Martin Sonenberg

Stimulus-secretion coupling in isolated adrenal chromaffin cells: calcium channel activation and possible role of cytoskeletal elements.

Abstract: The catecholamine secretory function of a preparation of isolated bovine adrenal chromaffin cells has been further characterized under conditions designed to elucidate the mechanism of calcium channel activation and the possible role of cytoskeletal elements in stimulus‐secretion coupling. Three related sets of data were obtained: (1) Differences in kinetics, Ca dependence, strength, and additivity of the secretory response to acetylcholine (ACh) versus excess K; (2) the effects on secretion of the Ca channel‐blocking agents, Ni, Mg, and verapamil; and (3) the Ca dependence of vinblastine action on ACh‐ and K‐evoked secretion. The results suggest that a major portion of the Ca influx required for catecholamine release enters the cell via voltage‐dependent Ca channels with some additional Ca influx via the ACh receptor channel. Comparison of the present secretion data with corresponding known electrophysiological properties of isolated chromaffin cells provides added evidence for a role of chromaffin cell action potentials in regulation of Ca influx and the secretory response. Elevated Ca concentrations enhanced K‐evoked secretion to levels comparable to that of ACh but did not induce a vinblastine block of K‐evoked release. This provides further evidence against a role of microtubules in the common exocytosis event per se. However, a role of cytoskeletal elements in directing the movement of secretory granules, or an action of vinblastine at cholinergic receptors, remain distinct possibilities.

Urinary Hydroxyproline and Calcium Metabolism in Patients with Cancer

IN the past few years an increasing interest has developed in the determination of urinary hydroxyproline as an index of the metabolism and turnover of collagen. Recent experimental1 , 2 and clinical studies3 4 5 6 7 8 9 10 11 12 13 14 15 16 performed in normal as well as in many endocrine, metabolic and bone diseases have indicated that bone collagen is the major source of this amino acid in urine. The present investigation was undertaken to study the excretion of total urinary hydroxyproline in patients with inoperable or widespread carcinoma, especially those with bone metastases or hypercalcemia or both. Our specific aim was to observe what relation exists between urinary excretion .xa0.xa0.

Modification by bovine growth hormone of liver plasma membrane enzymes, phospholipids, and circular dichroism☆

Abstract Liver plasma membrane phospholipid distribution, protein conformation, and 5′-nucleotidase, Mg 2+ -adenosine triphosphatase and (Na + + K + )-adenosine triphosphatase specific activities, were shown to depend on pituitary status and treatment with bovine growth hormone. In whole liver homogenates, hypophysectomy produced a decrease in the proportion of phosphatidyl serine, lysophosphatidyl choline, and phosphatidic acid and diphosphatidyl glycerol and an increased proportion of phosphatidyl ethanolamine. The phospholipid distribution in liver plasma membranes was the same for normal and hypophysectomized rats. Plasma membranes obtained from bovine growth hormone-treated hypophysectomized rats had approximately 50%, more phosphatidyl serine than membranes obtained from untreated hypophysectomized or normal rats. Plasma membranes from hypophysectomized rats had 75% of the 5′-nucleotidase, the same level of (Na + + K + )-adenosine triphosphatase, and twice the Mg 2+ -adenosine triphosphatase of membranes from normal rats. Twelve hours after administration of bovine growth hormone to hypophysectomized rats, (Na + + K + )-adenosine triphosphatase had almost doubled and Mg 2+ -adenosine triphosphatase decreased by 50%. 5′-Nucleotidase remained unchanged. Twenty-four hours after bovine growth hormone administration, both (Na + + K + )-adenosine triphosphatase and 5′-nucleotidase had increased. Mg 2+ -adenosine triphosphatase was 23% of the baseline level of untreated hypophysectomized rats. Treatment for 3 days or 5 days increased the 5′-nucleotidase 2-fold. Circular dichroism spectra of liver plasma membranes isolated from hypophysectomized rats consistently showed greater negative ellipticity in the far ultraviolet range (250-190 nm) than those from normal rats or rats treated with bovine growth hormone.

Alteration of liver plasma membrane protein conformation by bovine growth hormone in vitro

Abstract The response of liver plasma membranes prepared from hypophysectomized, bovine growth hormone-treated hypophysectomized, and normal rats to addition of bovine growth hormone in vitro were studied by circular dichroism and spectrofluorometry. Membranes of hypophysectomized but not normal or treated rats showed increased negative ellipticity in the presence of bovine growth hormone (10 −9 ) without any change in trough position; at higher concentrations (10 −7 −10 −6 , m ) there was less negative ellipticity. Membranes of hypophysectomized, normal, and growth hormone-treated rats all showed decreased emission of fluorescence and a small shift of the emission peak from 333 to 338 nm in the presence of bovine growth hormone (10 −17 −10 −7 , m ). The excitation of fluorescence was quenched by bovine growth hormone. Polarization of excitation of fluorescence was unchanged.

The effect of altered transmembrane ion gradients on membrane potential and aggregation of human platelets in blood plasma

Abstract The resting membrane potential of platelets suspended in blood plasma buffer mixture measured using tritiated triphenylmethylphosphonium was 48 ± 13 mV. Increases in extracellular potassium concentration caused depolarization of the platelet membrane. Ouabain (1.0 μM) produced a depolarization of 13 ± 5 mV. Depolarization with elevated extracellular potassium or ouabain potentiated the aggregating effects of adenosine diphosphate even in the presence of methysergide suggesting that the effects are not mediated through ion linked serotonin transport. These observations are consistent with the suggestion that transmembrane ion gradients and membrane potential can regulate platelet sensitivity to the aggregating agent adenosine diphosphate.

Decreased Incidence of Restraint-Stress Induced Gastric Erosions in Rats Treated with Bovine Growth Hormone

Summary Restraint-stress was induced in female CFE rats weighing between 150 and 200 g by stapling them in envelopes made of aluminum wire screening. Five mg of bovine growth hormone dissolved in 1 ml of buffer was administered subcutaneously immediately after stapling and again 10 hr later. Controls received injections of buffer or bovine serum albumin. The animals were sacrificed at 24 hr and the number of animals developing gastric erosions, the number per animal and the severity of erosions were determined with a dissecting microscope. Growth hormone provides partial but significant protection against the production of restraint-stress produced erosions, both in terms of incidence of erosions and their severity.

Growth hormone activity in man with components of tryptic digests of bovine growth hormone

Abstract Fractions of tryptic digests of bovine growth hormone previously demonstrated to have human growth hormone-like activity in hypopituitary humans were administered to hypopituitary human subjects, and effects on nitrogen metabolism similar to those of human growth hormone were demonstrated with five of the preparations. The nitrogen retaining property of the tryptic digests of bovine growth hormone fractions seems best correlated with components of intermediate mobility, as characterized by starch gel electrophoresis at pH 9.5. Four of the five preparations produced clinical side effects associated with human growth hormone administration.

Effects of gossypol on PAH transport in the rabbit kidney slice

The present work was carried out to investigate if gossypol, a toxic weak organic acid (pK = 7.2) contained in cottonseed, is secreted by the renal proximal tubule through the organic anion transport system in the rabbit. The slice uptake of p-aminohippurate (PAH), a prototypical organic anion, was significantly inhibited (by 30%) only when the medium concentration of gossypol was raised to 10(-4) M. However, gossypol at the latter concentration also induced a 30% inhibition of the slice uptake of tetraethylammonium (TEA), a prototypical organic cation. Moreover, gossypol at 10(-4) M significantly decreased the slice oxygen consumption (congruent to 30%) and Mg-ATPase (70%) and Na-K-ATPase (90%) activities of renal cortical microsomes, while it significantly decreased the intracellular (K+). These results indicate that gossypol inhibits PAH uptake through nonspecific nephrotoxic effects on cell metabolism and Na-K-ATPase activity rather than through its specific interaction with the organic anion transport system.

Growth hormone-induced alteration of morphology and tubulin expression in 3T3 preadipose cells

Effects of growth hormone on morphology and cytoskeletal protein expression were examined in 3T3-F442A preadipocytes in serum-free medium. Between 2 and 5 days of culture 2 nM methionyl human growth hormone converted 3T3-F442A cells from a flat fibroblastic morphology to a rounded form with numerous membrane convolutions. Growth hormone treated cultures manifested a 30-40% reduction in cell volume. Growth hormone induced changes in morphology and volume preceded and were independent of lipogenesis. In cells treated with growth hormone, expression of alpha and beta-tubulin as determined by Western blotting was found to increase approximately 50% within 72 h as compared to untreated cells. After 7 days, tubulin levels in growth hormone treated cells were approximately 40% of control levels. This indicated that morphological changes and alteration of tubulin expression were signatures of growth hormone action on 3T3-F442A cells.

Activity of growth hormone peptides bGH 96–133 and hGH 95–133 in 3T3-F442A cells

Chemically synthesized bovine growth hormone (bGH) bGH 96-133 and its human homologue, hGH 95-133, have similar in vitro biological activities. Unlike native GH, bGH 96-133 and hGH 95-133 were completely without adipogenic or anti-insulin activity at doses up to 10 microM. bGH 96-133 had insulin-like activity, with a 100% increase in glucose uptake at 10 microM. bGH was anti-mitogenic and bGH 96-133 and hGH 95-133 were mitogenic (EC50 approximately 180 nM and maximal response at 1-2 microM). Only bGH 96-133 and hGH 95-133 displaced [125I]hGH 95-133 binding from 3T3-F442A fibroblasts with a Kd between 60-120 nM. bGH, hGH, insulin and IGF-I were without effect on [125I]hGH 95-133 binding. bGH 96-133 and hGH 95-133 did not significantly inhibit [125I]hGH or [125I]IGF-I binding. These experiments indicate that GH containing peptides bGH 96-133 and hGH 95-133 have mitogenic and insulin-like activity without the adipogenic, anti-insulin or anti-mitogenic activity of bGH. These peptides have a specific binding site which appears to be distinct from the GH, insulin and IGF-I receptors.