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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 125I-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, Ca2+-sensitive, and displaceable with excess unlabeled CaM. Severe heat treatment (100 degrees C, 15 min), which results in a 75% reduction in phosphodiesterase activation by CaM, markedly decreased 125I-CaM binding to all protein bands. Secretory granule membranes showed enhancement for CaM-binding proteins with molecular weights of 184, 146, 131, 90, and 52,000. A specific, affinity purified antibody to chicken gizzard MLCK bound to the 146 kDa band in homogenates, centrifugal subcellular fractions, and secretory granule membrane. No such binding was associated with the granule contents. The enrichment of MLCK and other CaM-binding proteins in pituitary secretory granule membranes suggest a possible role for CaM and/or CaM-binding proteins in granule membrane function and possibly exocytosis.

PRL and GH are stored in bovine pituitary secretory granules in part as intermolecular disulfide-linked multimers (as many as 50 monomers/oligomer). In vitro incubation of granules with reduced glutathione (GSH) results in the production of monomeric hormones and increased hormone release. We tested the hypothesis that increases in intragranular osmolality due to conversion of oligomers to lower mol wt forms are key to granule rupture. Hormone release was measured after granule incubations in medium made hyperosmotic by the addition of raffinose or sucrose. PRL release at pH 7.8 with 0.7 M raffinose was reduced to 43.5% of the control value, whereas GH release ranged from 70-100% of the control value. Augmented release stimulated by 2 mM GSH was also diminished by raffinose, to 17.9% (PRL) and 75.6% (GH) of control values. The IC50 for raffinose inhibition of PRL release was 0.62 M; this shifted to 0.38 M in the presence of 2 mM GSH. Raffinose also blunted the granule response to GSH at all other GSH concentrations tested and decreased stimulated release at pH 9 as well as at 7.8. Interference with the ability to maintain an osmotic gradient, a maneuver accomplished by partial disruption of granule membranes with 0.5% deoxycholate treatment, reduced or abolished the raffinose inhibition. Molecular sizing of granule fractions on Superose-6 (Pharmacia) indicated that PRL monomers remaining in the pelletable granule fraction increased with raffinose about 2-fold under both basal and GSH-stimulated conditions. These data support the concept that thiol:disulfide equilibria may be important in PRL and GH storage and secretion by their influence on intragranular osmolality. In exocytosis in vivo, osmotically mediated granule rupture may be triggered by thiols during granule:plasma membrane fusion.

Divalent cations inhibited in vitro release of growth hormone (GH) and prolactin (PRL) from bovine adenohypophysial secretory granules. Zinc, nickel, and cadmium were most potent, exerting 50% inhibition of protein release near 0.1 mM; relative potency was Ni2+ greater than or equal to Zn2+ greater than Cd2+ much greater than Mn2+ greater than Co2+ greater than Cu2+ much greater than Mg2+ greater than Ca2+. The pH optimum for inhibition, 8.0, was lower than that for stimulation of release by thiols. EDTA augmented release and reversed metal inhibition. Both immunoassay and polyacrylamide gel electrophoresis results indicated that metals inhibited both PRL and GH release in a dose-related fashion, and that PRL was more sensitive to all cations tested. With zinc present, known stimulators of release (reduced glutathione, ATP, and bicarbonate) restored GH release, but only ATP restored PRL release. Bicarbonate potently stimulated GH release, but only affected PRL when Mg2+ and ATP were present. We suggest that divalent cations influence GH and PRL release in a reversible fashion and at multiple sites. Some loci may be common to both lactotrope and somatotrope granules; however, the different sensitivities to metals and differential reversal by stimulators of release indicate that metal-protein interactions may also be specific for either granule, or for the hormones themselves.

To assess the role in hormone release of the recently characterized anion-sensitive Mg+2-ATPase of pituitary secretory granules, three types of evidence were accumulated. First, granule suspensions were incubated with varying quantities of MgCl2 and ATP, and the effects on ATPase activity and release were measured. The main stimulatory influence on ATPase activity was the concentration of the complex between Mg+2 and ATP (MgATP), although very high concentrations of complex inhibited. In contrast, release of hormone was nearly totally independent of MgATP. Rather, release was primarily controlled by free Mg+2, which inhibited release at concentrations as low as 0.1 mM and reduced basal release by approximately 65-75% at concentrations approximating 2.0 mM or higher. Free ATP had small consistent inhibitory effects on ATPase activity, but stimulated protein release. Second, granules were incubated with other nucleotides and related compounds. Incubation with GTP, ITP, CTP, TTP, and UTP resulted in augmentation of hormone release duplicating that seen with ATP. Some increase was also seen with the nonhydrolyzable ATP derivative 5'-adenylylimido-diphosphate, whereas adenosine was inhibitory. Since the catalytic activity of the granule ATPase demonstrates purine nucleotide substrate specificity, these results provide additional evidence for the dissociation of ATPase activity from hormone release. Third, granules were incubated with several ATPase inhibitors. Though all inhibited ATPase activity to a comparable extent, only tri-n-butyltin inhibited hormone release; oligomycin, efrapeptin, and other tin compounds were inactive. Taken together, these data indicate that the conditions that influence ATPase differ strikingly from those affecting hormone release. Whatever the role of the anion-sensitive granule membrane ATPase, we suggest that it is not involved in the process of hormone release from isolated granules.

We investigated the possibility that thiol-disulfide interchange mechanisms are involved in depletion-transformation (loss of tissue PRL detectability) and release of PRL from adenohypophyses (AP) of lactating rats. The influence of pH, bicarbonate, and Triton X-100 as well as thiol-related compounds on these processes was assessed. Tissue PRL was depleted-transformed by the use of three different conditions: 1) 30 min of suckling after 8 h of nonsuckling; 2) in vitro incubation of APs for 2 h; or 3) in vivo cysteamine (CSH) treatment. Lactating rats nonsuckled for 8 h served as controls (no depletion-transformation). The depletion-transformation phenomenon was unchanged by extraction with Tris-HCl-0.1% Triton X-100 buffers but reversed either by extraction with bicarbonate buffer (pH 8.2 or 9.7) or by incubation of pH 8.2 homogenates for 3 h at 37 C. Reduced glutathione (GSH) added to these homogenates further enhanced PRL detectability. At pH 6.5, however, incubation with or without GSH had the opposite effect and decreased PRL detectability. In AP incubations, depletion was increased in a dose- and time-dependent fashion by the aminothiol CSH, and by GSH, dithiothreitol, or mercaptoethanol but not sodium ascorbate. These agents also inhibited PRL release. Similar results were obtained after injection of CSH (20-120 mg/kg BW) 4 h before death. Depletion and release of PRL in incubated APs were prevented by iodoacetamide and N-ethylmaleimide (0.1-5 mM); GSH or CSH counteracted these effects. In contrast to the alkylating agents, oxidized glutathione and 5,5'-dithio-2-nitrobenzoic acid inhibited PRL depletion but stimulated PRL release. Thus, thiols and aminothiols may preferentially lead to depletion-transformation of PRL, whereas disulfides may inhibit depletion and facilitate PRL release. Although in some experiments increased depletion was dissociated from increased release, nonetheless the data support the concept that shifts in PRL detectability during depletion-transformation, repletion, and release involve thiol-disulfide interchange mechanisms.

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.

Cysteamine [2-aminoethanethiol (CySH)] reduces measurable PRL concentrations in vivo and in vitro. Since secretion is also inhibited, CySH may block conversion from a poorly assayable hormone storage form(s) to readily assayable, releasable PRL. This would represent a previously unrecognized mechanism for secretory regulation. We undertook the present study to identify the sites involved in the loss of measurable PRL (depletion) induced by cysteamine. The disulfide cystamine was ineffective on secretory granules unless combined with reduced glutathione, indicating the generation of the active CySH-thiol form. Pretreatment of granules with thiol-blocking agents resulted in dose-dependent enhancement of CySH inhibition, achieving nearly complete inhibition with 5 mM iodoacetamide. In contrast, pretreatment with reduced glutathione or dithiothreitol, respectively, impaired or abolished the CySH effect. Similar results were obtained when tissues were exposed to thiols and/or alkaline conditions after CySH treatment; for example, exposure to 50 mM glutathione reduced the lowering of measured pituitary homogenate PRL from 76% to 37%. These data suggest that the mechanism by which CySH causes PRL depletion is mediated by granule disulfides and the -SH of CySH. The regulation of thiol:disulfide equilibria appears to be an important determinant of the detectability of PRL storage forms and of their secretion.

Little is known about the structure of prolactin (PRL) within secretory granules. Evidence from our previous studies in bovine tissue preparations suggests that control of secretion may reside, in part, in the conversion of storage hormone to releasable PRL. The conversion can be monitored by measuring changes in immunodetectability since the oligomeric, storage form is poorly recognized by antisera raised against monomeric PRL. Since many investigators use rats to study the secretory process and changes in detectability of rat pituitary PRL occur during lactation ("depletion-transformation"), we undertook the present immunodetectability studies to gain insight into the storage structure of rat (r) PRL. Cysteamine and zinc inhibited tissue PRL immunoassayability in male rat pituitary homogenates and also in partially purified secretory granules as they had inhibited bovine (b) PRL; however, zinc inhibited the rodent hormone less potently than the bovine. In vitro incubation of rat tissue samples without additions resulted in increases in rPRL detectability of up to 84% after 180 minutes; such incubation of bovine samples had no significant effect. A striking additional difference between the species was that exposure to reduced glutathione (GSH), cysteine, homocysteine, mercaptoethanol, and dithiothreitol inhibited rPRL by up to 44%. This compared to thiol stimulation of bPRL by as much as 450%. The inhibitory GSH effect on rPRL was abolished when 0.5% sodium dodecyl sulfate (SDS) was included; in contrast, the stimulatory GSH effect on bPRL did not change with added SDS. SDS alone had no effect on rat homogenate PRL, and only increased rat granule rPRL by 23% compared to its ability to increase bPRL assayability by 44%.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

We have identified an anion-sensitive Mg2+-ATPase in adenohypophyseal secretory granule membranes. This enzyme is unaffected by sodium, ouabain, and calcium. By electron microscopic morphology, sedimentation properties, nucleotide substrate utilization, and marker enzyme studies, this activity is clearly shown to be intrinsic to the granule membranes. The kinetics for ATP saturation were complex, as curvilinear Lineweaver-Burk plots were obtained with 2 mM magnesium. However, an approach to linearity was obtained (Km for ATP, approximately 0.27 mM) with low concentrations of free magnesium. Many anions and anion-transport blockers significantly influenced enzyme activity. Stimulatory anions in decreasing order of potency were bisulfite greater than sulfite greater than isethionate greater than bicarbonate; Ka values were 2.5 mM for sulfite and 10.8 mM for bicarbonate. Acetate, borate, chloride, citrate, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 2-(N-morpholino)ethanesulfonic acid, nitrite, oxalate, 1,3-piperazinediethanesulfonic acid, and sulfate were without major effect. Inhibitory anions in decreasing potency order were azide greater than thiocyanate greater than fluoride greater than nitrate. Anionic stimulation of the granule membrane Mg2+-ATPase linearized the Lineweaver-Burk plots by shifting the enzyme to its higher Km state. In addition, sulfite competitively reversed the produce inhibition exerted by ADP. Anion transport-blockers inhibited the enzyme; of those tested, the most potent was 4-acetamido-4-isothiocyano-stilbene-2,2'-disulfonic acid, with a Ki of 0.17 mM; pyridoxal phosphate, sulfisoxazole, and ethacrynic acid also inhibited enzyme activity. The protein-binding dye p-sulfobenzene-azo-o-sulfobenzene-azo-beta-naphthol-3,6-disulfonic acid, structurally similar to transport blockers, was a potent inhibitor, with a Ki of 2.8 mM. These data on pituitary secretory granule ATPase raise the possibility that the granule membranes may function in anion or proton transport, perhaps in relation to exocytosis and hormone secretion.

Depletion-transformation of PRL is a decrease in tissue PRL detectability which precedes and may often be required for increased PRL release. The present studies were designed to determine whether thiol-disulfide interchange mechanisms may be involved in PRL transformation and release by the pituitary of the lactating rat. Thirds of 8-h nonsuckled lactating rat adenohypophyses were incubated with or without thiols (reduced glutathione, the aminothiol cysteamine, or mercaptoethanol), in the presence of known inhibitors of transformation and release such as dopamine (DA), stalk median eminence (SME) extract, or Zn++. PRL concentrations in pre- and postincubated tissues, as well as the amount of released PRL, were determined by polyacrylamide gel electrophoresis and densitometry. In 30-min incubations without additions, 12-22% of the tissue PRL was depleted; however, in the presence of 17-50 microM DA, all doses of SME extracts tested (0.5-2.0 eq), or 0.1 mM Zn++, depletion was partially or totally prevented and PRL release was inhibited 25-60%. On the other hand, when thiols were added in addition to the above agents, a complete, dose-related, restoration of PRL depletion was obtained. In 120-min incubations, thiols similarly reversed the effects of 0.05 mM DA on depletion, but thiols did not reverse the inhibition of PRL release caused by DA, SME, or Zn++. Other data indicate that thiols alone may inhibit rat PRL release and also facilitate or induce PRL depletion; in bovine PRL granules, thiols reverse Zn++ inhibition of PRL release and detectability. These data suggest that thiol-disulfide interchange reactions may be importantly involved in both depletion-transformation and in secretion. The precise thiol sensitivity of the two processes does not appear identical, secretion being more sensitive to DA and less sensitive to thiols than depletion-transformation.