Alternative splicing determines sensitivity of murine calcium-activated potassium channels to glucocorticoids.

1. Large-conductance Ca(2+)- and voltage-activated potassium (BK) channels are important regulators of cellular excitability. Here, we present a patch-clamp electrophysiological analysis of splice-variant-specific regulation by the synthetic glucocorticoid dexamethasone (DEX) of BK channels consisting of cloned STREX or ZERO alpha-subunit variants expressed in human embryonic kidney (HEK 293) cells. 2. STREX channels in isolated membrane patches were inhibited by protein kinase A (PKA) and this was blocked on pre-treatment of intact cells with DEX (100 nM) for 2 h. 3. The effect of DEX required the synthesis of new mRNA and protein. Furthermore, it required protein phosphatase 2A (PP2A)-like activity intimately associated with the channels, as it was blocked by 10 nM okadaic acid but not by the specific protein phosphatase-1 inhibitor peptide PPI-2. 4. ZERO variant channels that lack the STREX insert were activated by PKA but were not influenced by DEX. ZERO channels containing a mutant STREX domain (S4(STREX)A) were also activated by PKA. Importantly, DEX blocked PKA activation of S4(STREX)A channels in a PP2A-dependent manner. 5. Taken together, the STREX domain is crucial for glucocorticoid regulation of BK channels through a PP2A-type enzyme. Moreover, glucocorticoids appear to induce a generic set of proteins in different types of cells, the actions of which depend on the expression of cell-specific targets.

Expression of adenylyl cyclase type IX and calcineurin in synapses of the central nervous system.

Distribution of type IX adenylyl cyclase and protein phosphatase calcineurin in the brain and in cultured hippocampal neurons from albino rat was immunohistochemically studied. Both enzymes were detected simultaneously in all synaptic structures of most cerebral neurons except for presynaptic sites, where calcium-inhibited type IX adenylyl cyclase was absent.

Characterisation of human adenylyl cyclase IX reveals inhibition by Ca(2+)/Calcineurin and differential mRNA plyadenylation.

The functional diversity of adenylyl cyclases provides for different modes of cyclic AMP signalling in mammals. This study reports the cloning and functional characterisation of a cDNA encoding human adenylyl cyclase IX (ACIX). The data show that human ACIX is a Ca(2+)/calcineurin-inhibited adenylyl cyclase prominently expressed in vital organs, including brain, heart, and pancreas. ACIX mRNA was detected in several brain regions, including neocortex, hippocampus, striatum, and cerebellum. By in situ hybridisation, ACIX mRNA was localised to pyramidal and granule cells of the hippocampus, indicating that it is expressed predominantly in nerve cells. Further analysis of ACIX mRNA expression revealed two major forms of ACIX mRNA that arose through tissue-specific differential mRNA polyadenylation. Taken together, the data show that (a) human ACIX is under inhibitory control by Ca(2+) through calcineurin, (b) ACIX may be involved in higher brain functions, and (c) post-transcriptional regulation of ACIX gene expression is a species-specific control mechanism that may enhance the versatility of cyclic AMP signalling in humans.

Molecular diversity of cyclic AMP signalling.

Several neuroendocrine control systems are prominently controlled by G-protein coupled receptors that activate the cAMP signal transduction pathway. The discovery of multiple genes that encode the molecular machinery of cAMP metabolism has revolutionized our knowledge of cAMP mediated processes. This perhaps all too familiar second messenger can be generated by nine different membrane enzymes in the context of varied levels of activation of G proteins as well as Ca(2+)- and protein kinase C-dependent processes. The amplitude, length and subcellular distribution of the cAMP signal are further modulated by over twenty functionally distinct isotypes of cAMP-degrading phosphodiesterases in a cell- and stimulus-specific manner. The present review summarizes the key properties of the molecular machinery that generates the cAMP signal and highlights how it is deployed in neuroendocrine systems.

Molecular components of large conductance calcium-activated potassium (BK) channels in mouse pituitary corticotropes.

Large-conductance calcium- and voltage- activated potassium (BK) channels play a fundamental role in the signaling pathways regulating mouse anterior pituitary corticotrope function. Here we describe the cloning and functional characterization of the components of mouse corticotrope BK channels. RT-PCR cloning and splice variant analysis of mouse AtT20 D16:16 corticotropes revealed robust expression of mslo transcripts encoding pore-forming alpha-subunits containing the mouse homolog of the 59-amino acid STREX-1 exon at splice site 2. RT-PCR and functional analysis, using the triterpenoid glycoside, DHS-1, revealed that native corticotrope BK channels are not functionally coupled to beta-subunits in vivo. Functional expression of the STREX-1 containing alpha-subunit in HEK 293 cells resulted in BK channels with calcium sensitivity, single-channel conductance, and inhibition by protein kinase A identical to that of native mouse corticotrope BK channels. This report represents the first corticotrope ion channel to be characterized at the molecular level and demonstrates that mouse corticotrope BK channels are composed of alpha-subunits expressing the mouse STREX-1 exon.

Circadian changes of type II adenylyl cyclase mRNA in the rat suprachiasmatic nuclei.

Circadian functions of the suprachiasmatic nuclei (SCN) are influenced by cyclic AMP (cAMP). Adenylyl cyclase type II (AC-II) is a cAMP-generating enzyme which, in the context of activation by Gsalpha, is further stimulated by protein kinase C or G protein betagamma subunits. Using in situ hybridization we have found a biphasic variation in AC-II mRNA within the rat SCN during the light-dark cycle (peaks at Zeitgeber time 6 and 18) and also in constant darkness (peaks at circadian time 2 and 14). The cingulate cortex showed no such variation. These findings suggest that circadian changes in AC-II expression may be pertinent to the rhythmic functions of the SCN.

Ca2+/calcineurin-inhibited adenylyl cyclase, highly abundant in forebrain regions, is important for learning and memory.

Activation of cAMP synthesis by intracellular Ca2+ is thought to be the main mode of cAMP generation in the brain. Accordingly, the Ca2+-activated adenylyl cyclases I and VIII are expressed prominently in forebrain neurons. The present study shows that the novel adenylyl cyclase type IX is inhibited by Ca2+ and that this effect is blocked selectively by inhibitors of calcineurin such as FK506 and cyclosporin A. Moreover, adenylyl cyclase IX is inhibited by the same range of intracellular free Ca2+ concentrations that stimulate adenylyl cyclase I. Adenylyl cyclase IX is expressed prominently in the forebrain. Substantial arrays of neurons positive for AC9 mRNA were found in the olfactory lobe, in limbic and neocortical areas, in the striatum, and in the cerebellar system. These data show that the initiation of the cAMP signal by adenylyl cyclase may be controlled by Ca2+/calcineurin and thus provide evidence for a novel mode of tuning the cAMP signal by protein phosphorylation/dephosphorylation cascades.

Depolarization counteracts glucocorticoid inhibition of adenohypophysical corticotroph cells.

In AtT20 mouse corticotroph tumour cells large conductance Ca2+-activated K+-channels (BK-channels) have an essential role in the early glucocorticoid inhibition of adrenocorticotrophin (ACTH) secretion evoked by corticotrophin-releasing factor. The present study examined whether or not BK-channels are also pivotal to glucocorticoid inhibition of normal rat anterior pituitary cells. A membrane-permeant, non-metabolizable cyclic AMP analogue, 8-(4-Chlorophenylthio)adenosine-3',5'-cyclic-monophosphate (CPT-cAMP) was used as the primary secretagogue stimulus, as this mimics the increase of intracellular cyclic AMP caused by corticotrophin-releasing factor, but is not subject to the complex Ca2+-dependent regulation of cyclic AMP metabolism that is evident in corticotroph cells. Experiments in AtT20 cells showed that ACTH secretion stimulated by 1 mM CPT-cAMP was suppressed to 34+/-1.5% (n = 12) of the control stimulus by a maximal dose of 100 nM dexamethasone. The ACTH secretion evoked by the combination of 1 mM CPT-cAMP with either 5 microm (-)BayK8644 (L-type Ca2+-channel activator) or 5 mM TEA (K+-channel blocker) was respectively 69.1+/-7.6% and 69.3+/-11.8% of control after 2 h preincubation with 100 nM dexamethasone (P<0.05 vs CPT-cAMP). The ACTH response elicited by 5 microM (-)BayK8644 and 5 mM TEA given together was completely resistant to inhibition by 100 nM dexamethasone. Furthermore, TEA and (-)BayK8644 given together synergistically stimulated ACTH release in combination with 0.1 mM or 1 mM CPT-cAMP, and these ACTH responses were not inhibited by 100 nM dexamethasone. In primary cultures of rat anterior pituitary cells, TEA (up to 20 mM), charybdotoxin (30 nM) or apamin (100 nM) failed to modify the glucocorticoid inhibition of 0.1 mM CPT-cAMP-induced ACTH release. The combination of 5 mM TEA and 5 microM (-)BayK8644 elicited a small but significant increase in ACTH secretion but did not modify the inhibition of 0.3 mM CPT-cAMP-induced ACTH secretion by 100 nM dexamethasone. In primary cultures of rat anterior pituitary cells, depolarization of the membrane potential with 40 mM KCl enhanced the ACTH response to CPT-cAMP and markedly reduced the maximal inhibitory effect of dexamethasone to 55+/-1.2% as well as that of corticosterone to 33+/-2.1% vs 100+/-2.5% and 100+/-1.9% inhibition respectively, when 0.1 mM CPT-cAMP was used alone. Introduction of 5 microM (-)BayK8644 with 40 mM KCl in this system had no additional effect on glucocorticoid inhibition. No glucocorticoid inhibition of ACTH release to any of the stimuli applied was observed in cells pretreated with the mRNA synthesis inhibitor, 5,6-dichloro-furanosyl-benzimidazole riboside (DRB) (0.1 mM) or the protein synthesis blocker, puromycin (0.1 mM). In summary, early glucocorticoid inhibition of stimulated ACTH release by cultured rat anterior pituitary cells was dependent on the synthesis of new mRNA and protein. Depolarization of the membrane potential potentiated CPT-cAMP-induced ACTH secretion in AtT20 cells as well as cultured rat corticotrophs and this was associated with a resistance to the early inhibitory effect of glucocorticoids. Glucocorticoid inhibition in rat anterior pituitary corticotrophs was unaltered by TEA, charybdotoxin as well as apamin, and hence it is unlikely to involve predominantly BK-or SK-type Ca2+-activated K+-channels. These results support the thesis that a prime target of glucocorticoid feedback inhibition in anterior pituitary corticotrophs is the membrane potential and indicate that glucocorticoid-induced proteins regulate the activities of several distinct plasma membrane ion channels.

Involvement of calyculin A inhibitable protein phosphatases in the cyclic AMP signal transduction pathway of mouse corticotroph tumour (AtT20) cells.

1. The role of non-calcineurin protein phosphatases in the cyclic AMP signal transduction pathway was examined in mouse pituitary corticotroph tumour (AtT20) cells. 2. Blockers of protein phosphatases, calyculin A and okadaic acid, were applied in AtT20 cells depleted of rapidly mobilizable pools of intracellular calcium and activated by various cyclic AMP generating agonists. Inhibitors of cyclic nucleotide phosphodiesterases were present throughout. The accumulation of cyclic AMP was monitored by radioimmunoassay, phosphodiesterase activity in cell homogenates was measured by radiometric assay. 3. Neither calyculin A nor okadaic acid altered basal cyclic AMP levels but cyclic AMP formation induced by 41 amino acid residue corticotrophin releasing-factor (CRF) was strongly inhibited (up to 80%), 1-Norokadaone was inactive. Similar data were also obtained when isoprenaline or pituitary adenylate cyclase activating peptide1-38 were used as agonists. 4. Pertussis toxin did not modify the inhibition of CRF-induced cyclic AMP production by calyculin A. 5. Pretreatment with calyculin A completely prevented the stimulation of cyclic AMP formation by cholera toxin even in the presence of 0.5 mM isobutylmethylxanthine (IBMX) and 0.1 mM rolipram. Cholera toxin mediated ADP-ribosylation of the 45 K and 52 K molecular weight Gs alpha isoforms in membranes from calyculin A-pretreated cells was enhanced to 150-200% when compared with controls. 6. Cholera toxin-induced cyclic AMP was reduced by calyculin A within 10 min when calyculin A was applied after a 90 min pretreatment with cholera toxin. Under these conditions the effect of calyculin A could be blocked by the combination of 0.5 mM IBMX and 0.1 mM rolipram, but not by 0.5 mM IBMX alone. 7. Phosphodiesterase activity in AtT20 cell homogenates showed a significant, 2.7 fold increase after treatment with calyculin A. In control cells phosphodiesterase activity was blocked by 80% in the presence of IBMX (0.5 mM), or IBMX plus rolipram (0.1 mM). In calyculin A-treated cells phosphodiesterase activity was also strongly inhibited by IBMX, but because of the stimulating effect of calyculin A, the activity remaining was still 55% of that found in control homogenates. This activity was reduced to 5% of control by using IBMX and rolipram in combination. Assay of phosphodiesterase in Ca2+ free conditions showed that calyculin A markedly increases the activity of rolipram sensitive (type 4) phosphodiesterase. 8. Taken together, blockers of protein phosphatases (PPases) impaired signal transduction through Gs-mediated pathways and activated cyclic AMP degrading phosphodiesterase(s), indicating that PPases 1 and/or 2A are essential for agonist-mediated regulation of cyclic AMP levels in AtT20 cells, and are thus important in maintaining the secretory phenotype of the cells.

Calcium regulation of adenylyl cyclase relevance for endocrine control.

A fundamental process in the hormonal regulation of body functions is the conversion of the intercellular signal into an intracellular signal. The first recognized intracellular messengers mediating the actions of hormones were calcium ions (Ca(2+)) and adenosine 3':5' monophosphate (cAMP), which is synthesized from ATP by adenylyl cyclase. Recent work on the structure of adenylyl cyclases has shown that these enzymes are individually tailored molecular machines controlled by diverse Ca(2+)-dependent mechanisms. These include allosteric regulation of enzyme activity through the Ca(2+)-receptor protein calmodulin, apparently direct actions of Ca(2+)on the cyclase catalytic moiety and phosphorylation/dephosphorylation by Ca(2+)-regulated protein kinases and protein phosphatases. This article is a brief review of the recent developments in the area of cyclase control that forecast a major revival of the interest in cAMP-Ca(2+)interactions. (c) 1997, Elsevier Science Inc. (Trends Endocrinol Metab 1997;8:7-14).

Mortyn Jones Memorial Lecture--1995. Calcium checks cyclic AMP--corticosteroid feedback in adenohypophysial corticotrophs.

This paper summarizes a particular aspect of the stress response-the negative feedback control of anterior pituitary adrenocorticotrophin secretion with special focus on the mechanism of action of protein(s) rapidly induced by glucocorticoids. The main thesis is that the principal intracellular mechanism underlying corticosteroid inhibition of corticotroph secretory function is the opposition of cAMP-mediated activation by calcium ions. An increase of intracellular cAMP levels in corticotrophs produces a rise in intracellular free Ca2+ known to be essential for triggering hormone secretion. In parallel, calcium regulates agonist-induced cAMP accumulation through inhibition of adenylyl cyclase and the stimulation of cAMP-degrading phosphodiesterase. Furthermore, a key action of cAMP is the inhibition of a slow, sustained potassium current which is activated by calcium ions. Collectively, the actions of calcium constitute a powerful intracellular feedback inhibition of cAMP-induced cellular activation. Analysis of corticosteroid action in mouse corticotroph tumour (AtT20) cells indicates that the essence of corticosteroid feedback inhibition is the amplification of intracellular calcium feedback. A common mediator of the inhibitory actions of calcium may be the calcium receptor protein calmodulin the de novo synthesis of which is rapidly stimulated by glucocorticoid hormones. Targets of glucocorticoid-induced calmodulin may include the protein phosphatase calcineurin, calmodulin-activated phosphodiesterase(s), and BK-type potassium channels. The net result of calcium feedback inhibition is a reduction of Ca2+ available for the facilitation of secretory activity i.e. calcium-induced desensitization. It is proposed that the intracellular calcium feedback loop outlined above also operates in the CNS components of negative corticosteroid feedback. A personal note: Professor Mortyn Jones introduced me to this field of research. His open-minded and critical approach to experimental work has always remained a guiding principle for my own efforts, and I hope that this paper which is dedicated to his memory will be found worthy of its purpose.

Glucocorticoids block protein kinase A inhibition of calcium-activated potassium channels.

Adrenal corticosteroids have well known and profound effects on neurons and neuroendocrine cells, but the underlying cellular mechanisms are poorly understood. The present study analyzed membrane currents and ACTH release in AtT20 mouse pituitary corticotrope tumor cells. Patch-clamp analysis revealed a significant and selective inhibition of calcium-activated (BK-type) potassium channels upon activation of protein kinase A by corticotropin-releasing factor or 8-chlorophenylthio-cAMP. The synthetic glucocorticoid dexamethasone had no effect on potassium currents evoked by depolarization but prevented the inhibitory effect of protein kinase A activators. The action of dexamethasone had the hallmarks of protein induction, i.e. a lag time and sensitivity to inhibitors of DNA transcription and mRNA translation. In parallel, the specific BK channel blocker iberiotoxin abolished early glucocorticoid inhibition of corticotropin-releasing factor-stimulated ACTH secretion. In summary, the present data show that glucocorticoid-induced proteins render BK-type channels resistant to inhibition by protein kinase A and that this action of the steroid is pivotal for its early inhibitory effect on the secretion of ACTH.

Calcineurin feedback inhibition of agonist-evoked cAMP formation.

The effects of immunosuppressant blockers of calcineurin (protein phosphatase 2B) on cAMP formation and hormone release were investigated in mouse pituitary tumor (AtT20) cells. Immunosuppressants enhanced corticotropin-releasing factor- and isoproterenol-evoked cAMP production in proportion with their potency to block calcineurin. Further analysis of cAMP production revealed that intracellular Ca2+ derived through voltage-regulated calcium channels reduces cAMP formation induced by corticotropin releasing-factor or beta 2-adrenergic stimulation and that this effect of Ca2+ is inhibited by blockers of calcineurin. AtT20 cells were found to express at least three species of adenylyl cyclase mRNA-encoding types 1 and 6 as well as a novel isotype, which appeared to be the predominant species. In two cell lines expressing very low or undetectable levels of the novel cyclase mRNA (NCB20 and HEK293 cells respectively), corticotropin-releasing factor-induced cAMP formation was not altered upon blockage of calcineurin activity. These data identify calcineurin as a Ca2+ sensor that mediates the negative feedback effect of intracellular Ca2+ on receptor-stimulated cAMP production. Furthermore, the effect of calcineurin on cAMP synthesis appears to be associated with the expression of a novel adenylyl cyclase isotype, which is highly abundant in AtT20 cells.

Control of a novel adenylyl cyclase by calcineurin.

The molecular complex formed by the immunosuppressant FK506 and the immunophilin protein FKBP12 potently inhibits the Ca2+/calmodulin-activated protein phosphatase calcineurin. This mechanism appears to be common to all types of cell, implying that fundamental physiological modes of calcineurin regulation are exploited by immunosuppressants. The present paper describes a novel adenylyl cyclase regulated by calcineurin that contains an FKBP12-like domain and may thus constitute a physiologically relevant calcineurin docking site mimicked by immunosuppressant-immunophilin complexes. The enzyme messenger RNA is particularly enriched in the cerebral cortex, striatum and hippocampus, where it is localized to neuronal perikarya, indicative of an important role in neuronal function.

Selective enhancement of an A type potassium current by dexamethasone in a corticotroph cell line.

Perforated patch recording was used to examine the effect of the synthetic steroid dexamethasone on the whole cell potassium (K+) current, in the mouse corticotroph tumour cell line AtT20/D16-16. In 15 out of 52 control cells (29%) there was a rapidly-activating, rapidly-inactivating K+ current of the A type, the amplitude of which was strongly dependent on the holding potential in use prior to its activation by depolarising voltage pulses, and which was blocked by 1 mM 4-aminopyridine (4-AP, n = 5). The effect of dexamethasone (100 nM, 2 h, 37 degrees C) was that the A current increased in prevalence (24 out of 31 cells, 77%), lost its dependence on holding potential (over the range studied), and as a result became significantly larger than in controls, for certain voltage steps (peak A current density was 18.5 +/- 2.4 pA/pF (n = 12) for control cells and 26.3 +/- 3.9 pA/pF (n = 18) for dexamethasone treated cells, for a step to +30 mV from -60 mV, values are mean +/- SEM). All cells exhibited a slowly-activating, sustained K+ current, which was unaffected by changes in the holding potential, unaffected by 4-AP and consisted of at least 3 components: one blocked by 30 mM tetraethylammonium(TEA) or 100 nM charybdotoxin (CTX); a second blocked by 100 nM apamin; and a third not blocked by TEA, CTX, apamin, clofilium (100 nM) or niflumic acid (0.1 mM). Dexamethasone produced no change in the slowly-activating, sustained current nor in any of its individual components. The effect of dexamethasone on the A current was completely blocked by 0.1 mM puromycin, a protein synthesis blocker, while puromycin alone did not affect the size or frequency of the A current, nor alter the slowly-activating, sustained current. Secretion studies using 4-AP confirmed that the A current has a role in stimulated adrenocorticotrophic hormone (ACTH) secretion. In summary, AtT20 cells contain at least four types of K+ current: an A current and 3 currents contributing to the slowly-activating current. Selective enhancement of the A current by dexamethasone, shown here to require synthesis of new protein, is one of the mechanisms whereby glucocorticoids exert inhibitory control on ACTH secretion.

Inhibitory role for calcineurin in stimulus-secretion coupling revealed by FK506 and cyclosporin A in pituitary corticotrope tumor cells.

The properties of the calcium/calmodulin-dependent protein phosphatase calcineurin and its potential role in stimulus-secretion coupling were examined in AtT20 mouse pituitary corticotrope tumor cells. Protein phosphatase activity was assayed by measuring the liberation of 32P from 32P-casein, adrenocorticotropin secretion was measured by radioimmunoassay. About 60% of the total phosphatase activity was inhibited by 500 nM okadaic acid, suggesting the presence of protein phosphatases 1 and/or 2A. A further 25-30% reduction of phosphatase activity was achieved by chelating free calcium. Addition of the EF-hand protein blocker trifluoperazine or a calcineurin autoinhibitory peptide fragment markedly reduced okadaic acid resistant and calcium-dependent protein phosphatase activity indicating that calcium-dependent 32P release is largely due to calcineurin (protein phosphatase 2B). The remaining 10-15% of total activity was Mg2+ dependent and blocked by NaF, hence possibly due to protein phosphatase 2C. Calcineurin activity was inhibited by the immunosuppressants FK506 and cyclosporin A, either when added to the cell lysates or after preincubation of intact cells with the drugs for 30 min at 37 degrees C. When added to lysates, cyclosporin A inhibited calcium/calmodulin-dependent phosphatase more effectively than FK506. However, when tested on intact cells, FK506 proved 10-fold more potent than cyclosporin A. Both immunosuppressive agents enhanced the calcium-dependent release of adrenocorticotropic hormone into the medium, once more, FK506 was 10-fold more potent than cyclosporin A. Taken together, these data suggest that calcineurin is an inhibitory element in the signal transduction pathway controlling exocytotic secretion in pituitary cells that express voltage-operated calcium channels. This is in direct contrast with leukocytes where voltage-operated calcium channels are not found, and calcineurin is an important element for agonist-induced activation.

Specific oxytocin agonist stimulates prolactin release but has no effect on inositol phosphate accumulation in isolated rat anterior pituitary cells.

We have previously characterized specific oxytocin receptors in the rat anterior pituitary gland, using a highly selective oxytocin receptor antagonist as radioligand. The aim of the present study was to examine whether occupation of these receptors by oxytocin produces a stimulation of prolactin release and a rise in the accumulation of total inositol phosphates in the rat adenohypophysis. Anterior pituitary cells harvested from randomly cycling and diethylstilboestrol (100 micrograms s.c.)-treated rats were perifused with Dulbecco's minimal essential medium at a rate of 0.3 ml/min. Oxytocin and the specific oxytocin agonist [Thr4-Gly7]-oxytocin (TG-OT) both stimulated a significant prolactin release at concentrations of 10(-6) and 10(-7) M. Oestrogen treatment did not affect the response to oxytocin, indicating that there is no straightforward correlation between receptor number and prolactin secretory response in the rat pituitary gland. The involvement of phosphoinositide hydrolysis was investigated in dispersed anterior pituitary cells and uterine tissue from randomly cycling rats. Oxytocin and arginine-vasopressin stimulated a significant (P < 0.05) and dose-related increase in total inositol phosphates, vasopressin being more potent. The specific oxytocin agonist TG-OT had no effect on total inositol phosphate production in pituitary cells, but when tested in uterine tissue it significantly (P < 0.05) stimulated the accumulation of total inositol phosphate at all concentrations tested (10(-5) to 10(-9) M). In conclusion, the data show that oxytocin has prolactin-releasing activity, acting on specific receptors in the anterior pituitary gland.(ABSTRACT TRUNCATED AT 250 WORDS)