A "20K" form of growth hormone in the murine pituitary gland

Extracts of mouse and rat adenohypophyses have been analyzed for a low-molecular-weight variant of growth hormone (GH) known to occur in humans, the so-called "20K"-GH (mol wt = 20,000 vs 22,000 for traditional human GH). Pituitary proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunostained with an antiserum raised against murine GH. Reactive and unreactive bands in close vicinity of the major GH band were fingerprinted by a peptide-mapping technique that reveals only the tyrosine-containing peptides, but can be applied to fingerprint single protein bands within gels. We found a protein band 2000 mol wt smaller than the major murine GH in both mouse and rat pituitary glands whose fingerprint resembled that of major GH. The peptide-mapping results are consistent with the interpretation that the internal deletion of amino acid residues most likely is in the same region of the molecule as in the human 20K-GH. Unlike the human 20K-GH, however, the murine counterpart showed no cross-reactivity with an antiserum raised against 22K-GH in the test system used here.     

Cortactin-binding protein 90 (CBP90) expression in the mouse mammary glands during prolactin-induced lobuloalveolar development

We have previously performed suppression subtractive hybridization to identify genes that were induced during prolactin (PRL)-driven lobuloalveolar development of the mammary gland. This suggested that cortactin-binding protein 90 (CBP90), which is known to be a brain-specific protein that binds to cortactin, was expressed under the regulation of PRL in the mammary glands (preliminary observation). In this study, the expression of CBP90 was examined in the mammary glands of mice under manipulated hormonal circumstances. PRL treatment by 9 days of pituitary grafting induced CBP90 expression in the normal mammary glands but not in the cleared fat pads, while cortactin was expressed constitutively in both the normal mammary glands and the cleared fat pads. Unlike milk proteins, longer treatment with PRL (36 days of pituitary grafting) did not increase the expression level of CBP90 mRNA, while it slightly increased the cortactin mRNA level. Mammary CBP90 mRNA expression was induced by pituitary grafting but not by progesterone treatment in PRL-deficient mice, while pituitary grafting induced mammary CBP90 expression in ovariectomized PRL-deficient mice only when estrogen and progesterone were appropriately supplemented to permit the formation of alveolar buds. The CBP90 protein was detected by immunohistochemistry in the luminal epithelium of the alveolar buds and more faintly in the ductal epithelium. Thus, from the unique expression pattern, CBP90 may be useful as a molecular marker for the hormone-stimulated development of mammary alveolar buds.     

Comparison by electrophoresis of proteins characteristic of the lateral line and skin of Xenopus laevis

In experiments aimed at determining acousticolateralis marker proteins, fractions of lateral-line organs and skin of Xenopus laevis were analyzed by one- and two-dimensional polyacrylamide-gel electrophoresis. A protein fraction of approximately 44K mol. wt (K = 1000 daltons) and isoelectric pH 6.3, consisting of at least two components, was enhanced in lateral-line neuromast tissue (containing hair cells) and was decreased in tactile organs and skin (lacking hair cells). This "neuromast-marker-protein" fraction had a mol. wt close to that of actin but was shown to be different from actin. Two other major proteins, at mol. wts 16 and 28K, were present in gels of skin and absent in gels of lateral-line tissue. These proteins were shown to be due to secretion of the amphibian granular glands and were designated "negative marker proteins".     

Effects of in vivo pretreatment with D-Trp-6-LH-RH on prolactin and LH secretion by pituitary glands in vitro

In order to study a possible direct action of LH-RH analogs on the pituitary lactotrophs, we investigated the effect of long-term in vivo pretreatment with D-Trp-6-LH-RH on in vitro secretion of PRL and luteinizing hormone (LH) by the pituitary glands from male and female rats. In vivo pretreatment with D-Trp-6-LH-RH (50 micrograms/day, SC) for 15 days greatly reduced basal in vitro PRL release (p less than 0.01) in female, but not in male pituitary glands. TRH-stimulated PRL secretion was not affected by pretreatment with D-Trp-6-LH-RH in female rats, but was impaired in male pituitaries. Acute in vitro exposure to D-Trp-6-LH-RH did not modify PRL secretion by female pituitary glands pretreated in vivo with the analog. However, this same in vivo pretreatment greatly decreased PRL release from male pituitaries (p less than 0.01). Basal in vitro LH release by male pituitary glands was partially lowered by in vivo pretreatment with D-Trp-6-LH-RH, as compared to controls (p less than 0.01), while basal LH release in female pituitaries remained at control levels. Finally, D-Trp-6-LH-RH-induced stimulation of in vitro LH release was severely impaired in female pituitaries (p less than 0.01) but only slightly reduced in the males.     

Effect of cyproterone acetate on prolactin secretion in the female rhesus monkey

Summary Adult female rhesus monkeys were given cyproterone acetate orally in doses of 0.04, 0.4, 4 and 40mg per kg per day for 12 weeks. Its effects were assessed on serum prolactin (PRL) concentration, the morphology of the PRL cells, and the development of the mammary glands. Serum PRL was relatively unchanged in the control animals from the fourth through the twelfth weeks of the study. In contrast, PRL was significantly elevated in each group of drug-treated animals during the same time periods. There was no development of the mammary glands nor was there any evidence of milk secretion in the control animals; however, in the monkeys given cyproterone acetate the mammary glands had extensive lobuloalveolar growth and milk-like secretion that could be expressed as early as the fourth or fifth week of the study. By immunocytochemistry and differential light microscopic staining techniques, the PRL cells in the pituitary glands of the experimental animals were found to be more numerous and much larger than those present in the controls. They displayed a well developed Golgi complex and had an abundance of cytoplasmic RNA. These data suggest that PRL secretion is markedly enhanced by cyproterone acetate.Supported in part by USPHS Grant AM12583     

Protein phosphorylation in normal and neoplastic development. Phosphorylation of proteins endogenous to foetal tissues and tumours.

The abilities of proteins endogenous to normal and neoplastic tissues to serve as substrates in a protein-phosphorylation reaction in vitro were compared. After the tissue extracts were incubated with [gamma-32P]ATP, the phosphorylated proteins were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and the dried gels were subjected to radioautography. Considerable incorporation of 32P into a protein of mol.wt. 135000 was observed with extracts from foetal tissues and tumours, but only minimal incorporation into this protein occurred when extracts from adult tissues were used. The ability of this protein to become phosphorylated in vitro may be related to cell proliferation. When ascites cells were incubated with [32P]Pi, one of the major phosphoproteins migrated on sodium dodecyl suphate/polyacrylamide gels at mol.wt. 135000, suggesting that this protein can be phosphorylated both in intact cells and broken-cell preparations. A protein of mol.wt. 87000 was highly phosphorylatable in extracts from solid tumours, but was not phosphorylated in extracts from ascites tumours, foetal or adult tissues. The phosphorylation pattern of these two proteins can thus distinguish solid neoplasms and normal adult tissues from ascites tumours and from foetal tissues. A protein of mol.wt. 49000, which was the most labelled protein in adult tissues, was also one of the major phosphoproteins in foetal and neoplastic tissues. Numerous mechanisms are postulated to explain how the extent of 32P incorporation into a protein could vary as a function of biological state.     

Biosynthesis and axonal transport of rat neurohypophysial proteins and peptides

35S-cysteine injected adjacent to the supraoptic nucleus (SON) of the rat is rapidly incorporated into proteins. These 35S-cysteine-labeled proteins in the SON (1-24 h after injection) were separated by polyacrylamide gel electrophoresis, and the distribution of radioactive proteins on the gels was analyzed. 1 h after injection, about 73% of the radioactivity appeared in two peaks (both about 20,000 mol wt). With time, these peaks (putative precursors of neurophysin) decreased, as a 12,000 mol wt peak (containing two distinct neurophysins) increased in radioactivity. Both the 20,000- and 12,000-mol wt proteins are transported into the axonal (median eminence) and nerve terminal (posterior pituitary) regions of the rat hypothalamo-neurohypophysial system. Conversion of the larger precursor protein to the smaller neurophysin appears to occur, in large part, intra-axonally during axonal transport. Six distinct 35S-cysteine-labeled peptides (less than 2500 mol wt), in addition to arginine vasopressin and oxytocin, are also synthesized in the SON and transported to the posterior pituitary where they are released together with labeled neurophysin by potassium depolarization in the presence of extracellular calcium. These data provide support for the hypothesis that the neurohypophysial peptides (vasopressin and oxytocin) and neurophysins are derived from the post- translational clevage of protein precursors synthesized in the SON, and that the conversion process can occur in the neurosecretory granule during axonal transport.     

Dopamine inhibition of prolactin release but not synthesis in the male Syrian hamster: in vitro studies

The hypothalamic mechanisms controlling prolactin (PRL) cell function in the male Syrian hamster are unclear. Equally unclear is the role of dopamine (DA) in regulating lactotrophic cell activity in long photoperiod-exposed hamsters particularly with respect to PRL synthesis and release. The synthesis of PRL, as measured by the incorporation of 3H-leucine into newly synthesized PRL, by anterior pituitary glands from male hamsters is linear over a five h incubation period. Approximately two-fold more 3H-PRL remained in the pituitary glands than in the medium by the end of the incubation period. The incubation of hamster hemipituitaries with DA at concentrations of either 5 X 10(-7) M or 5 X 10(-5) M, resulted in a 77% to 83% inhibition of the release of immunoreactive PRL into the medium as compared with controls. Similarly, the release of 3H-PRL into the medium was inhibited by 71% to 76% as compared with controls; however, the synthesis of PRL was virtually the same among the experimental and control groups. These results suggest that DA may be an important regulator of short-term PRL release but not synthesis in the long photoperiod-exposed male hamster.     

Two separate receptors for prolactin in the rabbit mammary gland

Rabbit mammary gland PRL receptors in the microsome fraction were solubilized with the zwitterionic detergent Chaps, and were separated into two fractions (Fr. A and B) by ion-exchange chromatography. The number of receptors in Fr. B was about 2.2 times greater than in Fr. A. In sucrose gradient centrifugation analysis, PRL receptors in Fr. A and Fr. B sedimented at different positions. After binding 125I-PRL, the apparent molecular weight (mol wt) of the PRL receptor in Fr. A changed from 42,400 to 65,500 and that in Fr. B changed from 89,400 to 108,000, suggesting that each binding subunit interacts with one PRL molecule. Cross-linking 125I-PRL to receptors revealed little change following SDS-PAGE, in the autoradiogram patterns of the microsome PRL receptors, either in the presence or absence of dithiothreitol. Both the microsome and the Chaps extract contained two major binding subunits (mol wt, 83,200 and 36,800) and one minor subunit (mol wt, 20,800). The mol wt of the dominant PRL receptors in Fr. A and Fr. B were 36,800 and 83,200, respectively. The latter form did not dissociate into a 36,800 mol wt form, suggesting that the rabbit mammary gland contains two independent binding subunits with mol wt of 36,800 and 83,200. Data showed that PRL receptors in the rabbit mammary gland are mostly the high Kd type receptor with a mol wt of 83,200.     

Studies of the cell surface of Paramecium. Ciliary membrane proteins and immobilization antigens.

We have developed a procedure to isolate the ciliary membranes of Paramecium and have analysed the membrane proteins by electrophoresis on polyacrylamide gels containing either Triton X-100 or sodium dodecyl sulphate. The electrophoretic pattern on gels containing sodium dodecyl sulphate showed 12-15 minor bands of mol.wt. 25 000-150 000 and on major band of mol.wt. 200 000-300 000 that contained approximately three-quarters of the total membrane protein. 2. We present evidence that the major membrane protein is related to, but not identical with, the immobilization antigen (i-antigen), which is a large (250 000 mol.w.), soluble, surface protein of Paramecium. The similarity of the i-antigen and the major membrane protein was shown by immunodiffusion and by the electrophoretic mobilities in sodium dodecyl sulphate of these two proteins from Paramecium of serotypes A and B. The non-identity of these two proteins was shown by their different electrophoretic mobilities on Triton X-100 containing gels and their different solubilities. 3. We propose that the major membrane protein and the i-antigen have a precursor-product relationship.     

Detection of xanthine oxidase and immunologically related proteins in fractions from bovine mammary tissue and milk after electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate.

A solid-phase immunoassay was used to detect xanthine oxidase in fractions from bovine mammary glands after electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. Under these conditions the major proportion of xanthine oxidase in either mammary tissue or mild could be recovered as a protein of mol.wt. 150 000. In mammary tissue approx. 80% of the enzyme was in a soluble form and the remainder was accounted for in either 'mitochondrial' or microsomal fractions after tissue homogenization and fractionation. Affinity chromatography of either detergent-solubilized microsomal membranes or postmicrosomal supernatants on immobilized antibody to xanthine oxidase yielded a single protein that cross-reacted with antibody to the enzyme. In milk presumptive degradation products of the enzyme were detected in minor quantities with mol.wts. of 43 000 in the whey fraction and 90 000 in fat-globule membrane. Only the undegraded enzyme was present in the skim-milk membrane fraction. Xanthine oxidase is therefore synthesized and secreted as a protein with a monomeric mol.wt. of 150 000 and is not subjected to extensive proteolytic degradation during the storage of milk in mammary alveoli. The significance of the results is discussed in relation to the overall protein composition of the membranes of milk-fat globules and skim milk.     

Stimulatory action of prolactin on gonadotropin secretion in vitro

The action of prolactin (PRL) on the secretion of gonadotropin was investigated by means of a cell culture system of rat anterior pituitary gland. Anterior pituitary glands were removed from Wistar male rats, enzymatically digested and cultured. Luteinizing hormone (LH) release into medium was increased by adding PRL dose-dependently in the range between 10 ng/ml and 1 microgram/ml. This effect of PRL was further augmented by the presence of either gonadotropin-releasing hormone or estradiol. The intracellular LH concentration was also increased by PRL. PRL also caused an increase in follicle-stimulating hormone release into medium dose-dependently. In conclusion, PRL was shown to stimulate the secretion of gonadotropin at the pituitary level, thus suggesting a paracrine mode of PRL action in the anterior pituitary gland.     

Studies of anterior pituitary-grafted rats: I. Abnormal prolactin response to thyrotropin releasing hormone,clonidine, insulin,and fasting

Although the rat implanted with extra anterior pituitary glands (AP) under the kidney capsule has been widely used as a model of chronic hyperprolactinemia, its hormonal status has not been fully characterized. Using conscious, unrestrained female pituitary-grafted rats and sham-operated littermates, we investigated prolactin (PRL) secretion in response to the following stimuli: thyrotropin releasing hormone (TRH), clonidine, insulin, and fasting. The AP-implanted rats had a greater and more sustained rise in serum PRL after TRH than control rats, reflecting a direct effect of TRH on the ectopic lactotropes. In contrast after clonidine, which acts via the hypothalamus, the serum PRL rose to much higher levels in sham-operated rats than in rats bearing ectopic pituitary tissue. Both insulin-induced hypoglycemia and fasting decreased serum PRL in control rats, but the AP-implanted animals manifested a rise in serum PRL in response to these stimuli. Thus, the AP-implanted rat is not only a valid model of excess and abnormal PRL secretion, but it may also be useful for distinguishing between stimuli requiring an intact hypothalamic-pituitary unit and agents which act directly on the pituitary gland.     

Discovery of a Novel Prolactin in Non-Mammalian Vertebrates: Evolutionary Perspectives and Its Involvement in Teleost Retina Development

Background

The three pituitary hormones, viz. prolactin (PRL), growth hormone (GH) and somatolactin (SL), together with the mammalian placental lactogen (PL), constitute a gene family of hormones with similar gene structure and encoded protein sequences. These hormones are believed to have evolved from a common ancestral gene through several rounds of gene duplication and subsequent divergence.

Principal Findings

In this study, we have identified a new PRL-like gene in non-mammalian vertebrates through bioinformatics and molecular cloning means. Phylogenetic analyses showed that this novel protein is homologous to the previously identified PRL. A receptor transactivation assay further showed that this novel protein could bind to PRL receptor to trigger the downstream post-receptor event, indicating that it is biologically active. In view of its close phylogenetic relationship with PRL and also its ability to activate PRL receptor, we name it as PRL2 and the previously identified PRL as PRL1. All the newly discovered PRL2 sequences possess three conserved disulfide linkages with the exception of the shark PRL2 which has only two. In sharp contrast to the classical PRL1 which is predominantly expressed in the pituitary, PRL2 was found to be mainly expressed in the eye and brain of the zebrafish but not in the pituitary. A largely reduced inner nuclear layer of the retina was observed after morpholino knockdown of zebrafish PRL2, indicating its role on retina development in teleost.

Significance

The discovery of this novel PRL has revitalized our understanding on the evolution of the GH/PRL/SL/PL gene family. Its unique expression and functions in the zebrafish eye also provide a new avenue of research on the neuroendocrine control of retina development in vertebrates.     

The met-enkephalin analog FK 33-824 directly inhibits ACTH release by the rat pituitary gland in vitro

Systematic administration of the enkephalin analog FK 33-824 was previously shown to stimulate PRL secretion and to inhibit ACTH secretion in man. Naloxone prevented the effect on PRL release, but not on ACTH release. In this study, the direct action of this analog on hormone release by rat anterior pituitary lobes $\text{in}$$\text{vitro}$ were investigated. 1 uM FK 33-824 inhibited basal ACTH secretion by anterior pituitary glands in vitro, while 0.1 uM and 1 uM attenuated the lysine vasopressin stimulated ACTH release. Naloxone did not reverse the inhibitory action of the analog on ACTH release. β-Endorphin (0.01 - 1 uM) did not directly affect ACTH release. Basal and dopamine-induced inhibition of PRL release by anterior pituitary glands was neither influenced by FK 33-824 (0.1 and 1 uM), nor by β-endorphin (0.1 and 1 uM) with or without bacitracin. This study shows that the long-acting met-enkephalin analog FK 33-824 differentially affects PRL and ACTH secretion by the pituitary gland. It seems to stimulate PRL release at a suprapituitary site and this action probably involves u opiate receptors, because naloxone prevents these stimulatory effects. The inhibitory effect of FK 33-824 on ACTH release, however, is mediated via a direct effect at the pituitary level, which does not involve u receptors, as naloxone did not prevent this effect. In this respect, its action differs from that of β-endorphin, which does not directly affect ACTH release by the anterior pituitary gland.     

Inhibition of prolactin secretion by gastrin releasing peptide (GRP) in the rat

Synthetic gastrin releasing peptide (GRP) injected intraventricularly (1 microgram/rat), but not intravenously, suppressed rat prolactin (PRL) release induced by a Met-enkephalin analog, FK33-824 (10 micrograms/100 g body wt., iv). GRP also blunted PRL release induced by a dopamine antagonist, domperidone (1 microgram/100 g body wt., iv). In contrast, GRP did not suppress elevated plasma PRL levels sustained by a large dose of domperidone (10 micrograms/100 g body wt., iv). GRP (10(-5) M) had no effect on PRL release from superfused pituitary cells in vitro. These results suggest that GRP inhibits PRL secretion in the rat by acting through the brain to stimulate the dopaminergic mechanism.     

Estrogen modulates neuromedin B effects on thyrotropin and prolactin release in vitro

Neuromedin B(NB), a bombesin-like peptide, has been shown to inhibit thyrotropin (TSH) release in pituitary explants of male rats and to stimulate Prolactin (PRL) release in male pituitary cell cultures. We investigated the effect of estrogen status of female rats on the response of thyrotrophs and lactotrophs to neuromedin B (NB) in vitro. Ovariectomized rats were treated with near-physiological or high doses of 17beta estradiol benzoate (0.7 or 14 EB microg/100 gBW/daily, 10 days) or with vehicle (OVX). EB treatment induced a dose-dependent increase in serum prolactin and an increase in pituitary NB content, measured by specific RIA, that was similar in both EB groups (P < 0.05). TSH release from isolated hemipituitaries of OVX rats was significantly reduced (P < 0.05) in the presence of 10(-7) M NB. OVX + EB0.7 glands responded to NB with a not statistically significant dose-dependent decrease in TSH release. However, glands from hyperestrogenized rats (OVX + EB14) required a higher dose (10(-5) M) of NB to inhibit TSH release (P < 0.05). PRL release was highly increased (p < 0.001) by the presence of 10(-5) M NB only in glands of hyperestrogenized rats, while no effect of NB was observed in the other groups. In conclusion, estrogen status of female rats modulates the inhibitory effect of NB on TSH release in vitro and hyperestrogenism is required for stimulatory effect of NB on PRL release in vitro. It is suggested that the induction of PRL release by neuromedin B is a pharmacological rather than a physiological effect, but neuromedin B may contribute to the increased release of PRL associated with hyperestrogenism.     

Studies of anterior pituitary-grafted rats: II. Normal growth hormone secretion

Of the various animal models used to study chronic hyperprolactinemia, the otherwise intact rat implanted with extra anterior pituitary glands (AP) under the kidney capsule is assumed to be normal except for excess circulating prolactin (PRL). Since the ectopic glands contain numerous somatotropes in addition to abundant and active lactotropes, it was important to assess growth hormone (GH) secretion as well in this model of hyperprolactinemia. The structural and functional similarities of PRL and GH are such that it is necessary to demonstrate that metabolic abnormalities noted in AP-implanted rats are due to hyperprolactinemia and not to altered GH secretion. AP-implanted female rats have significantly higher resting serum PRL concentrations when compared to sham-operated control rats, but baseline serum GH levels are similar in normal and pituitary-grafted rats. Suppression of GH by insulin and clonidine is comparable in AP-implanted and control rats. The intrasellar pituitary GH concentration is also similar (ca. 20 μg/mg wet weight) in hyperprolactinemic and normal rats. We conclude that GH secretion is normal in the non-hypophysectomized AP-implanted rat, in contrast to the hypophysectomized AP-implanted rat model which has been reported to have diminished GH secretion. Despite the presence of recognizable somatotropes, the ectopic anterior pituitary does not appear to secrete significant amounts of GH, making the intact rat bearing multiple pituitary grafts an excellent model of chronic hyperprolactinemia.     

Molecular organization of prolactin granules. II. Characterization of glycosaminoglycans and glycoproteins of the bovine prolactin matrix

Prolactin (PRL) granules can be isolated from the anterior pituitary gland of adult cows in nearly 50% yield by use of a procedure previously developed for the fractionation of the rat pituitary. Treatment of the isolated bovine granules with 0.2% Lubrol PX results in the solubilization of most membranes present in the fractin but has only a limited effect on the matrices, which remain aggregated and can be recovered and purified by gradient centrifugation. These membraneless PRL granules, studied in detail by morphological and biochemical techniques, were found to contain only small amounts of contaminants (primarily growth hormone granules and small membrane fragments). SDS polyacrylamide gel electrophoresis revealed that, in comparison with other fractions isolated from the bovine pituitary, the membraneless granules have a simpler polypeptide composition including PRL (approximately 85%), growth hormone (approximately 8%), as well as approximately 13 minor bands with apparent mol wt ranging from 80,000 go 45,000. Many of these minor bands are accounted for by glycoproteins, as revealed by their binding of 125I-concanavalin A, and two of these are also stained blue by the stains-all procedure, a reaction specific for acidic glycoconjugates. Chemical analyses of the membraneless granule fractin revealed the presence of a heterogeneous mixture of complex carbohydrates. Among glycosaminoglycans, the major component is heparan sulfate, while hyaluronic acid and chondroitin sulfate ar present in smaller amounts. Moreover, some of the glycoproteins are sulfated and account for over 50% of the nondialyzable 35S radioactivity found in the fraction isolated from labeled slices. Although the concentration of glycosaminoglycans and glycoproteins is relatively low in membraneless granules, the possibility that their presence in the fraction is largely due to cross- contamination and/or artifactual adsorption could be excluded on two grounds. These are: (a) electron microscope radiautography of preparations obtained from [35S]sulfate- and D-[6-3H]glucosamine- labeled slices showed a significant labeling of PRL granules in both intact cells and membraneless granule pellets, and (b) a mixing experiment showed that membraneless granules contain very little macromolecular sulfate radiactivity adsorbed from the soluble glycoconjugates present in the pituitary homogenate.     

Cortisol rapidly suppresses intracellular calcium and voltage-gated calcium channel activity in prolactin cells of the tilapia (Oreochromis mossambicus)

Cortisol was previously shown to rapidly (10-20 min) reduce the release of prolactin (PRL) from pituitary glands of tilapia (Oreochromis mossambicus). This inhibition of PRL release by cortisol is accompanied by rapid reductions in (45)Ca(2+) and cAMP accumulation. Cortisol's early actions occur through a protein synthesis-independent pathway and are mimicked by a membrane-impermeable analog. The signaling pathway that mediates rapid, nongenomic membrane effects of glucocorticoids is poorly understood. Using the advantageous characteristics of the teleost pituitary gland from which a nearly pure population of PRL cells can be isolated and incubated in defined medium, we examined whether cortisol rapidly reduces intracellular free calcium (Ca(i)(2+)) and suppresses L-type voltage-gated ion channel activity in events that lead to reduced PRL release. Microspectrofluorometry, used in combination with the Ca(2+)-sensitive dye fura 2 revealed that cortisol reversibly reduces basal and hyposmotically induced Ca(i)(2+) within seconds (P < 0.001) in dispersed pituitary cells. Somatostatin, a peptide known to inhibit PRL release through a membrane receptor-coupled mechanism, similarly reduces Ca(i)(2+). Under depolarizing [K(+)], the L-type calcium channel agonist BAY K 8644, a factor known to delay the closing of L-type Ca(2+) channels, stimulates PRL release in a concentration-dependent fashion (P < 0.01). Cortisol (and somatostatin) blocks BAY K 8644-induced PRL release (P < 0.01; 30 min), well within the time course over which its actions occur, independent of protein synthesis and at the level of the plasma membrane. Results indicate that cortisol inhibits tilapia PRL release through rapid reductions in Ca(i)(2+) that likely involve an attenuation of Ca(2+) entry through L-type voltage-gated Ca(2+) channels. These results provide further evidence that glucocorticoids rapidly modulate hormone secretion via a membrane-associated mechanism similar to that observed with the fast effects of peptides and neurotransmitters.