Laurence S. Jacobs

The regulation of insulin-induced and sleep-related human growth hormone secretion: A review

Abstract (1) A series of studies evaluating pharmacological alterations of insulin-induced and sleep-related GH secretion are presented. (2) In several cases, the effects of the same pharmacological agent on the two forms of secretion were very different. (3) It may be, then, that the neurotransmitter pathways which mediate the GH response to insulin differ from those that regulate sleep-related secretion.

Pharmacological evidence for opioid and adrenergic mechanisms controlling growth hormone, prolactin, pancreatic polypeptide, and catecholamine levels in humans☆

A group of 14 healthy subjects received 50 mg/kg body weight of 2 deoxy-D-glucose (2DG) IV in a 20-minute infusion to induce glucoprivation and stimulate the release of growth hormone (GH), prolactin (PRL), pancreatic polypeptide (hPP), and catecholamines. Six subjects having spontaneously high GH baseline levels (greater than 8 ng/mL) failed to mount a GH response to 2DG-induced glucoprivation while eight subjects having low GH baseline levels (less than 8 ng/mL) all had increases (greater than 10 ng/mL) of GH levels after 2DG (P less than 0.05). Baseline level of GH was a reliable predictor of subsequent GH response to 2DG. Administration of the alpha 2-adrenoreceptor agonist clonidine (0.5 mg po) reliably increased GH levels (P less than 0.05). Elevated GH levels following clonidine administration abolished GH responses to subsequently infused 2DG (P less than 0.05). While these data do not exclude the possibility of a short loop feedback control of GH secretion, they strongly suggest that the direction of the GH response to a provocative stimulus is determined by the antecedent GH level and that an alpha-adrenoreceptor mechanism is involved in such a biphasic modulation of GH levels. Clonidine administration significantly reduced total catecholamine, pancreatic polypeptide, and prolactin response to 2DG while opiate receptor blockade with naloxone (10 mg IV bolus followed by 2 mg/hr) did not affect catecholamine and pancreatic polypeptide response but did slightly attenuate the GH and PRL response to glucoprivation. We conclude that alpha adrenoreceptor mechanisms are of major importance while opiate receptor mechanisms are of relatively minor importance in modulating the effects of glucoprivation on sympathetic outflow and hPP, GH, and PRL levels.

Distribution of calmodulin and calmodulin-binding proteins in bovine pituitary: association of myosin light chain kinase with pituitary secretory granule membranes.

Calcium is necessary for secretion of pituitary hormones. Many of the biological effects of Ca2+ are mediated by the Ca2+-binding protein calmodulin (CaM), which interacts specifically with proteins regulated by the Ca2+-CaM complex. One of these proteins is myosin light chain kinase (MLCK), a Ca2+-calmodulin dependent enzyme that phosphorylates the regulatory light chains of myosin, and has been implicated in motile processes in both muscle and non-muscle tissues. We determined the content and distribution of CaM and CaM-binding proteins in bovine pituitary homogenates, and subcellular fractions including secretory granules and secretory granule membranes. CaM measured by radioimmunoassay was found in each fraction; although approximately one-half was in the cytosolic fraction, CaM was also associated with the plasma membrane and secretory granule fractions. CaM-binding proteins were identified by an 251-CaM gel overlay technique and quantitated by densitometric analysis of the autoradiograms. Pituitary homogenates contained nine major CaM-binding proteins of 146, 131, 90, 64, 58, 56, 52, 31 and 22 kilodaltons (kDa). Binding to all the bands was specific, Cat+-sensitive, and displaceable with excess unlabeled CaM. Severe heat treatment (100°C, 15 min), which results in a 75% reduction in phosphodiesterase activation by CaM, markedly decreased 251I-CaM binding to all protein bands. Secretory granule membranes showed enhancement for CaM-binding proteins with molecular weights of 184, 146, 131, 90, and 52000. A specific, affinity purified antibody to chicken gizzard MLCK bound to the 146 kDa band in homogenates, centrifugal subcellular fractions, and secretory granule membranes. No such binding was associated with the granule contents. The enrichment of MLCK and other CaM-binding proteins in pituitary secretory granule membranes suggests a possible role for CaM and/or CaM-binding proteins in granule membrane function and possibly exocytosis.

Inhibitor studies with adenohypophyseal granule membrane ATPase. Evidence for a membrane environment which modulates sensitivity to inhibitors.

The limiting membranes of pituitary growth hormone and prolactin secretory granules contain a Mg2+-ATPase sensitive to anions. This enzyme is in many ways similar to mitochondrial ATPase. The enzyme was potently inhibited by oligomycin (Ki 6.5 X 10(-9) M), and was much more sensitive to the inhibitor than pituitary mitochondrial ATPase (Ki 2.7 X 10(-7) M). In contrast, the enzyme activity of intact secretory granules was only sparingly inhibited by oligomycin (maximal inhibition close to 30% at 5 X 10(-4) M). However, oligomycin (5 microM) did diminish to basal levels the enhanced granule ATPase activity observed in the presence of a stimulatory anion (25 mM sodium sulfite). Other compounds known to inhibit the proton translocating mitochondrial ATPase were also tested for their ability to inhibit the secretory granule ATPase. A similar pattern of limited inhibition in granules and greater sensitivity in isolated membranes was seen with the inhibitors N,N-dicyclohexylcarbodiimide and efrapeptin. In contrast, tri-n-butyltin chloride was a potent inhibitor of the ATPase of intact granules, and the susceptibility of the enzyme to inhibition by this compound was less after isolation of membranes. These observations suggest that pituitary secretory granule membrane ATPase may have a proton pumping function similar to that of the mitochondrial enzyme. In addition, the data imply that the inhibitor binding site(s) may be masked, inaccessible, or ineffective in intact granules, but exposed (or activated) in isolated membranes. The greater sensitivity of granule ATPase to tri-n-butyltin chloride, in contrast to the greater sensitivity of membrane ATPase to the other inhibitors, indicates that the tin compound may be effective at a membrane site(s) distinct from the others, or that the mechanism of inhibition is different.

Regulation of hormonal and secretory granule membrane disulfides by adenohypophysial gluthion: disulfide oxidoreductase

Abstract An NADPH-dependent glutathione: disulfide oxidoreductase (thiol-transferase) has been identified in and partially purified (12.3-fold) from adenohypophysial cytosol. The enzyme is specific for NADPH and reduced glutatione, but the disulfide substrates include a wide size range (glutathione, cystine, RNase, oxytocin, vasopressin, monomeric and oligomeric growth hormone and prolactin). It also utilizes secretory granule membrane proteins. Substrate specificity studies (including utilization of cystine and failure to utilize insulin) and physico-chemical properties (M.W. 180,000) distinguish this enzyme from other glutathione: disulfide oxidoreductases. This thioltransferase may play a regulatory role in the hormone secretory process by control of the thiol: disulfide oxidation state of disulfide-bonded oligomers or of granule membrane proteins.

Characterization and partial purification of a cytoplasmic glutathione : disulfide oxidoreductase (thioltransferase) from adenohypophysis

A glutathione-dependent thioltransferase (thiol : disulfide oxidoreductase) has been partially purified (70-fold) from anterior pituitary cytosol, and characterized. Purification was effected by differential centrifugation, precipitation between 30 and 60% (NH4)2SO4, and sequential chromatography on Sepharose 6B, DEAE-cellulose, and CM-cellulose. Enzyme activity, monitored by the disappearance of NADPH, was associated with a protein of molecular weight 170 000 both by gel filtration and by polyacrylamide gel electrophoresis in SDS. There was apparent charge heterogeneity after the gel filtration step, and only the major DEAE-cellulose peak was further purified on CM-cellulose. When SDS-polyacrylamide gel electrophoresis was carried out in the presence of mercaptoethanol, the two predominant bands seen in its absence were converted to five major bands, all of different apparent molecular weights from the originals. Isoelectric focusing yielded two major peaks of enzyme activity, at pI 7.0 and pI 4.5-5.0. These peaks were shown to be interconvertible upon reelectrofocusing. Both low- and high-molecular weight disulfides could be reduced. The pH optimum was sharp, at pH 8.2. The Km values for glutathione and cystine (the standard assay disulfide) were 0.57 and 0.062 mM, respectively, each in the presence of saturating concentrations of the other substrate. N-Ethylmaleimide at 0.1 and 1.0 mM inhibited enzyme activity non-competitively, suggesting a non-catalytic role of enzyme thiol(s) for maintenance of optimal activity.