Divalent Cation Inhibition of Hormone Release from Isolated Adenohypophysial Secretory Granules*

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+ 2 Zn2+ > Cd2+ >> Mn2+ > Co2+ > Cu2+ >> Mg2+ > Ca2+. The pH optimum for inhibition, 8.0, was lower than that for stimulation of release by thiols. EDTA aug-mented 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 stim- ulated GH release, but only affected PRL when Mg2+ and ATP were present. We suggest that divalent cat- ions 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.

Secretory systems require calcium for maintenance of normal responses to secretagogues (1)(2)(3). Similarly, calcium is required in the response of target tissues to hormonal actions (3,4). Many cellular sites and biochemical processes may be influenced by calcium; the cell membrane (5-7), microtubules (8,9), and secretory granules (10, 11) are included among the cellular loci where calcium may play important roles in secretion. Recent observations have suggested that high calcium concentrations might play a role in modulating the inhibitory effects of somatostatin on hormone secretion, at least in the pancreas (12,13). In the pituitary, in contrast, excess calcium tends to suppress release of GH' (14), thus mimicking the effect of somatostatin rather than counteracting it. Excess calcium alone has been reported either to suppress GH release (15) or to be without effect (16). Under most experimental * This research was supported in part by Grants AM-21783 and AM-31326 from the National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases and Grant RR-00044 from the Division of Research Resources, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisernent" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
conditions, extracellular calcium reduces in vitro secretion of PRL and GH, but is apparently not required for maintenance of the stimulatory effect of theophylline on both hormones, or for expression of the inhibitory effect of dopamine on prolactin secretion (17). Recent studies have suggested that at least one secretagogue for PRL, thyrotropin-releasing hormone, does not induce influx of extracellular calcium but rather mobilizes calcium into GHs cell cytosol from intracellular sites; secretion is still ultimately dependent upon calcium availability (18).
Our interest in examining the effects of calcium and other divalent cations on hormone release from secretory granules developed out of previous granule studies dealing with a Mg2+dependent, anion-sensitive granule membrane ATPase (19). In the course of establishing that optimal conditions for enzyme activity differed substantially from conditions augmenting hormone release, we noted that Mg2' was a potent inhibitor of GH and PRL release (20). ' We wondered whether the inhibition observed was specific for Mg', and if it reflected one or more regulatory phenomena which might play important roles in the control of secretion in uiuo. In order to address these questions, we have employed our recently developed secretory granule incubation system (21). This in vitro hormone release system may simulate the events of the final stage of secretion, the dissolution of stored granule hormone into the extracellular space following fusion of the granule and plasma membranes. The present studies, using this method, indicate that a wide variety of divalent cations share with magnesium the ability to inhibit GH and PRL release from isolated secretory granules, and that this inhibitory effect is reversible with EDTA, and also counteracted by ATP, reduced glutathione, and bicarbonate.

DISCUSSION
The data reported here indicate that a number of divalent cations are potent inhibitors of the release of GH and PRL from suspensions of secretory granules. No apparent relationship existed between the inhibitory potency of these metals and their size or orbital structures, or their electrophilicity.
The independent results of the protein measurements, radioimmunoassay determinations, and polyacrylamide gel patterns corroborated each other. Results in Fig. 1 and Table I showed that metal inhibition was dose-dependent and that PRL release was more sensitive than GH. Data in Table  I also showed that EDTA stimulated release of both hormones in the absence of added metals. It should be pointed out that neither metals nor EDTA had any direct effect on the hormonal radioimmunoassays at the concentrations used in the studies. The greater sensitivity of PRL than of GH to metal inhibition was also observed in polyacrylamide gel electrophoresis studies (not shown); for example, a t zinc concentrations of 100 ~L M , only the PRL band became undetectable. Previously, we had also shown that PRL was more sensitive than GH to the stimulatory effects of thiols on hormone release (21). In addition, granule PRL immunoactivity was tremendously enhanced by thiols, whereas that of GH was only modestly affected (21). This information suggests that the proteins in the membranes of each granule type have different sensitivities, or that the intragranular storage form of PRL is more sensitive to metals and thiols. If the hormones interact directly with the cations, the disulfide near the NH, terminus of the molecule, present only in PRL, might be involved, in part, in metal-thiol complexation. (Both PRL and GH have a midmolecule large disulfide loop and a small COOH terminus disulfide.) Results from the studies in which granules were incubated with a combination of two cations (e.g. zinc and magnesium) were also in agreement with the suggestion that PRL and GH granules have different sensitivities to divalent cations and contain several metal-sensitive sites. First, PRL release was again more potently inhibited than was GH release under all conditions. Second, the observed inhibition a t submaximal Zn2+ and Mg2+ concentrations was equal to the additive effects of each alone, whether protein or immunoassay data were employed to evaluate the results (Figs. 2 and 3). Although inferences regarding the similarities or differences of mechanisms of inhibition by the two cations cannot be made from these studies, several sites with different sensitivities are implied since, otherwise, competition with less than additive effects, would have been expected.
Like thiol stimulation of hormone release from granules, the divalent cation inhibition varied with the pH of the medium, being attenuated in the acidic range, and maximal a t alkaline values. Thiol-stimulated and basal hormone release were maximal at pH 8.8 in these studies ( Fig. 4; also Ref. 21), but inhibition by zinc was maximal at pH 8.0 and decreased somewhat at the higher pH value. While the mechanism of metal inhibition cannot be ascertained from these studies, the metals may interact with critical thiol groups. We have previously suggested that thio1:disulfide interchange reactions may be critical for the release process (21,27,28), involving thiols present either in granule membranes or in hormone storage forms. Divalent cations may inhibit these directly or they may inhibit enzymes involved in the interchange process. For example, a pituitary GSH:disulfide oxidoreductase is known which is capable of catalyzing disulfide interchange between GSH and secretory granule protein disulfides (29,30) and which is inhibitable by metals (30). Stimulation of release by thiols may be under enzymatic control at or below pH 8, whereas nonenzymatic disulfide interchange, being increased in the alkaline range, may account for the increased basal and thiol-stimulated release at pH 8.8. These data, of course, are also consistent with separate mechanisms for thiol stimulation and for metal inhibition of release.
The reversibility of the inhibitory effect of metals by EDTA is clearly shown in Figs. 5 and 6. Although reversibility became less complete as prior incubation time with zinc was prolonged, nonetheless, over 70% of the maximal hormone release was still observed even after 30 min of exposure to zinc. Also, the effect of 1 h of contact with metal, as shown in Fig. 6, is virtually completely reversible in a second incubation with EDTA. Such prompt and substantial reversal of inhibition indicates that a nonspecific denaturation or precipitation of the hormones by metal is quite unlikely. This was also substantiated by electron microscopic studies which indicated that granules incubated with zinc were indistinguishable from those incubated without metal^.^ One of the interesting observations in the two-stage incubation experiment was the failure of EDTA to augment hormone release when it was present only during the second incubation (Fig. 6, top pair of  bars). This suggests the possibility that, during the first hour of control incubation, a change may take place in the granule or its contained hormone which renders it indifferent to the stimulatory effects of EDTA. This stands in striking contrast to the marked stimulation of release observed when EDTA was present from the start of the incubation (Fig. 5 ) .
Electrophoretic evidence (Fig. 7) and radioimmunoassay data (Table I1 and text) indicated that the inhibitory effects of divalent cations could be counteracted by stimulators of release other than EDTA. The mechanisms by which ATP, HCOs-, and thiols stimulate release have not been elucidated in detail, nor do we know conclusively from these data whether the stimulators and metals were acting at the same site(s). However, the finding that GH release and PRL release were similar at comparable GSH:zinc ratios even when absolute concentrations of GSH and zinc varied by 10-fold suggested that these ligands were in fact competing for the same site(s) rather than complexing with each other. Differences in responsiveness of GH and PRL granules were apparent. GSH and HC03-, added to inhibitory concentrations of zinc, influenced primarily GH release whereas ATP attenuated the zinc inhibition of both hormones. These differences may relate to differences in numbers or characteristics of granule-binding sites for these agents, or differences in the molecular structures and organization of the granule membranes or the hormones. Since bicarbonate had previously been shown to stimulate the release or production of PRL (but not GH) by hemipituitaries or pituitary cells (31)(32)(33), additional experiments were carried out with bicarbonate in the presence of magnesium. These results, shown in Fig. 8, indicated that magnesium and bicarbonate could modulate each other's effect on protein, GH, and PRL release. PRL was more sensitive to M e , while GH was more sensitive to HCO,-. In fact, virtually no effect of HCOs-on PRL was seen unless Mg2+ was present, thus implying that different mechanisms were operational for GH and PRL release under these conditions. The divalent cation environment might be critical for expression of the bicarbonate stimulatory effects in PRL granules but such a requirement may not be a major factor in GH release mechanisms.
The absence or diminution of stainable protein bands with zinc exposure (Fig. 7), despite a comparable Lowry protein load in each lane of the gel, coupled with increases in stainable protein at the gel origin, constitutes strong circumstantial evidence for an induced increase in molecular size under the influence of metals, or an inhibition of conversion to smaller forms. Such an effect might be expected to cause a reduction in immunoassayable hormone even if these large forms are released, since very high molecular weight disulfide oligomers of both GH and PRL have been noted to have poor immu-M. Y. Lorenson and L. S. Jacobs, unpublished observations. noactivity on a protein basis (27,28,34). This information from the nondenaturing gel runs shown in Fig. 7 was buttressed by identical results when supernatants from metalexposed granules were electrophoresed in sodium dodecyl sulfate (not shown). Other investigators have reported that nickel inhibits the secretion of PRL but not that of other adenohypophysial hormones, from pieces of tissue incubated in uitro (35, 36). The present data, which show that nickel potently inhibits GH as well as PRL release from isolated granules, may not necessarily contradict the prior data, since the systems used are so different. Obviously, many other sites are potentially available in whole tissues including plasma membrane sites which may complex the cations, thus preventing their internalization.
In summary, these experiments demonstrate that divalent cations strikingly inhibit GH and PRL release from incubated secretory granules in a reversible and dose-related fashion. Given the differences observed with incubation time and sequence of addition of effective compounds, the interactions with glutathione, ATP, and bicarbonate, the additivity of combinations of cations, the differential magnitude of the effects on PRL and GH, and the electrophoretic evidence for changes in hormonal molecular size with cations, it seems that a multiplicity of sites may be involved in the expression of these effects, as is the case with thiol stimulation of hormone release (21).