Dispersed Rat Anterior Pituitary Cells Research Articles

Neuropeptide W stimulates adrenocorticotrophic hormone release <i>via</i> corticotrophin-releasing factor but not <i>via</i> arginine vasopressin

Neuropeptide W (NPW) was isolated as an endogenous ligand for NPBWR1, an orphan G protein-coupled receptor localized in the rat brain, including the paraventricular nucleus. It has been reported that central administration of NPW stimulates corticosterone secretion in rats. We hypothesized that NPW activates the hypothalamic-pituitary-adrenal (HPA) axis via corticotrophin-releasing factor (CRF) and/or arginine vasopressin (AVP). NPW at 1 pM to 10 nM did not affect basal or ACTH-induced corticosterone release from dispersed rat adrenocortical cells, or basal and CRF-induced ACTH release from dispersed rat anterior pituitary cells. In conscious and unrestrained male rats, intravenous administration of 2.5 and 25 nmol NPW did not affect plasma ACTH levels. However, intracerebroventricular (icv) administration of 2.5 and 5.0 nmol NPW increased plasma ACTH levels in a dose-dependent manner at 15 min after stimulation (5.0 vs. 2.5 nmol NPW vs. vehicle: 1802 ± 349 vs. 1170 ± 204 vs. 151 ± 28 pg/mL, respectively, mean ± SEM). Pretreatment with astressin, a CRF receptor antagonist, inhibited the increase in plasma ACTH levels induced by icv administration of 2.5 nmol NPW at 15 min (453 ± 176 vs. 1532 ± 343 pg/mL, p<0.05) and at 30 min (564 ± 147 vs. 1214 ± 139 pg/mL, p<0.05) versus pretreatment with vehicle alone. However, pretreatment with [1-(β-mercapto-β, β-cyclopentamethylenepropionic acid), 2-(Ο-methyl)tyrosine]-arg-vasopressin, a V1a/V1b receptor antagonist, did not affect icv NPW-induced ACTH release at any time (p>0.05). In conclusion, we suggest that central NPW activates the HPA axis by activating hypothalamic CRF but not AVP.

Resistin Regulates Pituitary Somatotrope Cell Function through the Activation of Multiple Signaling Pathways

The adipokine resistin is an insulin-antagonizing factor that also plays a regulatory role in inflammation, immunity, food intake, and gonadal function. Although adipose tissue is the primary source of resistin, it is also expressed in other tissues and organs, including the pituitary. However, there is no information on whether resistin, as described previously for other adipokines such as leptin and adiponectin, could regulate this gland. Likewise, the molecular basis of resistin actions remains largely unexplored. Here we show that administration of resistin to dispersed rat anterior pituitary cells increased GH release in both the short (4 h) and long (24 h) term, decreased mRNA levels of the receptor of the somatotrope regulator ghrelin, and increased free cytosolic Ca(2+) concentration in single somatotropes. By means of a pharmacological approach, we found that the stimulatory action of resistin occurs through a Gs protein-dependent mechanism and that the adenylate cyclase/cAMP/protein kinase A pathway, the phosphatidylinositol 3-kinase/Akt pathway, protein kinase C, and extracellular Ca(2+) entry through L-type voltage-sensitive Ca(2+) channels are essential players in mediating the effects of resistin on somatotropes. Taken together, our results demonstrate for the first time a regulatory role for resistin on somatotrope function and provide novel insights on the intracellular mechanisms activated by this protein.

Cocaine- and Amphetamine-Regulated Transcript Is Localized in Pituitary Lactotropes and Is Regulated during Lactation

Cocaine- and amphetamine-regulated transcript (CART) is a highly expressed peptide implicated in the regulation of feeding, reward and reinforcement, and stress-related behaviors. CART has been localized to discrete cell populations in the brain, gut, adrenal gland, and pancreas. In contrast, CART-producing cell types in the pituitary gland remain ill defined. In the present study, double-label immunohistochemistry, employing a high-affinity antiserum we generated against CART-(62-102), was used to identify CART-producing cells in the pituitary gland. In the anterior pituitary, the majority of CART immunoreactivity (-ir) was localized in lactotropes; minor populations of CART-ir cells were identified as somatotropes and corticotropes. In the posterior pituitary, CART-ir extensively colocalized with oxytocin-containing fibers; in contrast, only a few vasopressin fibers contained CART-ir. As expected, CART colocalized with oxytocin in magnocellular neurons of the supraoptic nucleus. The effects of bromocriptine, a potent dopamine receptor agonist, were examined to determine whether CART mRNA expression and protein release are regulated in a similar fashion as prolactin. Similar to prolactin, CART mRNA expression and protein release were significantly decreased after bromocriptine treatment of dispersed rat anterior pituitary cells in culture. To explore the putative physiological role of pituitary CART, we compared levels of CART mRNA expression in lactating and nonlactating female rats. CART mRNA levels were significantly increased in the anterior pituitary and supraoptic nucleus of lactating rats. Furthermore, levels of CART in the systemic circulation were significantly elevated at the onset of lactation, peaked on d 10 of lactation and returned to baseline values 10 d after pups were weaned. The current study describes the cellular localization and regulation of CART expression and protein release from the rat pituitary gland. These findings suggest a putative role for CART in lactation.

Sonic hedgehog regulates CRH signal transduction in the adult pituitary

Sonic hedgehog (Shh) is a signaling protein that binds to Patched and mediates its effects through Gli transcription factors. Shh is important in regulating survival and growth in both the embryo and the adult. It is known to be involved in pituitary development, but its role in the adult pituitary has not been investigated. Here, we show Shh and Gli1 immunoreactivity in adult human corticotroph cells. Administration of Shh (5 microg/ml) alone and in combination with corticotrophin-releasing hormone (CRH; 100 nM) in dispersed rat anterior pituitary and AtT-20 mouse corticotrophinoma cells increased corticotrophin (ACTH) secretion and pro-opiomelanocortin (POMC) promoter activity. Shh and CRH act additively in increasing CRH receptor 1 (CRH-R1). Unexpectedly, we found that CRH on its own increased Gli-dependent transcription, which in turn stimulated POMC transcription. Gli1 is necessary for CRH signaling, since knocking down Gli1 by RNA interference abolished the stimulatory effect of CRH on POMC. Taken together, our results demonstrate a new role for Shh and Gli1 in corticotroph function and provide a new link between Shh and CRH signaling pathways.

Cocaine- and amphetamine-regulated transcript activates the hypothalamic-pituitary-adrenal axis through a corticotropin-releasing factor receptor-dependent mechanism.

Cocaine- and amphetamine-regulated transcript (CART) is a highly expressed hypothalamic transcript that is concentrated in areas associated with the stress response. There is evidence for a role of CART in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis. However, it is not clear whether CART regulates activity of the HPA axis by directly stimulating ACTH release from pituitary corticotropes or through interaction with hypothalamic factors. To address this issue, the effects of central and peripheral administration of CART on the HPA axis were compared. Central administration of CART(55-102) (1 microg) significantly increased circulating levels of ACTH (481 +/- 122 vs. 93 +/- 14 pg/ml; CART vs. vehicle) and corticosterone (460 +/- 29 vs. 179 +/- 62 ng/ml; CART vs. vehicle). In contrast, iv injection of CART(55-102) (0.09-9.0 nmol/kg) did not significantly affect circulating levels of ACTH or corticosterone. The corticotropin-releasing factor (CRF) receptor antagonist Astressin B was used to determine whether CART(55-102) elicits ACTH secretion via a CRF receptor-dependent mechanism. Injection of Astressin B (50 microg/kg, iv) inhibited CART(55-102)-induced ACTH and corticosterone responses. The effects of CART(55-102) on CRF and arginine vasopressin (AVP) expression were also examined in static hypothalamic explants. RT-PCR analysis revealed a significant up-regulation of CRF and AVP mRNA levels after CART(55-102) (10 nm and 1 microm) treatment. Last, the effects of CART(55-102) on CRF- and AVP-mediated ACTH release was investigated in dispersed rat anterior pituitary cells. Incubation of CART(55-102) (10-100 nm) did not significantly affect ACTH release from anterior pituitary cells. Findings from the present study suggest that CART regulates activity of the HPA axis through a CRF-dependent central mechanism and not by means of direct interaction with pituitary corticotropes.

Short-term plasticity of cyclic adenosine 3',5'-monophosphate signaling in anterior pituitary corticotrope cells: the role of adenylyl cyclase isotypes.

Anterior pituitary corticotropes show a wide repertory of responses to hypothalamic neuropeptides and adrenal corticosteroids. The hypothesis that plasticity of the cAMP signaling system underlies this adaptive versatility was investigated. In dispersed rat anterior pituitary cells, depletion of intracellular Ca2+ stores with thapsigargin combined with ryanodine or caffeine enhanced the corticotropin releasing-factor (CRF)-evoked cAMP response by 4-fold, whereas reduction of Ca2+ entry alone had no effect. CRF-induced cAMP was amplified 15-fold by arginine-vasopressin (AVP) or phorbol-dibutyrate ester. In the presence of inhibitors of cyclic nucleotide phosphodiesterases and phorbol-dibutyrate ester, the depletion of Ca2+ stores had no further effect on CRF-induced cAMP accumulation. Adenohypophysial expression of mRNAs for the Ca2+-inhibited adenylyl cyclases (ACs) VI and IX, and the protein kinase C-stimulated ACs II and VII was demonstrated. ACIX was detected in corticotropes by immunocytochemistry, whereas ACII and ACVI were not present. The data show negative feedback regulation of CRF-induced cAMP levels by Ca2+ derived from ryanodine receptor-operated intracellular stores. Stimulation of protein kinase C by AVP enhances Ca2+-independent cAMP synthesis, thus changing the characteristics of intracellular Ca2+ feedback. It is proposed that the modulation of intracellular Ca2+ feedback in corticotropes by AVP is an important element of physiological control.

In vitro effects of oestradiol on galanin gene expression in rat anterior pituitary cells.

While the pituitary galanin gene is highly responsive to oestrogen stimulation in vivo, in vitro effects of oestrogens on pituitary galanin gene expression have been less studied. We therefore examined the short-term effects of 17beta-oestradiol on galanin synthesis by dispersed rat anterior pituitary cell cultures and investigated the mechanisms by which oestrogens may modulate galanin gene expression. 17beta-oestradiol increased galanin mRNA expression in a dose-dependent manner, with a maximal increase observed at a concentration of 10-7 M. The 17beta-oestradiol (10-7 M)-induced increase in galanin mRNA expression varied from 3- to 20-fold (average 12-fold) depending upon the experiments and was also time-dependent, reaching significance after 6 h. A 1-h exposure of anterior pituitary cells to 17beta-oestradiol was sufficient to induce markedly galanin mRNA expression after 24 h (by 16-fold) and 48 h (by 25-fold). Tamoxifen administered simultaneously with or following 17beta-oestradiol treatment completely abolished the oestrogen-induced increase of galanin mRNA levels. Cycloheximide (10 microg/ml), a protein synthesis inhibitor, also blocked 17beta-oestradiol-induced galanin gene expression. Using transcription blockade by actinomycin D, we observed similar decreases of pituitary galanin mRNA concentrations, in the presence and absence of 17beta-oestradiol, implying no oestrogen effect on mRNA stability. We conclude that oestrogens stimulate rat pituitary galanin gene expression, mainly through a transcriptional mechanism, and that this effect requires persistent binding of the hormone to its nuclear receptor and newly synthesized protein intermediates.

Calcitonin Inhibits Anterior Pituitary Cell Proliferation in the Adult Female Rats

Previous studies have shown that CT-like immunoreactive peptide(s) (pit-CT) is synthesized by the anterior pituitary (AP) gland, and exogenously added salmon(s) CT inhibits PRL release and PRL gene transcription in cultured AP cells. Anti-sCT serum, which immunoreacts with pit-CT, stimulates PRL secretion, suggesting pit-CT is a physiologically relevant PRL-inhibiting hormone. Using proliferating cell nuclear antigen (PCNA) staining and 5-bromo-2′deoxyuridine (BrdU) incorporation into newly replicated DNA, the effect of calcitonin (CT) on cellular proliferation in the rat anterior pituitary gland (AP) was examined. CT significantly attenuated PCNA-immunopositive as well as BrdU-positive AP cell populations in dispersed rat AP cells. A second series of experiments tested the effects of CT on AP cell proliferation in vivo. OVX + E2 rats were injected with 200 μg CT (iv), the rats killed at various time points, and the APs were processed for BrdU staining. CT inhibited BrdU incorporation at all time points up to 15 h after the injection, and this inhibitory effect was reversed at later time points. The effect of CT was concentration dependent, and a maximal inhibition was observed 10 h after the CT injection. Subsequent experiments identified CT-responsive AP cell populations using double immunofluorescence for BrdU and either PRL or FSH. The number of BrdU-labeled lactotropes in the AP gland declined by 74% in the CT-treated rats. Neutralization of endogenous pit-CT by passive immunization with anti-sCT serum caused a 2-fold increase in AP cell proliferation. These results suggest an important role for the endogenous pit-CT in regulation of lactotrope population of the AP gland.

Calcitonin inhibits anterior pituitary cell proliferation in the adult female rats.

Previous studies have shown that CT-like immunoreactive peptide(s) (pit-CT) is synthesized by the anterior pituitary (AP) gland, and exogenously added salmon(s) CT inhibits PRL release and PRL gene transcription in cultured AP cells. Anti-sCT serum, which immunoreacts with pit-CT, stimulates PRL secretion, suggesting pit-CT is a physiologically relevant PRL-inhibiting hormone. Using proliferating cell nuclear antigen (PCNA) staining and 5-bromo-2'deoxyuridine (BrdU) incorporation into newly replicated DNA, the effect of calcitonin (CT) on cellular proliferation in the rat anterior pituitary gland (AP) was examined. CT significantly attenuated PCNA-immunopositive as well as BrdU-positive AP cell populations in dispersed rat AP cells. A second series of experiments tested the effects of CT on AP cell proliferation in vivo. OVX + E2 rats were injected with 200 microg CT (iv), the rats killed at various time points, and the APs were processed for BrdU staining. CT inhibited BrdU incorporation at all time points up to 15 h after the injection, and this inhibitory effect was reversed at later time points. The effect of CT was concentration dependent, and a maximal inhibition was observed 10 h after the CT injection. Subsequent experiments identified CT-responsive AP cell populations using double immunofluorescence for BrdU and either PRL or FSH. The number of BrdU-labeled lactotropes in the AP gland declined by 74% in the CT-treated rats. Neutralization of endogenous pit-CT by passive immunization with anti-sCT serum caused a 2-fold increase in AP cell proliferation. These results suggest an important role for the endogenous pit-CT in regulation of lactotrope population of the AP gland.

Characterization and localization of lipocortin 1-binding sites on rat anterior pituitary cells by fluorescence-activated cell analysis/sorting and electron microscopy.

Lipocortin 1 (LC1) is an important mediator of glucocorticoid action in the anterior pituitary gland, where it appears to act via cell surface binding sites to suppress peptide release. We have exploited a combination of fluorescence-activated cell (FAC) analysis/sorting and electron microscopy to detect, characterize, and localize LC1-binding sites on the surface of dispersed rat anterior pituitary cells, using human recombinant LC1 (hu-r-LC1) as a probe. High affinity (Kd = 14 +/- 3 nM) hu-r-LC1-binding sites were detected on approximately 80% of anterior pituitary cells dispersed with collagenase. The binding characteristics of the ligand resembled those observed in leukocytes, in that it was saturable; concentration, Ca2+, and temperature dependent; and abolished by trypsin. Functional studies demonstrated an excellent correlation between the presence of the cell surface binding protein and the capacity of an anti-LC1 monoclonal antibody to abrogate the inhibitory actions of dexamethasone (10 nM) on the release of ACTH initiated in vitro by CRH-41 (1 nM). Morphological analysis of cells harvested by FAC sorting showed that 1) somatotrophs, corticotrophs, lactotrophs, thyrotrophs, and gonadotrophs were all included in the population expressing LC1 binding sites; and 2) the LC1-binding sites assume a punctate distribution across the cell surface. These data show that anterior pituitary cells express high affinity surface LC1-binding protein(s); they thus provide further evidence for a specific membrane mechanism of action of LC1 in regulating the endocrine function of the anterior pituitary.

Effect of oxytocin on growth hormone release in vitro

The effect of oxytocin (OT) on growth hormone (GH) secretion was investigated using dispersed rat anterior pituitary cells. OT dose-dependently inhibited GH secretion as well as GHRH-stimulated GH release. The inhibitory actions of OT on GH release were totally abolished by pretreatment with the OT-antagonist VAP 259. The peptides galanin and cholecystokinin did not affect the OT-induced inhibition on basal or GHRH-stimulated GH release. Several possible mechanisms by which OT may influence GH release are discussed.

Cloning and characterization of human urocortin.

Urocortin, a new member of the CRF peptide family which also includes urotensin I and sauvagine, was recently cloned from the rat midbrain. The synthetic replicate of urocortin was found to bind with high affinity to type 1 and type 2 CRF receptors and, based upon its anatomic localization within the brain, was proposed to be a natural ligand for the type 2 CRF receptors. Using a genomic library, we have cloned the human counterpart of rat urocortin and localized it to human chromosome 2. Human and rat urocortin share 95% identity within the mature peptide region. Synthetic human urocortin binds with high affinity to CRF receptor types 1, 2 alpha, and 2 beta, stimulates cAMP accumulation from cells stably transfected with these receptors, and acts in vitro to release ACTH from dispersed rat anterior pituitary cells. In addition, the CRF-binding protein binds human urocortin with high affinity and can prevent urocortin-stimulated ACTH secretion in vitro. The inhibitory effect of the CRF-binding protein on human urocortin can be blocked by biologically inactive CRF fragments, such as CRF(9-33).

Cyclic GMP Stimulates Growth Hormone Release in Rat Anterior Pituitary Cells

In this study, the role of cGMP on growth hormone (GH) release was examined using a static monolayer culture prepared from dispersed rat anterior pituitary cells. Treatment with 8-bromo-cGMP (1 μM to 1 mM) stimulated GH release up to 3.8-fold in a concentration-dependent manner. Elevating cGMP with nitroprusside or the C-type natriuretic peptide was also effective in stimulating GH release. The increase in GH release by cGMP-elevating agents occurred without a concomitant increase in cAMP. Unlike cAMP which increased intracellular Ca 2+ concentration, 8-bromo-cGMP caused a small reduction in intracellular Ca 2+ concentration. Taken together, these results indicate that i) cGMP appears to be another mechanism that regulates GH release, ii) activation of cytosolic or membranous guanylyl cyclase is equally effective in stimulating GH release; and iii) the cGMP-induced GH release appears to be through a mechanism distinct from that of cAMP.

Insulin, insulin-like growth factor I (IGF-I) and IGF-II enhance basal and gonadotrophin-releasing hormone-stimulated luteinizing hormone release from rat anterior pituitary cells in vitro

The role of insulin-like growth factor I (IGF-I) and IGF-II on luteinizing hormone (LH) release is still unclear. The present study was performed to investigate modifications of basal and gonadotrophin-releasing hormone (GnRH)-stimulated (10(-9) mol/l) LH release, induced by 4-h and a 24-h incubation with physiological doses of IGF-I (1, 5 and 10 nmol/l) and IGF-II (5, 10 and 15 nmol/l) in comparison with insulin (0.0017, 0.1722 and 1.722 nmol/l), from primary cultures of male rat anterior pituitary cells. Both during the 4-h and the 24-h incubation, basal and GnRH-stimulated LH release were increased by IGF-I, IGF-II and insulin in a dose-dependent fashion. Present data confirm insulin's capability of potentiating anterior pituitary LH release from dispersed rat anterior pituitary cells in vitro, and reveal similar effects for physiological doses of IGF-I and IGF-II.

Oxytocin-stimulated release of adrenocorticotropin from the rat pituitary is mediated by arginine vasopressin receptors of the V1b type.

Previous work showed the existence of receptors for arginine vasopressin (AVP) in the anterior pituitary; these receptors were classified as belonging to a distinct AVP receptor subtype, referred to as AVP-V1b receptors, and are thought to mediate the well documented ACTH-releasing activity of AVP. In the present work, high affinity receptors for another neurohypophyseal hormone, oxytocin (OT), were also shown to be present within the rat anterior pituitary; to this end, [125I]d(CH2)5[Tyr(Me)2Thr4Tyr-NH2(9)]OVT was used as a ligand in receptor binding studies. Experiments on dispersed rat anterior pituitary cells in a superfusion system confirmed earlier reports that OT acts as an additional secretagogue of ACTH, with significant effects first seen at concentrations as low as 1 nM. Further studies addressed the question of whether stimulation of ACTH release is mediated by OT receptors or whether receptors for AVP (V1b receptors) might serve this role. For this, highly selective agonist and antagonist ligands of the OT receptor and nonselective agonist and antagonist ligands of the V1b receptor were employed. Neither the OT receptor agonist Thr4Gly7OT nor the OT receptor antagonist des-Gly(NH2)9d(CH2)5-[Tyr(Me)2 Thr4]OVT displayed any influence on basal ACTH release, and des-Gly(NH2)9d(CH2)5-[Tyr(Me)2Thr4]OVT did not interfere with OT-induced ACTH release; these results indicated that OT promotes ACTH release through a receptor(s) other than the OT receptor itself. Evidence for the involvement of AVP V1b receptors was provided by the observation that the AVP receptor antagonist dP[Tyr(Me2)]AVP completely abolished OT-elicited increases in ACTH release. Thus, AVP V1b receptors mediate the actions of two structurally related peptides on ACTH secretion; the role of OT receptors in adenohypophyseal function remains to be determined.

Evidence that Ca 2+-activated K + channels participate in the regulation of pituitary prolactin secretion

We evaluatedthe role of Ca 2+-activated K + channels in the regulation of prolactin (PRL) secretion with a perifusion system using acutely dispersed rat anterior pituitary cells. Apamin, which blocks Ca 2+-activated K + channels, induced PRL secretion in a dose-dependent fashion between 1 and 300 nM ( r = 0.99, P < 0.01). Charybdotoxin, another Ca 2+-activated K + channel-blocker, also induced PRL secretion at 20 nM concentration. These were not non-specific toxic effects, since stimulation of PRL secretion by 10 nM thyrotropin-releasing hormone (TRH) was not different before and after applying the channel-blockers. Both 10 μM dopamine and 2 μM nifedipine significantly, but incompletely, depressed PRL secretion induced by 100 nM apamin; 10 μM dopamine completely blocked PRL secretion induced by 20 nM charybdotoxin. Our data indicate that Ca 2+-activated K + channels may play an important role in the regulation of PRL secretion.

Dopamine Inhibits TRH-Induced MAP Kinase Activation in Dispersed Rat Anterior Pituitary Cells

We recently reported the existence of two separate pathways for thyrotropin-releasing hormone (TRH)-induced mitogen-activated protein (MAP) kinase activation in GH3 pituitary tumor cells. To test the role of MAP kinase in TRH action, we examined the effect of dopamine (DA) on TRH-induced MAP kinase in primary cultures of rat anterior pituitary cells. 1 μM of DA attenuated 1 μM TRH-induced MAP kinase activity and phosphorylation. 100 ng/ml of islet-activating protein (IAP) blocked these inhibitory effects of DA. These results suggest that crosstalk exists between the DA signaling pathway and the TRH-stimulated MAP kinase activating pathway in rat anterior pituitary cells.

Demonstration of biological activity of a growth hormone-releasing hormone-like substance produced by a pheochromocytoma

The biological characteristics of a growth hormone-releasing hormone (GHRH)-like substance produced by a pheochromocytoma were studied. Analysis by gel filtration chromatography combined with the use of two distinct GHRH antisera that recognize the N- and C-termini of authentic GHRH(1-44)NH2 indicated molecular heterogeneity of the immunoreactive GHRH in the tumor extract, but a component corresponding to GHRH(1-44)NH2 was the predominant form. The biological activity of this immunoreactive component was assessed in vitro by measuring its ability to induce growth hormone release from dispersed rat anterior pituitary cells. At concentrations of 0.125-2.0 nmol/l, the test materials induced a dose-related increase in growth hormone release from the cells into the incubation medium (range 992 +/- 68-1872 +/- 32 ng.1.7 x 10(5) cells-1 x 3 h-1), similar to that observed with synthetic GHRH(1-44)NH2. (control value 640 +/- 30 ng.1.7 x 10(5) cells-1 x 3 h-1). This suggests that immunoreactive GHRH in the tumor has almost the same biological activity as the synthetic product and that a combination of pheochromocytoma and acromegaly is not always fortuitous because both diseases may be caused by a single neoplasm.

Effect of protein kinase-C depletion on inositol trisphosphate-mediated and cyclic adenosine 3',5'-monophosphate-dependent protein kinase-mediated adrenocorticotropin secretion.

We studied the effect of Ca2+/phospholipid-dependent protein kinase-C (protein kinase-C) down-regulation by chronic exposure to phorbol 12-myristate 13-acetate (PMA) on ACTH secretion by dispersed male rat anterior pituitary cells in a microperifusion system. Preincubation for 24 h and preperifusion for 3 h with 0.1 and 1 microM PMA significantly inhibited (by 85% and 91%, respectively) the specific cell binding of [3H]phorbol 12,13-dibutyrate, an index of protein kinase-C concentration, and significantly reduced (by 101% and 20%, respectively) the sustained plateau (final 15-min) phase of the ACTH response to arginine vasopressin (AVP) and (by 56% and 54%, respectively) the sustained (full 20-min) response to dioctanoylglycerol (DOG), both of which are mediated by protein kinase-C activation. In contrast, the spike (initial 5-min) phase of the response to AVP, which is mediated by intracellular Ca2+ release from inositol 1,4,5-trisphosphate (InsP3)-sensitive stores, was significantly increased (by 112% and 99%, respectively), but the spike-type response to ionomycin, which releases intracellular Ca2+ by an InsP3-independent mechanism, was unaffected. AVP significantly stimulated inositol bisphosphate and InsP3, but not inositol monophosphate, accumulation, and PMA pretreatment significantly enhanced their AVP-stimulated accumulation (by 86%, 34%, and 78%, respectively), an effect that was abolished by simultaneous preperifusion with PMA and cycloheximide to inhibit new protein synthesis. Enhancement of the spike phase response to AVP and AVP-stimulated InsP3 accumulation were lost within 1 h of PMA removal, but [3H]phorbol 12,13-dibutyrate binding and the sustained responses to AVP and DOG remained suppressed after 3 h. Pretreatment with 0.1 and 1 microM PMA slightly reduced the sustained responses to CRF (by 29% and 16%, respectively) and 8-bromo-cAMP (by 8% and 12%, respectively), which are mediated by protein kinase-A activation and extracellular Ca2+ influx via L-type voltage-sensitive Ca2+ channels, but not the response to KCl, which is mediated by extracellular Ca2+ influx via all types of voltage-sensitive Ca2+ channels. The sustained response to CRF was still suppressed 1 h after PMA removal, but returned to the control level by 3 h. When new protein synthesis was inhibited by preperifusion with cycloheximide alone for 3 h after 24-h PMA pretreatment, recovery from the effects of PMA was abolished. Three-hour exposure to cycloheximide without PMA pretreatment inhibited the sustained responses to CRF, AVP, and DOG, but not the spite response to AVP.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of a high-affinity galanin receptor in the rat anterior pituitary: absence of biological effect and reduced membrane binding of the antagonist M15 differentiate it from the brain/gut receptor.

Structure-activity studies demonstrate that galanin fragments 1-15 and 2-29 are fully active, whereas fragment 3-29 has been reported to be inactive, in a number of different in vivo models. M15, a chimeric peptide comprising galanin 1-13 and substance P5-11, has recently been found to be a potent galanin antagonist. Direct effects of galanin at the level of the pituitary have been defined, yet, paradoxically, a number of studies have been unable to demonstrate galanin binding to an anterior pituitary receptor. Porcine galanin stimulated prolactin release from dispersed rat anterior pituitary cells up to 180% +/- 12% (mean +/- SEM) of control secretion. The addition of a specific galanin antiserum caused a profound inhibition of basal prolactin release, maximal inhibition being 12% +/- 0.5% of control secretion. Addition of M15 produced no effect on basal or galanin-stimulated prolactin release. Galanin fragment 3-29 was fully active when compared to galanin 1-29. Fragments 5-29 and 8-29 stimulated prolactin release to a lesser extent and galanin 1-15, 10-29, and 20-29 had no significant prolactin-releasing activity. Using [mono(125I)iodo-Tyr26]galanin or porcine 125I-labeled Bolton-Hunter [mono(125I)iodo-Lys25]galanin, no anterior pituitary membrane binding was observed. In contrast, 125I-labeled Bolton-Hunter N-terminally labeled galanin allowed characterization of a single high-affinity anterior pituitary galanin receptor with a Kd of 4.4 +/- 0.34 nM and a Bmax of 79 +/- 8.3 fmol/mg of protein. The IC50 for porcine galanin was 0.51 +/- 0.04 nM but for M15 was in excess of 10 microM. Galanin 3-29 fully displaced the label with an IC50 of 0.96 +/- 0.7 nM. The IC50 for galanin 5-29 was 200 nM, whereas 8-29 and 1-15 were > 1 microM. Galanin 10-29 and 20-29 failed to displace the label. These data suggest the presence of a high-affinity pituitary galanin receptor, designated GAL-R2, in which region 3-10 and amino acid 25 are crucial for membrane binding and biological activity, in contrast to the known gut/brain galanin receptor (designated GAL-R1). A number of tissues known to bind or respond to galanin were screened. GAL-R2 would appear to be expressed only in the anterior pituitary and hypothalamus.