “vaccine MCMV” killed only 3/18 (P < 0.05) and was thus shown to be highly attenuated even in immunosuppressed animals. 4 mo after subcutaneous inoculation of vaccine MCMV, no infectious virus was detectable in the tissues of normal C3H mice. However, immunosuppression with anti-lymphocyte serum and cortisone caused MCMV reactivation, dissemination, and wide-spread cytomegalic inclusion disease in 19 of 20 animals. Characterization of the reactivating virus recovered from salivary glands indicated that reversion to virulence had occurred. Thus, vaccine MCMV, although markedly attenuated initially, established latent infection, reactivated after immunosuppression, and reverted to virulence, at least in salivary gland tissue. These data from the murine model substantiate the need for careful surveillance and virologic study of patients given experimental CMV vaccine.

Authors

M. Colin Jordan

Motility and Adhesiveness in Human Neutrophils: REDISTRIBUTION OF CHEMOTACTIC FACTOR-INDUCED ADHESION SITES

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Abstract

Human peripheral blood neutrophils obtained from healthy adults were examined in vitro. We assessed the effects of sequential stepwise increases in the concentration of the chemotactic dipeptide N-formyl-l-methionyl-l-phenylalanine (f-Met-Phe) on neutrophil attachment to serum-coated glass, detachment from serum-coated glass and the distribution on the cell surface of binding sites for albumin-coated latex beads. The initial exposure to f-Met-Phe resulted in increased adhesiveness and binding of latex beads in a random pattern over the cell surface. The second exposure to f-Met-Phe resulted in decreased adherence, detachment of neutrophils from serum-coated glass, and movement of binding sites for latex beads to the uropod. Enhanced adhesiveness and redistribution of binding sites were blocked by 0.1 mM N-α-p-tosyl-l-lysine chloromethyl ketone, a concentration that did not reduce the change in cellular shape caused by f-Met-Phe. Cytochalasin B (5 μg/ml) blocked the redistribution of binding sites as well as the change in shape. The third exposure to f-Met-Phe was given along with the latex beads. The stimulus was stopped after 2 min by fixing cells in suspension with glutaraldehyde. If the third exposure was at a concentration higher than the second, the beads were bound in the region of the lamellipodia in 70% of the cells. If lower, binding to the lamellipodia was found in a significantly smaller proportion of cells (13%). The results support the concept that neutrophils develop a polarized distribution of f-Met-Phe-induced adhesion sites in response to increasing concentrations of f-Met-Phe, and these sites flow from the region of the lamellipodia to the uropod.

Authors

C. Wayne Smith, James C. Hollers

Determinants of Chronic Carbon Dioxide Retention and Its Correction in Humans

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Abstract

17 patients with chronic ventilatory failure (including 14 with chronic obstructive pulmonary disease) were studied to determine the causes of carbon dioxide retention and the chronic effect of medroxyprogesterone acetate on ventilatory drive and acid-base status. Carbon dioxide retention in patients with high mechanical loads occurred concomitantly with a higher than normal inspiratory effort (mouth occlusion pressure) and normal minute ventilation to carbon dioxide production ratio (V̇e/V̇co2); but with shortened inspiratory time (1.3±0.1 vs. 1.8±3 s), increased breathing frequency (17±1 vs. 14±1 breaths/min), low tidal volume (0.57±0.03 vs. 0.88±0.04 L), and high dead space to tidal volume ratio (0.63±0.02 vs. 0.39±0.07). Using a randomized application of treatment and placebo conditions, it was shown that 4 wk of medroxyprogesterone acetate caused significant reductions in Paco2 (from 51±1 to 42±1 mm Hg) in 10 of 17 patients. This “correction” of Paco2 in these patients was associated with increases in mouth occlusion pressure (14%), tidal volume (11%), and alveolar ventilation (15%) compared to placebo, although inspiratory time remained shortened. Arterial and lumbar cerebrospinal fluid pH was alkaline compared to placebo in patients who “corrected” Paco2. No change was noted in lung mechanics or core temperature. Common prerequisites for correction of Paco2 with medroxyprogesterone acetate treatment were the ability to significantly lower Paco2 upon acute voluntary hyperventilation and to increase tidal volume rather than breathing frequency in response to the drug. We attribute chronic CO2 retention in these patients to alterations in respiratory cycle timing and to a neuromuscular inspiratory effort which is adequate for the level of tissue CO2 production, but inadequate in the presence of mechanical and ventilation-perfusion abnormalities to normalize arterial blood gases.

Authors

James B. Skatrud, Jerome A. Dempsey, Praful Bhansali, Charles Irvin

Effects of melanotropic peptides on fetal adrenal gland.

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Abstract

Adrenal glands from early, mid, and late fetuses of rabbit, guinea pig, and rat, and from newborn animals of each species, were incubated for 1-4 h with and without 0.1 nM-1 microM ACTH, alpha- or beta-melanocyte-stimulating hormone (alpha MSH or beta MSH). The effects of the peptides were measured on production of glucocorticoids, and on incorporation of labeled thymidine or leucine into DNA or protein, respectively. The findings were similar in all three species. ACTH stimulated synthesis of glucocorticoids throughout fetal life. Potency increased progressively, as reflected by declining minimal effective dose and rising maximal response. In early and mid fetus alpha MSH and beta MSH caused a modest glucocorticoid steroidogenic effect. ACTH and alpha MSH stimulated DNA and protein synthesis in the early and mid fetal gland. alpha MSH was more potent than ACTH in these respects, minimal effective dose being generally 10 times less and maximal response 25-200% greater. The effects diminished or disappeared in the late fetal and newborn gland. These data indicate that alpha- and beta MSH possess steroidogenic or growth-promoting properties, or both, for the fetal adrenal gland.

Authors

D Rudman, B M Hollins, N C Lewis, R K Chawla

Familial combined hyperlipoproteinemia. Evidence for a role of growth hormone deficiency in effecting its manifestation.

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Hyperlipidemia associated with an isolated deficiency of growth hormone was investigated in 10 subjects with hypercholesterolemia consistently present over a 10-yr period. 8 of these 10 had serum triglyceride concentrations greater than 185 mg/dl. 13 growth hormone-deficient patients with normal serum lipids, 28 age-matched controls, and 6 families possessing both growth hormone-deficient and hormonally normal members were also studied. Hyperlipidemia occurred with growth hormone deficiency only in families in which hormonally normal subjects likewise exhibited hyperlipidemia. However the elevation of serum lipids, particularly cholesterol, was invariably greater in the growth hormone-deficient members of these families. Studies were most consistent with the classification of this trait as familial combined hyperlipoproteinemia. Basal serum concentrations of insulin, glucose, and free fatty acids were similar in all groups. After oral glucose (1.5 g/kg of body wt) both hyperlipidemic and normolipidemic dwarfs exhibited a similar degree of glucose intolerance associated with insulinopenia. Sensitivity to insulin, assessed after the intravenous injection of insulin (0.05 U/kg of body wt), increased and was virtually identical in the two dwarf groups. Administration of 5 mg of human growth hormone twice a day for 1 wk to five subjects did not alter serum lipid patterns. The data provide no conclusive evidence concerning a direct effect of growth hormone deficiency on hyperlipoproteinemia. We postulate that in some individuals growth hormone deficiency may unmask an underlying defect in lipoprotein metabolism.

Authors

T J Merimee

Biphasic Adrenergic Modulation of β-Adrenergic Receptors in Man: AGONIST-INDUCED EARLY INCREMENT AND LATE DECREMENT IN β-ADRENERGIC RECEPTOR NUMBER

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Abstract

β-Adrenergic receptors in mononuclear leukocyte preparations were assessed with (−)[3H]-dihydroalprenolol binding studies during the infusion of adrenergic agonists into normal human subjects. During the infusion of isoproterenol into seven subjects, mean (±SE) (−)[3H]dihydroalprenolol binding increased from 25±3 fmol/mg protein to 47±8 fmol/mg protein (P < 0.02) at 0.5 h and 40±3 fmol/mg protein (P < 0.01) at 1 h and decreased to 12±1 fmol/mg protein (P < 0.01) at 4-6 h. During the infusion of epinephrine into three subjects, mean (−)[3H]dihydroalprenolol binding increased from 32±3 to 63±3 fmol/mg protein (P < 0.01) at 0.5-1 h. By Scatchard plot analysis, these changes were attributable to changes in the number of available binding sites rather than changes in binding affinity. The observed changes in the number of (−)[3H]dihydroalprenolol binding sites were not paralleled by changes in total mononuclear cell counts or in T lymphocyte, B lymphocyte, and monocyte distributions. Thus, we conclude that adrenergic agonists modulate the number of available β-adrenergic receptors on circulating mononuclear cells in a biphasic manner, with an early increment and a late decrement, in man. Further, the finding that the increase in pulse rate in response to a “pulse” infusion of isoproterenol was significantly greater after 0.5-1 h of agonist infusion suggests that the observed early agonist-induced increment in β-adrenergic receptor number on circulating cells is paralleled by increments in extra-vascular β-adrenergic receptor sensitivity.

Authors

Jack F. Tohmeh, Philip E. Cryer

Human Pulmonary Endothelial Cells in Culture: ACTIVITIES OF CELLS FROM ARTERIES AND CELLS FROM VEINS

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Endothelial cells were cultured from various different human vessels, including aortas, pulmonary, ovarian, and umbilical arteries, and pulmonary, ovarian, and umbilical veins. The cultured cells were identified as endothelial cells by the presence of Factor VIII antigen and antiotensin I converting enzyme (kininase II). They retained these markers for at least five passages in culture, and some cells had them for seven passages or more. Endothelial cells from the various vessels were compared with respect to their ability to metabolize angiotensins I and II and bradykinin. Cells from arteries had three to five times the angiotensin I converting enzyme activity as cells from veins. The activity of angiotensinase A (aspartyl aminopeptidase) had a similar distribution, and cells from arteries were consistently more active than cells from veins. Cultures of endothelial cells from pulmonary and umbilical vessels formed prostacyclin in response to mechanical stimulation. Media from cell monolayers that were subjected to a change of medium and gentle agitation inhibited aggregation of human platelets. This inhibitory activity was generated within 2-5 min, and it was not formed by cells that were treated with indomethacin or tranylcypromine. Addition of prostaglandin (PG)H2 to indomethacin-treated cells restored the ability to form the inhibitor, but cells treated with tranylcypromine were not responsive to PGH2. In experiments where [14C]arachidonic acid was added to the cells before stimulation, the major metabolite identified by thin-layer chromatography was 6-keto PGF1α. Thus, it appears that pulmonary endothelial cells, as well as umbilical cord cells, can form prostacyclin. In experiments comparing the ability of arterial and venous cells to form prostacyclin, arterial cells were more active than venous cells. These studies of cells from various human vessels suggest that the vascular origin of cultured endothelial cells determines how they metabolize vasoactive peptides and form prostacyclin.

Authors

Alice R. Johnson

Reversal of Ethinyl Estradiol-induced Bile Secretory Failure with Triton WR-1339

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Abstract

The effects of Triton WR-1339 and phenobarbital on ethinyl estradiol bile secretory failure were examined to determine the mechanism responsible for decreased bile salt excretion. When administered to ethinyl estradiol-treated rats, Triton WR-1339 restored bile salt independent bile flow and maximum taurocholate transport, whereas phenobarbital corrected bile flow only. Ethinyl estradiol decreased the activities of Na+-K+-ATPase, 5′-nucleotidase, while increasing the activities of Mg++-ATPase and alkaline phosphatase. In contrast to these heterogeneous changes in surface membrane enzyme activities, the number and affinity of [14C]cholic acid carriers were not altered. When administered in vivo or added directly to surface membrane fractions Triton WR-1339 restored the activities of Na+-K+-ATPase and Mg++-ATPase of rats treated with ethinyl estradiol through a process that did not require protein synthesis (unaffected by cycloheximide). Phenobarbital also restored the activity of Na+-K+-ATPase to control levels, but, unlike Triton WR-1339 it did not correct the defect responsible for reduced bile salt secretion. Ethinyl estradiol increased the concentration of cholesterol esters in surface membrane fractions. When administered to ethinyl estradiol-treated rats, Triton WR-1339 restored cholesterol ester concentrations to normal, whereas phenobarbital did not. These combined data suggest that decreased or altered bile salt carriers or reduced sodium driving forces resulting from impaired activity of Na+-K+-ATPase are not responsible for decreased bile salt excretion in ethinyl estradiol-treated rats. It is proposed that the diverse changes in surface membrane function, which are associated with ethinyl estradiol bile secretory failure, may be the result of a generalized alteration in membrane lipid structure.

Authors

Francis R. Simon, Manuel Gonzalez, Eileen Sutherland, Luigi Accatino, Roger A. Davis

Enhanced Contractile Response and Protein Kinase Activation to Threshold Levels of β-Adrenergic Stimulation in Hyperthyroid Rat Heart

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The contractile response measured as maximum rate of force development to a near threshold concentration of isoproterenol (1 nM) was enhanced in perfused interventricular septa from hyperthyroid (128±4% control) compared with euthyroid rats (105±2%, P < 0.01). This enhanced contractile response was accompanied by a significant activation of cyclic (c)AMP-dependent protein kinase (protein kinase activity ratio increased from 0.159±0.008 to 0.218±0.019, P < 0.005, although no significant changes from base line occurred in euthyroid septa, 0.152±0.007-0.179±0.012). No difference between hyperthyroid and euthyroid hearts was observed in the contractile response to 0.1 mM dibutyryl cAMP (126.5±2.5% and 122.0±9.2% in hyperthyroid and euthyroid, respectively), and the magnitude of the response to dibutyryl cAMP was comparable with that observed in the hyperthyroid group with 1 nM isoproterenol. These results suggest that the mechanism for enhanced protein kinase activation and contractile response to low concentrations of isoproterenol in the hyperthyroid heart is at or proximal to cAMP generation. The maximum contractile response to isoproterenol (0.5 μM), however, was decreased in hyperthyroid myocardium (192±13%) compared with euthyroid (291±37%, P < 0.05). Both protein kinase activity ratio (0.356±0.017 and 0.344±0.013) and the maximum contractile response to Ca++ (335±15 and 340±12% control in hyperthyroid and euthyroid, respectively) were similar, suggesting that the mechanism of the diminished maximum response was distal to protein kinase activation but not a function of an altered Ca++-troponin interaction. The diminished maximum rate of force development response in the hyperthyroid hearts was accompanied by significantly less shortening of the contraction duration that was 85.6±2.1% control in hyperthyroid vs. 66±2.8% control in euthyroid, P < 0.001. Although the basal rate of Ca++ accumulation was greater in microsomes isolated from hyperthyroid than from euthyroid hearts, there was significantly less additional stimulation of Ca++ accumulation in response to exogenous cAMP and protein kinase in hyperthyroid compared with euthyroid hearts. This reduction may explain the diminished effect of isoproterenol on the shortening of contraction duration in hyperthyroid compared with the euthyroid myocardium, and may explain, at least in part, the diminished maximum contractile response to isoproterenol.

Authors

Thomas Guarnieri, Charles R. Filburn, Elsie S. Beard, Edward G. Lakatta

Cellular and Humoral Immunity in the Pathogenesis of Recurrent Herpes Viral Infections in Patients with Lymphoma

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Abstract

86 patients with lymphoma were evaluated prospectively for clinical and laboratory evidence of recurrent varicella-zoster, herpes simplex, and cytomegalovirus infections during the first 16 mo of treatment. Cellular immunity to the viral antigens was measured by in vitro lymphocyte transformation and interferon production. Antibody titers and nonspecific measures of cellular immunity, including T-cell quantitation and transformation to phytohemagglutinin, were also assessed. The patients treated with radiation and chemotherapy had the highest incidence of reactivation of each of the viruses (15-19%). Greater susceptibility to herpes viral reactivation in these patients correlated with suppression of cell-mediated immunity to the specific virus. In individual patients, suppression of cellular immunity to the specific herpes viral antigen preceded each episode of reactivation, but recurrent infection did not occur in all patients with diminished specific lymphocyte transformation. Absence of the response appears to be a necessary but not a sufficient condition for the recrudescence of latent infection. Better preservation of cellular immunity to herpes simplex antigen during treatment was associated with infrequent reactivation of herpes simplex. In 25 patients with acute herpes zoster, uncomplicated recovery from the infection was accompanied by the development of lymphocyte transformation and interferon production to varicella-zoster antigen. Quantitation of T-cell numbers and phytohemagglutinin transformation did not correlate with the presence of viral cellular immunity in treated patients. Responses returned while T-cell numbers were low, and the recovery of phytohemagglutinin transformation often preceded recovery of the responses to viral antigens. Although some patients had deficiencies in viral cellular immunity at diagnosis, the duration of the suppression of specific antiviral responses resulting from treatment appears to be the most important factor predisposing to the recurrence of herpes infections in lymphoma patients.

Authors

A. M. Arvin, R. B. Pollard, L. E. Rasmussen, T. C. Merigan

Selective noncompetitive assimilation of bovine testicular beta-galactosidase and bovine liver beta-glucuronidase by generalized gangliosidosis fibroblasts.

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Bovine liver beta-glucuronidase and testicular beta-galactosidase were assimilated by generalized gangliosidosis fibroblasts at respectively rates of 90 and 464 times the rate of assimilation of horseradish peroxidase. Assimilation of either of the two enzymes by the fibroblasts was saturable, suggesting the participation of receptor-mediated adsorptive endocytosis for internalization. The rate of assimilation of either enzyme was not affected by high levels of the other enzyme, suggesting that distinct receptors for each enzyme occur on the fibroblasts' cell surface. Furthermore, although assimilation of beta-galactosidase was inhibited by mannose, methyl mannosides, mannosyl alpha 1 leads to 2 mannose, and mannose-6-phosphate, these compounds did not detectably inhibit the assimilation of beta-glucuronidase. These results suggest that testicular beta-galactosidase was assimilated by the well-established phosphomannosyl recognition system. However, liver beta-glucuronidase was assimilated by a distinct, noncompeting, and as yet undefined, recognition system.

Authors

V Hieber, J Distler, R Myerowitz, R D Schmickel, G W Jourdian

Phenotypic and Genetic Variation in the Susceptibility of Haemophilus influenzae Type b to Antibodies to Somatic Antigens

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Abstract

Haemophilus influenzae type b (H.i.b) has been investigated with respect to phenotypic and genetic variations resulting in differential susceptibility to bactericidal antibody. Previous studies had shown that after growth in infected rats or in dialysate of rat serum, H.i.b strain Eag became more resistant to the bactericidal activity of antisomatic antibody. We now report that a similar phenotypic shift occurs when strain Eag is incubated with dialysate of human serum, that the increased resistance is to antibodies against determinants in the lipopolysaccharide not for the somatic antigens generally, and that most strains of H.i.b undergo the shift.

Authors

Porter Anderson, Alan Flesher, Stephen Shaw, A. Lynn Harding, David H. Smith

Calcitonin secretion in congenital nongoitrous cretinism.

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Plasma calcitonin (CT) was measured in the basal state and/or during provocative tests of hormone secretion in 11 children with congenital non-goitrous cretinism (CNC), in 1 girl with a lingual thyroid, and in 11 normal children. Basal and stimulated CT concentrations were significantly lower in the patients with CNC than in the normal subjects. Mean basal CT (+/- SE) was 41 +/- 4 pg/ml in the normal children, 24 +/- 3 pg/ml in the children with CNC, and 20 +/- 2 pg/ml in the patient with the lingual thyroid. The mean incremental CT responses to calcium infusion were 7.0 +/- 2 pg/ml in the children with CNC, 6.0 pg/ml in the patient with the lingual thyroid, and 146 +/- 47 pg/ml in the normal children. The children with CNC also demonstrated a significant delay in the return of the total serum calcium to basal level after the calcium infusion. The mean incremental CT response after infusion of pentagastrin was 7.6 +/- 2 pg/ml in the children with CNC, 10.0 pg/ml in the child with the lingual thyroid, and 34.4 +/- 11 pg/ml in the normal children. These data indicate that CT deficiency is present in children with CNC and suggest that the deficiency is a consequence of the defective embryologic development of the thyroid gland.

Authors

D E Carey, K L Jones, J G Parthemore, L J Deftos

Nonenzymatic glycosylation of erythrocyte membrane proteins. Relevance to diabetes.

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Nonenzymatic glycosylation of proteins of the erythrocyte membrane was determined by incubating erythrocyte ghosts with [3H]borohydride. The incorporation of tritium into protein provides a reliable assay of ketoamine linkages. The membrane proteins from 18 patients with diabetes incorporated twice as much radioactivity as membrane proteins from normal erythrocytes. After acid hydrolysis, amino acid analysis showed that the majority of radioactivity was localized to glucosyllysine. Autoradiograms showed that all of the major proteins of the erythrocyte membrane, separated by electrophoresis on sodium dodecyl sulfate gels, contained ketoamine linkages. No protein bands in either normal or diabetic erythrocytes showed significant preferential labeling. Erythrocyte membranes from three patients with hemolytic anemia showed reduced incorporation of tritium from [3H]-borohydride, indicating decreased nonenzymatic glycosylation. Two patients with diabetes and hemolytic anemia had incorporation of radioactivity similar to that of normal individuals. In these groups of patients the incorporation of tritium into erythrocyte membrane proteins correlated with levels of hemoglobin AIc. Thus the modification of membrane proteins like that of hemoglobin depends on blood glucose levels as well as erythrocyte age. These studies show that the enhanced nonenzymatic glycosylation of proteins in diabetics extends beyond hemoglobin to the proteins of the erythrocyte membrane and probably affects other proteins that have slow turnover and are exposed to high concentrations of glucose.

Authors

J A Miller, E Gravallese, H F Bunn

Consequences of Ventromedial Hypothalamic Lesions upon Insulin and Glucagon Secretion by Subsequently Isolated Perfused Pancreases in the Rat

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The existence of a relationship between the ventromedial hypothalamic area (VMH) and the activity of the endocrine pancreas has been shown previously. This relationship has been further tested and extended in the present study, using isolated perfused pancreases from rats previously lesioned (4-7 d) in the VMH. It was found that in isolated pancreases obtained from rats fed ad lib. for 4 d after VMH lesions (i.e., that were hyperphagic), the typical biphasic pattern of insulin secretion was observed following glucose stimulation (20 mM) and that the total insulin output was much greater than that of controls. The increased insulin output was not a result of hyperphagia because similar results were obtained using pancreases obtained from VMH-lesioned rats in which a food restriction matching exactly that of control rats was started either immediately of 3 d after the lesions. Pancreases from such food-restricted VMH-lesioned rats oversecreted insulin, when compared with controls fed the same amount, from 7 mM of glucose concentration in perfusion medium onwards. After the addition of arginine (10 mM), the total output of glucagon by pancreases from food-restricted VMH-lesioned rats was twice that of controls. Qualitatively, the arginine-induced glucagon secretion by pancreases from food-restricted VMH-lesioned rats retained its biphasic pattern. Similarly, epinephrine (0.1 μM) elicited a greater glucagon release by pancreases from food-restricted VMH-lesioned rats when compared with controls. These data further support the concept of a link (as yet undefined) between the hypothalamus and the endocrine pancreas, as lesions of the VMH area resulted in abnormal secretion not only of insulin, but of glucagon as well.

Authors

Françoise Rohner-Jeanrenaud, Bernard Jeanrenaud

Human Apolipoprotein A-IV: INTESTINAL ORIGIN AND DISTRIBUTION IN PLASMA

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The role of the human intestine has been explored as a site of synthesis of apoA-IV, a major apoprotein of human intestinal triglyceride-rich lipoproteins. Intestinal biopsies were performed on normal volunteers while fasting and after lipid ingestion. Indirect immunofluorescence demonstrated a marked increase in immunofluorescence for apoA-IV during lipid absorption consistent with an increased intracellular content. ApoA-IV comprised 10-13% of chylomicron apoprotein and 24-30% of intestinal very low density lipoprotein (VLDL) as assessed by densitometry of sodium dodecyl sulfate gels of lipoproteins from chylous urine (mesenteric lymphatic-urinary fistula) and thoracic duct lymph (postoperative fistula). After one subject with chyluria ingested 40 g of corn oil, triglyceride excretion in urine was accompanied by an increased excretion of apoA-IV. 11.5 g of triglyceride and 81 mg of apoA-IV were recovered in the urine. In chylous urine 56% of apoA-IV was in the triglyceride-rich lipoproteins (chylomicrons and intestinal VLDL) and 44% in the d > 1.006-g/ml fraction.

Authors

Peter H. R. Green, Robert M. Glickman, John W. Riley, Elaine Quinet

Cholesterol reduces the effects of dihydroxy bile acids and fatty acids on water and solute transport in the human jejunum.

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Jejunal perfusion studies were performed in 16 healthy volunteers to test the hypothesis that intraluminal cholesterol can mitigate the fluid secretion induced by dihydroxy bile acids and fatty acids. Fluid secretion in the presence of 5 mM taurodeoxycholate was somewhat reduced by 4 mM mono-olein which was used for the solubilization of cholesterol. Addition of 0.8 mM cholesterol reduced fluid secretion further (P less than 0.05). Fluid secretion induced by 4 mM oleic acid was changed to net absorption in a linear fashion with increasing cholesterol concentration in the perfusion solutions. 1 mM cholesterol reduced fluid secretion induced by 6 mM oleic acid (P less than 0.005), but had no effect on fluid secretion induced by 6 mM linolenic acid. Glucose absorption was generally affected in a similar manner as water transport. In vitro, 1 mM cholesterol reduced monomer activity of 6 mM oleic acid to 72.3 +/- 0.9% of control and that of linolenic acid to 81.1 +/- 1.7% of control. Although statistically significant (P less than 0.001), the difference in the effects of cholesterol on monomer activities of the two fatty acids was rather small and it is unlikely that changes in monomer concentration of fatty acids and bile acids account for the protective effect of cholesterol. The in vivo observations point to a new physiological role for biliary cholesterol: the modification of the response of the small intestine to the effects of dihydroxy bile acids and fatty acids.

Authors

S L Broor, T Slota, H V Ammon

Origin of platelet-derived growth factor in megakaryocytes in guinea pigs.

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Growth factor activity, as determined by the stimulation of [3H]thymidine incorporation into the DNA of quiescent 3T3 cells in culture, was found in lysates of guinea pig platelets and megakaryocytes. Quantitative dilution studies demonstrated that, of the cells present in the guinea pig bone marrow, only the megakaryocyte possessed quantitatively significant growth factor activity. The amount of activity present in one megakaryocyte was equivalent to that present in 1,000-5,000 platelets, a value approximately comparable to the number of platelets shed from a single megakaryocyte. It is suggested that guinea pig platelet-derived growth factor has its origin in the megakaryocyte.

Authors

A Chernoff, R F Levine, D S Goodman

pH gradient-stimulated transport of urate and p-aminohippurate in dog renal microvillus membrane vesicles.

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Abstract

The transport mechanism of urate and p-aminohippurate (PAH) was evaluated in microvillus membrane vesicles isolated from the renal cortex of the mongrel dog. Imposition of a transmembrane pH gradient (pHo less than pH1) markedly accelerated the uptake of [14C]urate and [3H]PAH and caused the transient accumulation ("overshoot") of each anion above its final level of uptake. The transport of urate and PAH under both stimulated (pHo less than pHi) and basal (pHo = pHi) conditions was insensitive to valinomycin-induced K+ diffusion potentials. The pH gradient-stimulated uptake of 25 microM [14C]urate and 1.0 microM [3H]PAH was significantly inhibited by 1.2 mM PAH, urate, furosemide, salicylate, or probenecid. The effect of each inhibitor on [14C]urate transport was identical to the effect of the same inhibitor on [3H]PAH flux. We conclude that the transport of urate and PAH in dog renal microvillus membrane vesicles occurs via a pH gradient-stimulated electroneutral carrier-mediated process such as 1:1 H+-anion cotransport or OH-anion exchange. Such a transport mechanism may possibly play a role in effecting uphill urate reabsorption in the proximal tubule. Moreover, this study demonstrates that secondary active solute transport in epithelial membranes may be coupled to the electrochemical gradient of an ion other than Na+.

Authors

J W Blomstedt, P S Aronson

Rapid Thyroxine to 3,5,3′-Triiodothyronine Conversion and Nuclear 3,5,3′-Triiodothyronine Binding in Rat Cerebral Cortex and Cerebellum

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Abstract

Thyroxine (T4) to 3,5,3′-triiodothyronine (T3) conversion was evaluated in vivo in cerebral cortex, cerebellum, and anterior pituitary of male euthyroid Sprague-Dawley rats. Tracer quantities of 125I-T4 and 131I-T3 were injected into controls and iopanoic acid-pretreated rats 3 h before isolation of nuclei from these tissues. Specifically-bound nuclear 131I-T3, denoted T3(T3); 125I-T3, denoted T3(T4); and 125I-T4 were extracted and identified by chromatography. Plasma iodothyronines were similarly quantitated. In control rats, nuclear T3(T3) (percent dose per milligram DNA × 10−4) was 174±31 in cerebral cortex, 50±9 in cerebellum, and 932±158 in pituitary (all values, mean±SEM). Nuclear T3(T4) (percent dose per milligram DNA × 10−4) was 23.3±3.3 in cortex, 3.5±0.6 in cerebellum, and 39.4±6.9 in pituitary. Two-thirds of nuclear T3(T4) derived from local T4 to T3 conversion. Nuclear T3(T4) in all tissues was reduced to less than 15% of its control value by iopanoic acid treatment and all of the residual nuclear T3(T4) could be accounted for by plasma T3(T4). Nuclear T3(T3) binding was not inhibited by iopanoic acid. These results indicate there is rapid local T4 to T3 conversion in rat brain and nuclear binding of the T3 produced. We have previously found that local T3(T4) production is the source of ∼50% of the T3 in rat anterior pituitary. The present observations that the ratio of locally derived nuclear T3(T4) to nuclear T3(T3) is much higher in cerebral cortex (0.1) and cerebellum (0.04) than in anterior pituitary (0.015) suggest that this locally produced T3(T4) is the predominant source of intracellular T3 in these portions of rat brain.

Authors

F. R. Crantz, P. R. Larsen

Sustained Oscillations of Insulin, Glucagon, and Somatostatin from the Isolated Canine Pancreas during Exposure to a Constant Glucose Concentration

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Abstract

Canine pancreata were perfused in vitro to examine whether hormone cycles could be demonstrated without hepatic or central nervous influence. Insulin, glucagon, and somatostatin demonstrated regular sustained cyclic secretion from the in vitro canine pancreas. Oscillations were noted for over 200 min during the infusion of a constant glucose concentration. Insulin demonstrated a 10-min period with a range of 8-12 min/cycle. Somatostatin had a 10-min period with a range of 8-11 min. Glucagon had a period of 8.6 min with range of 6-10 min. These periods do not allow glucagon to be consistently 90° out of phase with insulin and somatostatin.

Authors

John I. Stagner, Ellis Samols, Gordon C. Weir

Dietary Modification of Thyroxine Deiodination in Rat Liver is Not Mediated by Hepatic Sulfhydryls

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Abstract

The enzymatic deiodination of thyroxine (T4) is thiol dependent. Fasting (72 h) depresses hepatic T4 deiodination and lowers the hepatic content of nonprotein sulfhydryls (NP-SH) and reduced glutathione (GSH). It has been proposed that the fasting effect may be mediated through these alterations in hepatic sulfhydryls. To test the importance of tissue (hepatic) thiol content in the modification of T4 deiodination consequent to dietary manipulation, we examined the sequential deiodination of T4 to 3,5,3′-triiodothyronine (T3) (5′-deiodination) and 3,3′,5-triiodothyronine (reverse T3, rT3) (5-deiodination) in liver homogenates without added thiol from groups of rats fed Purina lab chow (P) (a protein-rich diet), glucose alone (G), or glucose plus cysteine (Gc) for 72 h or fasted (F) for the same period. The initial rate of each reaction was compared to the tissue concentrations of NP-SH and GSH.

Authors

Laurence A. Gavin, Francis A. McMahon, M. Moeller