Prolactin (PRL) is a zinc-binding protein. I. Zinc interactions with monomeric PRL and divalent cation protection of intragranular PRL cysteine thiols.

PRL in secretory granules is osmotically inert. Previous studies by us and others have suggested that this is due in part to hormone oligomerization. Data suggest intermolecular disulfide bridges and/or intermolecular ionic interactions, as thiols, urea, and chelators increase monomerization of the majority of granule PRL. Because of the inhibitory effect of zinc on PRL release from isolated granules and the effects of zinc on the specific packing of PRL within granules, we examined the possibility that zinc contributed to the stability and/or oligomerization of intragranular PRL. To do this, we first analyzed zinc binding to purified monomeric rat PRL in solution. Zinc binding was demonstrated using the chromogenic chelator 5,5'-nitrilodibarbituric acid (murexide) and was confirmed by matrix-assisted, time of flight mass analysis. Because these spectrophotometric methods were not applicable for intragranular PRL studies, we tested the influence of zinc on granule PRL indirectly. As hormone free thiols were potentially formed during PRL oligomerization and storage, these were possible sites for hormone-divalent cation interactions. By derivatization of thiols with 4-vinyl pyridine and isolation of the carboxyterminal region of granule PRL, we found that a proportion of the cysteines 189 and 197 occurred as thiols and not disulfides. These thiols were only detectable when EDTA was present in the granule incubations. It is proposed that binding of zinc stabilizes the intermolecularly bonded storage form of PRL, in part by protection of hormone free thiols. Removal of the divalent cation and exposure of free thiols could be what initiates the thiol-disulfide interchange necessary for conversion of intermolecular to intramolecular disulfide bonds before exocytosis. Experimentally, both urea and EDTA could remove zinc, thus initiating monomerization and explaining how these agents as well as thiols achieve monomerization.

Intragranular prolactin phosphorylation and kallikrein cleavage are regulated by zinc and other divalent cations.

Rat prolactin (PRL) secretory granules contain enzymes for proteolytic cleavage and serial phosphorylation, but hormone cleavage products and phosphorylated PRL are not detected until just prior to exocytosis. Similarly, although PRL is stored in granules, in part, as high-mol-wt oligomers, PRL is primarily monomeric in the circulation. PRL secretory granules contain zinc, calcium, and magnesium, which inhibit depolymerization and dissolution of granules. Divalent cations also protect cysteine free thiol residues in the carboxy-terminal region of the intragranular hormone. The present studies examined the effect of removal and replacement of divalent cations on kallikrein cleavage and phosphorylation of secretory granule PRL.Kallikrein cleavage was assessed utilizing two experimental protocols. First, granules were treated with or without 3 mM EDTA, free hormone thiols were alkylated, the PRL was cleaved by kallikrein, and the small kallikrein-cleavage peptides were assessed by reversephase HPLC. No differences in hormone cleavage owing to removal of divalent cations were observed at this concentration of EDTA. Second, divalent cations in granules were reduced/removed by 10 mM EDTA/ 3 mM o-phenanthroline (OP), followed by addition of either 5 mM zinc, magnesium, calcium, or additional EDTA. Kallikrein cleavage was then initiated. In this instance, the extent of proteolysis was analyzed by two-dimensional polyacrylamide gel electrophoresis (PAGE) of the larger remnant PRL pieces. After treatment with 10 mM EDTA/3 mM OP, results indicated that cleavage between R174 and R175 (site 1) was unaffected by added cations or additional EDTA. Recovery of site 2 cleaved PRL (L1-K185) and site 3 cleaved PRL (L1-R188) was∼40% reduced by zinc, but unaffected by calcium or magnesium. Additional EDTA resulted in increased recovery of site 2 cleaved PRL, but no change in site 3 recovery, suggesting the presence of tightly bound intragranular zinc around site 2, even after the initial EDTA/OP treatment.Phosphorylation of PRL at S177 was studied using the same protocols. Phosphorylation was increased by added EDTA, even at 3 mM, and decreased by divalent cations, with no marked specificity for zinc observed. An additional experiment studied phosphorylation without exposure to kallikrein. Comparisons between the plus and minus kallikrein experiments showed kallikrein to have no apparent preference for unmodified or phosphorylated PRL.From the kallikrein cleavage and phosphorylation studies and modeling of PRL, we suggest D181 as a likely site for intragranular zinc coordination. When C189 and C197 are present as free thiols in intragranular PRL, these may also contribute to binding. Zinc coordination in this region of the molecule apparently regulates proteolytic processing by kallikrein, as well as contributing to the stability of the hormone storage forms.

Measurement of serum L-asparagine in the presence of L-asparaginase requires the presence of an L-asparaginase inhibitor.

The antileukemic activity of L-asparaginase (ASNase), an important component of therapy for acute lymphoblastic leukemia, is thought to result from depletion of serum L-asparagine (Asn). In studies of the pharmacological effects of ASNase, investigators have reported prolonged reduction in the serum concentration of Asn after the administration of ASNase. Such measurements may not be valid because ASNase present in the blood sample may hydrolyze Asn before its determination. We examined recovery of [U-14C]Asn from blood samples with and without various concentrations of added ASNase. In the presence of greater than or equal to 0.01 IU/ml of ASNase, the amount of [U-14C]Asn recovered was less than 15% of that without ASNase. Utilizing this assay, we studied the effect of 2 known inhibitors of ASNase in an attempt to improve Asn recovery. In the presence of aspartic beta semialdehyde (ASA), or 5-diazo-4-oxo-L-norvaline (DONV), and up to 1.0 IU/ml ASNase, Asn levels remained at greater than 90% of control. ASA prevented the hydrolysis of exogenous Asn in blood samples drawn from patients after ASNase injection. We also developed a method to determine Asn in serum utilizing high pressure liquid chromatography. Using this method, we found that the Asn level was greater than 90% of a normal level in the presence of 40 mM DONV and 1.0 IU/ml ASNase. Examination of serum from 4 patients treated with ASNase showed that Asn is detectable 7-19 days sooner when DONV is present in the blood collection system than in its absence. We conclude that: (a) as little as 0.01 IU/ml ASNase can hydrolyze Asn added to blood; (b) continued hydrolysis of Asn by ASNase ex vivo can result in falsely low serum Asn measurements; (c) ASA or DONV present in the collection tubes obviates the problem of continued ASNase activity; and (d) the degree and duration of Asn depletion after ASNase therapy is much less than previously believed. Thus, for accurate measurements of the duration and degree of Asn depletion by ASNase, an ASNase inhibitor such as ASA or DONV should be present in the blood collection system.

Calcium release from pituitary secretory granules: modulation by thiols, disulfides, and dihydropyridine calcium channel blockers.

The distribution of calcium in isolated bovine pituitary secretory granules was studied by atomic absorption. The total granule calcium (in 26 preparations) averaged 14.5 nmol/mg protein, or 21.2 +/- 1.6% of the total pituitary homogenate calcium. Incubation of granules with KCl resulted in calcium release (78% at 15 mM and 100% at 50 mM, for example). Calcium release was also pH dependent, with greater release at acidic pH values; it was not influenced by either 500 microM strontium or 500 microM lanthanum. Release was augmented by reduced glutathione (GSH), with significant release observable at thiol levels as low as 10 microM. In addition to GSH, cysteine also stimulated release; mercaptoethanol and dithiothreitol were less potent. Interestingly, the disulfides cystine and oxidized glutathione also stimulated calcium release. Since the latter compounds are known to inhibit hormone release from granules, calcium and protein release appear to be regulated independently. A number of dihydropyridines were tested as potential blockers of calcium release from granules. Nimodipine inhibited basal calcium release at high concentrations and potently inhibited GSH-stimulated calcium release, with an apparent Ki in the 10-20 nM range; it also inhibited K(+)-stimulated release but to a lesser extent. Nimodipine, however, did not significantly influence protein or hormone release. GSH-stimulated calcium release was also inhibited by nifedipine, and this inhibition was qualitatively and quantitatively similar to that by nimodipine. Nisoldipine and nitrendipine, however, displayed no significant inhibition. In summary, it appears that the release of secretory granule calcium in vitro is independent of protein release. Thiols and some disulfides stimulate calcium release, and its inhibition by dihydropyridines suggests that granule membranes may contain specific ion channels. The role of granule calcium in the cell remains to be defined.

Alterations in in situ prolactin secretory granule morphology and immunoactivity by thiols and divalent cations.

The mechanisms involved in PRL storage in secretory granules are generally poorly understood. Recent studies with isolated granules, however, have suggested that granule storage forms may be relatively osmotically inactive due to oligomerization involving hormonal intermolecular disulfide bonds. Thus, expenditure of metabolic energy by the cell in order to maintain granule integrity would be reduced. When secretion is stimulated, oligomer depolymerization by thiol exchange mechanisms has been proposed to occur before or even concomitant with exocytosis. The present studies were designed to investigate the influence of metabolic inhibitors, thiols, and divalent cations on PRL storage in situ, rather than in isolated granules. The results suggest that 1) PRL granules require little energy to maintain their structure, since a combination of azide (10 mM), fluoride (10 mM), and cyanide (1 mM) had no effect on PRL granule morphology in normal anterior pituitary cells in primary culture; 2) disulfide linkages are involved in the osmotic activity of the PRL granule contents, since thiols induced granule swelling in lightly fixed cells; and 3) thiols and divalent cations are capable of altering the arrangement of stored hormone molecules, since PRL immunoactivity could be modified by these agents in glycol methacrylate-embedded exposed granule cores.

Preferential interaction of [35S]cysteamine with pituitary secretory granule storage forms of prolactin.

Cysteamine (CySH) inhibits the immunodetectability and bioactivity of prolactin (PRL), and we have proposed that it may act by impeding the conversion from secretory granule hormone storage forms to releasable and assayable hormone. This process appears to be dependent upon thiol:disulfide interchange reactions, which can be inhibited by the aminothiol. The present studies utilized [35S]CySH to determine whether preferential interactions could be demonstrated between CySH and bovine pituitary storage hormone forms as opposed to monomeric PRL. [35S]CySH was incubated with purified intact secretory granules, with granule 'core' preparations enriched in oligomeric forms by prior hypotonic exposure, with chromatographically isolated oligomers, and with monomeric PRL. Binding to granules was saturable and pH-dependent with greatest binding observed at pH 7.5-8.0. Binding to monomer was much less than binding to all other fractions, being 20% or less than that to any other form. HPLC studies of granules treated with [35S]CySH indicated that exposure to CySH was associated with a predominance of very high molecular weight oligomers. These forms were entrapped on the gel permeation columns, resulting in decreased protein and PRL recovery; as little as 6.6% of the PRL was eluted after 60 min of CySH exposure. CySH not only bound to storage forms of PRL but also to secretory granule membranes; whether the bioeffect is mediated through membrane modifications is unknown. Despite its relative ineffectiveness at altering growth hormone immunoactivity or secretion, CySH nonetheless also bound to growth hormone.(ABSTRACT TRUNCATED AT 250 WORDS)

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 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.

Osmotic pressure regulation of prolactin and growth hormone release from bovine secretory granules.

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.

Reversal by thiols of dopamine-, stalk-median eminence-, and zinc-induced inhibition of prolactin transformation in adenohypophyses of lactating rats.

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.

Thiol regulation of depletion-transformation and release of prolactin by the pituitary of the lactating rat.

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.

Cysteamine, zinc, and thiols modify detectability of rat pituitary prolactin: a comparison with effects on bovine prolactin suggests differences in hormone storage.

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)

In vitro conditions modify immunoassayability of bovine pituitary prolactin and growth hormone: insights into their secretory granule storage forms.

The amount of immunoassayable intracellular bovine (b) PRL and GH varies depending on treatment conditions. The present studies were designed to characterize the mechanisms involved and to compare immunoassayability of both hormones under similar conditions. Pituitary homogenate and secretory granule hormones displayed both time- and temperature-dependent increases when incubated at pH 10.5 with reduced glutathione; for example, 180 min values were 156% (GH) and 439% (PRL) at 22 C compared with control homogenate values. Under these conditions, PRL in granule cores (the sedimentable fraction after hypotonic lysis of granules) increased the most, changing 120-fold from 2.9 +/- 0.25 micrograms/ml (pH 8.3 control) to 349 +/- 22.8 micrograms/ml. Isolated oligomeric PRL increased significantly after exposure to mercaptoethanol, indicating that changes in assayability do not require nonhormonal granule components. No change in monomeric PRL, or GH in either form, was observed. Pretreatment with iodoacetamide resulted in a 33% decrease in maximal PRL values, indicating the presence and functional importance of granule thiols. Cysteamine inhibition of bPRL values in cores was modulated by urea, EDTA, and iodoacetamide. Though 5 M urea or 0.5% sodium dodecyl sulfate (SDS) increased granule PRL at pH 8.3, maximal values required thiols at pH 10.5. In contrast, maximal GH values were obtained with SDS at pH 8.3 without thiols. Changes in immunoassayability seem to reflect conversion from poorly immunoactive tissue hormone oligomers to monomeric hormone. The data indicate that oligomeric bPRL is stabilized primarily by intermolecular disulfide bonds, although it is also susceptible to urea, SDS, and EDTA; granule thiols may also influence the conversion to monomer. The storage form of bGH appears to be stabilized differently. Maneuvers demonstrated in these studies to influence immunoassayability correlate very well with their previously established effects on hormone release and secretion, strengthening the likelihood that a functional link exists between assayability and secretion.

Depletion of bovine pituitary prolactin by cysteamine involves a thiol:disulfide mechanism.

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.

Cysteamine inhibition of prolactin immunoassayability and secretion: studies with aminothiophenols and other analogs.

Analogs of the aminothiol cysteamine (CySH) have been studied to clarify the structural features required for exertion of inhibitory effects on PRL. The inhibited functions examined were the detectability of PRL by RIA and the release of PRL from suspensions of isolated secretory granules. The influence on GH assayability and release was also tested. The aromatic compounds 2-aminothiophenol, 3-aminothiophenol, and 4-aminothiophenol shared with CySH the ability to inhibit PRL assayability and release, but were all more potent. Derivatization of the thiol, as in 4-aminothioanisole, was associated with a substantial loss of inhibitory potency, whereas derivatization of the primary amine, as in N-dimethylaminoethanethiol, had no influence. Thiols such as mercaptoethanol, cysteine, glutathione, and others without nearby amino groups were stimulators of PRL assayability and release. The inhibitory effects of the aminothiols were highly pH dependent, being marked at pH 5.5, 6.5, and 7.5, but modest or marginal at 8.5. After CySH exposure, inhibition was reversible in part by extraction of samples with reduced glutathione or at pH 10.5. Though CySH and 4-aminothiophenol induced changes in the electrophoretic migration of granule PRL, similar changes occurred in the migration of standard purified hormone despite the known absence of immunochemical effects. There was close quantitative correlation between the potency of a compound to inhibit PRL assayability and its potency to inhibit PRL release. We conclude that the inhibitory aminothiol action on PRL requires the thiol rather than the sulfide form and involves a reversible interaction which diminishes the immunochemical recognition of granule PRL. This change also results in diminished secretion.

Cysteamine inhibition of bovine pituitary secretory granule prolactin immunoassayability and release.

Cysteamine [2-mercaptoethylamine (CySH)] displays a variety of neuroendocrine effects, the most potent being the depletion of immunoassayable tissue PRL. The present study used bovine adenohypophysial secretory granules to characterize this inhibition of measurable hormone (assayability). CySH decreased assayability in a dose-dependent manner at pH 7.4, with 50% inhibition observed close to 2 mM. Maximal inhibition was found between pH 6.0 and 6.5, whereas diminished (or no) inhibition occurred under alkaline conditions, depending on the buffer. In contrast, reduced glutathione (without CySH) increased assayability at pH 8-8.5, had little potency near neutrality, and inhibited assayability under acidic conditions. Electrophoretic studies under nondenaturing conditions demonstrated that CySH exposure of standard PRL resulted in additional charged species. With granules, CySH markedly decreased the staining of the major PRL band, no new bands were evident, and this effect was abolished by glutathione. Sodium dodecyl sulfate-electrophoretic patterns indicated that CySH resulted in higher apparent mol wt species of granule and standard PRL. This effect was nullified by mercaptoethanol. Never was there evidence for species smaller than monomeric. Depletion of PRL might involve interference with the conversion from oligomeric storage PRL to assayable PRL; 44-fold increases in PRL oligomer immunoactivity after alkali and thiol treatment were reduced to 6-fold increases when CySH was present. Reactions involved in production of assayable hormone appear relevant to secretion, since CySH also inhibited PRL release from granules, with maximal inhibition occurring under acidic conditions. Thus, CySH may be useful in investigating the physicochemical properties of tissue PRL and may also represent an approach to treatment of hyperprolactinemic states.

Secretory granule growth hormone and prolactin release: independence from granule membrane ATPase.

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.

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.

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+ 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.

Detectability of pituitary PRL and GH by immunoassay is increased by thiols and suppressed by divalent cations.

The amount of pituitary PRL detected by RIA in extracts of tissue and of secretory granules was augmented by thiols or EDTA in the extractant, and diminished by divalent cations or cysteamine. GH was affected by thiols and cations similarly, but to a lesser extent, and was not influenced by EDTA or cysteamine. For full immunologic detection of tissue PRL and GH, thiol-dependent mechanisms appear to be required to unmask immunoreactive sites from the poorly immunoreactive oligomeric granule storage forms. Interpretation of studies which rely on measurement of tissue PRL and GH content by RIA, immunoprecipitation, or electrophoretic detection should be reassessed in light of these observations.