Stimulation of growth hormone release by 5-hydroxytryptamine (5-HT) in cultured rat anterior pituitary cell aggregates: evidence for mediation by 5-HT2B, 5-HT7, 5-HT1B, and ketanserin-sensitive receptors

5-Hydroxytryptamine (5-HT) promotes the release of GH by a hypothalamic site of action. The present study explores a putative pituitary action in a perifused rat anterior pituitary aggregate cell culture system. In aggregates cultured with 1 nM estradiol for expression of the 5-HT4, -5, and -6 receptor (R), 5-HT promptly stimulated GH secretion with a dose dependency between 1 and 10 nM. The effect of 5-HT was partially blocked by methiothepin and methysergide; by SB-206553, a 5-HTR2B/C antagonist; SB-269970, a 5-HTR7/5A antagonist; and SB-224289, a 5-HTR1B antagonist. The GH response was fully blocked by combined administration of SB-206553+SB-269970 and SB-206553+ketanserin but not by SB-206553+spiperone. Culturing the aggregates without estradiol diminished the magnitude of the GH response to 5-HT as well as the impact of 5-HTR7/5 blockade on the response. Basal GH release was stimulated by the 5-HTR2 agonists 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane, m-chlorophenyl piperazine, and alpha-methyl 5-HT; 5-carboxytryptamine (agonist at 5-HTR1, -5, and -7); tryptamine (preferential 5-HTR7 agonist); and the selective 5-HTR1B agonist CP93129 but not the 5-HTR1A agonists 7-(dipropylamino)tetralin-1-ol-8-hydroxy-2-(di-n-propylamino)tetralin and the 5-HTR1B/D agonist sumatriptan. The selective 5-HTR2B agonist BW 723C86 stimulated GH release, and the selective 5-HTR2B antagonist SB-204741 attenuated the GH response to 5-HT. The present data suggest that 5-HT may release GH through a pituitary site of action, and that the 5-HTR2B, 5-HTR7 and 5-HTR1B mediate this response, with possibly an inhibitory component of the 5-HTR1D. The relative contribution of these receptors may be modulated by estrogen.     

Effect of serotonin 5-HT1, 5-HT2, and 5-HT3 receptor antagonists on the prolactin response to restraint and ether stress.

Serotonin (5-HT) appears to be involved in the central control of the prolactin (PRL) response to suckling and estrogen. Furthermore, 5-HT may participate in the mediation of stress-induced PRL release. In order further to elucidate the role of 5-HT and the type of 5-HT receptor(s) involved in the PRL response to stress, we investigated the effect of blockade of 5-HT1, 5-HT2 or 5-HT3 receptors on the restraint or ether stress-induced release of PRL in male rats. Pretreatment with the 5-HT1 + 2 receptor antagonist methysergide (0.5 or 2.5 mg/kg i.p.) inhibited or prevented the PRL response to restraint or ether stress. Pretreatment with the 5-HT2 receptor antagonists ketanserin or LY 53857 (0.5 or 2.5 mg/kg i.p.) inhibited the response to restraint or ether stress approximately 30 or 60%, respectively. Higher doses of both 5-HT2 receptor antagonists (10 mg/kg i.p.) had a minor inhibitory effect (5-30% for ketanserin and 50% for LY 53857). Prior intraperitoneal administration of the 5-HT3 receptor antagonists ICS 205-930 or GR 38032F (0.05-2.5 mg/kg i.p.) inhibited the restraint stress-induced PRL release dose-dependently. Both compounds inhibited the PRL response to ether stress, but only the effect of GR was dose-related. The maximal inhibitory effect (70% inhibition of the PRL response to restraint or ether stress) was obtained for both compounds at a dose of 0.1 mg/kg. We conclude that serotonergic neurons are involved in the mediation of the stress-induced PRL release by activation of 5-HT1, 5-HT2 as well as 5-HT3 receptors.     

Differential functional effects of two 5-HT4 receptor isoforms in adult cardiomyocytes

Serotonin 5-HT4 receptors are present in human atrial myocytes and have been proposed to contribute to the generation of atrial fibrillation. However, 5-HT4 receptors have so far been only found in human and pig atria and are absent from the heart of small laboratory animals, such as rat, guinea pig, rabbit and frog, which limits the experimental settings for studying their functional properties. In this study, we developed an adenovirus expression system to examine the properties of two human 5-HT4 receptor splice variants, h5-HT4(b) and h5-HT4(d), expressed in adult cardiomyocytes devoid of native 5-HT4 receptors. When expressed in the HL-1 murine cell line of atrial origin, both receptors caused specific binding of the 5-HT4 selective antagonist GR113808 and activated adenylyl cyclase in the presence of serotonin (5-HT, 1 microM). When expressed in freshly isolated adult rat ventricular cardiomyocytes, a stimulation of the L-type Ca2+ current (ICa,L) by 5-HT (100 nM) was revealed. Both effects were blocked by GR113808. In HL-1 cells, the h5-HT4(d) receptor was found to be more efficiently coupled to adenylyl cyclase than the h5-HT4(b). Pertussis toxin treatment (250 ng/ml for 5 h) potentiated the stimulatory effect of 5-HT on ICa,L in rat myocytes expressing the h5-HT4(b) but not the h5-HT4(d) receptor, indicating a likely coupling of the (b) isoform to both Gs and Gi/o proteins. Adenoviral expression of h5-HT4 receptor isoforms in adult cardiac myocytes provides a valuable means for the exploration of the receptor signaling cascades in normal and pathological situations.     

The serotonin 5-HT4 receptor and the amyloid precursor protein processing

A large body of evidence supports a major role for the serotonin 5-HT(4) receptor in learning and memory and it is suggested that 5-HT(4) agonists may be beneficial for memory disorders such as Alzheimer's disease (AD). The 5-HT(4) receptors are members of the G protein-coupled receptor superfamily and are positively coupled to adenylyl cyclase. In this communication we show that a neuronal isoform of the human 5-HT(4) receptor, h5-HT(4(g)) regulates the metabolism of the amyloid precursor protein (APP695). This process is observed in Chinese hamster ovary (CHO) cells stably coexpressing the neuronal h5-HT(4(g)) receptor isoform as well as the human APP695. The 5-HT(4) agonists strongly stimulate the release of the non-amyloidogenic soluble amyloid precursor protein sAPPalpha as detected by immunoblot. Prucalopride was more potent than serotonin (5-HT) with regard to enhanced of sAPPalpha secretion. This process was blocked by a selective 5-HT(4) antagonist, GR113808. Furthermore, 5-HT(4) ligands enhance sAPPalpha secretion via cAMP-dependent and PKA-independent signalling pathways indicating there are alternative pathways by which the h5-HT(4) receptor via cAMP regulates APP metabolism. Because the alpha-cleavage event may preclude the formation of amyloidogenic peptides, and secreted sAPPalpha has putative neuroprotective and enhancing-memory properties, our present data suggest the 5-HT(4) receptor as a novel target for the treatment of AD.     

Involvement of 5-HT1, 5-HT2, and 5-HT3 receptors in the mediation of the prolactin response to serotonin and 5-hydroxytryptophan.

Serotonin (5-HT) is involved in the neuroendocrine regulation of prolactin (PRL) secretion as a stimulator. Within the last decade several 5-HT receptor types have been identified, but their individual role in the mediation of the PRL response to 5-HT is only partly understood. We investigated in conscious male rats the effect of different 5-HT1, 5-HT2, and 5-HT3 receptor antagonists on the PRL response to 5-HT or to the 5-HT precursor 5-hydroxytrytophan (5-HTP) which was administered in combination with the 5-HT reuptake inhibitor fluoxetine. 5-HT (0.5-5.0 mg/kg BW i.v.) or 5-HTP (25-100 mg/kg i.p.) in combination with saline or fluoxetine (10 mg/kg i.p.) increased the plasma PRL concentration dose-dependently. Pretreatment with the 5-HT1+2 receptor antagonist methysergide (2.5 mg/kg i.p.) prevented the stimulatory effect of 5-HT or 5-HTP + fluoxetine. Pretreatment with the 5-HT2 receptor antagonists ketanserin or LY 53857 (2.5 mg/kg i.p.) inhibited the PRL response to 5-HT by approximately 80% and to 5-HTP + fluoxetine approximately 100%. A higher dose (10 mg/kg) of the 5-HT2 receptor antagonists possessed only 50% inhibitory effect. Pretreatment with the 5-HT3 receptor antagonists ICS 205-930 or GR 38032F (0.05-2.5 mg/kg i.p.) inhibited the PRL response induced by 5-HT or by 5-HTP + fluoxetine. The maximal inhibitory effect (approximately 80%) was obtained by a dose of 0.1 mg/kg of both compounds.(ABSTRACT TRUNCATED AT 250 WORDS)     

Beta-adrenergic stimulation of prolactin release from superfused pituitary cell aggregates

l-Isoproterenol (l-ISO), a specific agonist of beta-adrenergic receptors, evoked a prompt rise of prolactin (PRL) release from superfused anterior pituitary cell aggregates established in culture for 5 days. The effect was concentration-dependent between 1 and 100 nM. d-Isoproterenol was more than 2 orders of magnitude weaker than the l-isomer. When dopamine receptors were blocked with domperidone, PRL secretion was also stimulated by l-epinephrine (E) and l-norepinephrine (NE), the rank order of potency being l-ISO greater than E much greater than NE. Under the latter conditions dopamine and the alpha-adrenergic agonists, clonidine and phenylephrine, had no stimulatory effect at 1 microM. Stimulation of PRL release by l-ISO and E was blocked by the beta-receptor antagonist, propranolol, but not by the alpha-receptor blocker, prazosin.     

5-HT inhibits spontaneous contractility of isolated sheep mesenteric lymphatics via activation of 5-HT(4) receptors

Spontaneous isometric contractions were measured in rings of sheep mesenteric lymphatic vessels in vitro. 5-Hydroxytryptamine (5-HT) produced a concentration-dependent decrease in spontaneous contraction frequency and force which was not antagonised by either the nonspecific 5-HT(1)/5-HT(2) receptor antagonist methysergide (1 microM) or the 5-HT(3) receptor antagonist ondansetron (1 microM). The 5-HT(4) receptor agonist BIMU-8 mimicked the inhibitory effect of 5-HT and its effects were abolished by the 5-HT(4) receptor antagonist DAU 6285 (1 microM). DAU-6285 also abolished the inhibitory effect of 5-HT and unmasked a weak excitatory response, which was mimicked by the 5-HT(2) receptor agonist alpha-methyl-5-hydroxytryptamine maleate. This excitatory response was, in turn, blocked by the 5-HT(2) receptor antagonist pirenperone (1 microM). The results of this study suggest that sheep mesenteric lymphatics possess both 5-HT(4) receptors and 5-HT(2) receptors. The inhibitory 5-HT(4) receptor appeared to be the predominant subtype since the excitatory response to 5-HT could only be observed in the presence of the 5-HT(4) receptor antagonist DAU 6285.     

Serotonin receptor subtypes influence prolactin secretion in the turkey

Serotonin (5-HT) stimulation of prolactin (PRL) secretion is mediated through the dopaminergic (DAergic) system, with 5-HT ligands having no direct effect on pituitary PRL release. Infusion of 5-HT into the third ventricle (ICV) or electrical stimulation (ES) of the medial preoptic area (POM) or the ventromedial nucleus (VMN) induces an increase in circulating PRL in the turkey. These increases in PRL do not occur when a selective antagonist blocks the D₁ dopamine (DA) receptors in the infundibular area (INF). In this study, the ICV infusion of (R)(−)-DOI hydrochloride (DOI), a selective 5-HT_2A eceptor agonist, caused PRL to increase. Pretreatment with Ketanserin tartrate salt (KETAN), a selective 5-HT_2A receptor antagonist, blocked DOI-induced PRL secretion, attesting to the specificity of the response. DOI-induced PRL secretion was prevented when the D₁ DA receptors in the INF were blocked by the D₁ DA receptor antagonist, R(+)-SCH-23390 hydrochloride microinjection, suggesting that the DAergic activation of the vasoactive intestinal peptide (VIP)/PRL system is mediated by a stimulatory 5-HT_2A receptor subtype. The DOI-induced PRL increase did not occur when (±)-8-OH-DPAT (DPAT) was concurrently infused. DPAT is a 5-T_1A receptor agonist which appears to mediate the inhibitory influence of 5-HT on PRL secretion. When DPAT was microinjected directly into the VMN, it blocked the PRL release affected by ES in the POM. These data suggested that when 5-HT_2A receptors are activated, they influence the release of DA to the INF. When 5-HT_1A receptors are stimulated, they somehow inhibit the PRL-releasing actions of 5-HT_2A receptors. This inhibition could take place centrally, or it could occur postsynaptically at the pituitary level. It is known that D₂ DA receptors in the pituitary antagonize PRL-releasing effect of VIP. A release of DA to the pituitary, initiated by 5-HT_1A receptors, could effectively inhibit PRL secretion.     

5-HT1 and 5-HT2 receptor agonists blunt +/- -alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-stimulated GH secretion in prepubertal male rats

OBJECTIVE: Excitatory amino acids, gamma-amino butyric acid (GABA), serotonin and catecholamines are involved in the control of GH secretion. The actions of these neurotransmitters are interconnected, and recently we showed that the stimulatory effect of GABA was blocked by MK-801, an antagonist of N-methyl-D-aspartate receptors. The present experiments were carried out to analyze the interrelationships between +/- -alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors and serotoninergic and catecholaminergic pathways in the control of GH secretion in prepubertal (16-23-day-old) male rats. DESIGN AND METHODS: The GH response to AMPA was analyzed in animals pretreated with 5-hydroxytryptophan methyl ester (5-HTP) plus fluoxetine (a precursor of 5-hydroxytryptamine (5-HT) synthesis and a blocker of 5-HT re-uptake), R (+)-8-hydroxydipropylaminotetralin hydrobromide (8-OH-DPAT, an agonist of the 5-HT1 receptors), +/- -2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI) and alpha-methyl-5-hydroxytryptamine (agonists of 5-HT2 receptors), I-phenylbiguanide (an agonist of 5-HT3 receptors), or alpha-methyl-p-tyrosine (alpha-MPT) and diethyldithiocarbamate (DDC) (blockers of catecholamine synthesis). RESULTS: Basal GH secretion remained unchanged in prepubertal rats after activation of the serotoninergic system or blockade of catecholamine synthesis. The stimulatory effect of AMPA on GH secretion was blocked after activation of the serotoninergic system, through specific 5-HT1 and 5-HT2 receptor agonists. In contrast, activation of 5-HT3 receptors potentiated AMPA-stimulated GH secretion. CONCLUSIONS: Serotoninergic receptors modulate the stimulatory effect of AMPA on GH secretion in prepubertal male rats.     

Stimulation of growth hormone and prolactin release from rat pituitary cell aggregates by bombesin- and ranatensin-like peptides is potentiated by estradiol, 5 alpha-dihydrotestosterone, and dexamethasone

The effect of the bombesin-like peptides, gastrin-releasing peptide (GRP) and neuromedin-C (NMC), and the ranatensin-like peptides, neuromedin-B (NMB), neuromedin-B30 (NMB30), and neuromedin-B32 (NMB32), on pituitary GH and PRL release was studied in perifused anterior pituitary aggregate cell cultures from 9- to 12-week-old male rats cultured in serum-free defined medium supplemented with 0.05 nM T3 and 4 nM dexamethasone (DEX). All peptides stimulated PRL and GH release. GRP and NMC stimulated hormone release in a concentration-dependent manner between 0.1-10 nM. NMB was slightly more potent than NMB30 and NMB32, but was significantly less potent than GRP and NMC. The magnitude of the PRL response to GRP and NMC inversely correlated with that of the GH response. Cultures with relatively low PRL response levels displayed high GH responses, whereas the opposite was found in cultures with high PRL response levels. The stimulatory actions of GRP, NMC, and NMB were blocked by the bombesin receptor antagonist Leu13 psi (CH2NH) Leu14-bombesin, supporting the specificity of the findings. Addition of 1 nM estradiol (E2) to the culture medium provoked an impressive (4- to 10-fold) increase in the magnitude of the GH response to NMC without changing the EC50 value (0.5 nM). In contrast, E2 significantly decreased the stimulation of GH release by rat GH-releasing factor. In the E2-treated aggregates 3 nM NMC stimulated GH release to a comparable extent as 0.1 nM GRF. 5 alpha-Dihydrotesterone (10 and 100 nM) and DEX (80 nM) also enhanced the GH response to NMC, but to a much smaller extent than E2. E2 had also a stimulatory effect on the PRL response to NMC, particularly in cultures with a low intrinsic PRL response. The PRL response to NMC was decreased by DEX and slightly augmented by 5 alpha-dihydrotestosterone. It is concluded that bombesin- and ranatensin-like peptides have a stimulatory effect on GH and PRL release at the pituitary level. Since their action on GH release is strongly potentiated by E2 and much less so by glucocorticoids, these peptides clearly distinguish their activity and specificity from that of the protagonist releasing factor GH-releasing factor, suggesting a role in sex-related differences in GH release or in the control of GH secretion during sexual maturation.     

5-HT1 and 5-HT2 receptor activation reduces N-methyl-D-aspartate (NMDA)-stimulated LH secretion in prepubertal male and female rats

OBJECTIVE: Excitatory amino acids and serotonin are involved in the control of gonadotropin secretion. The actions of these neurotransmitters are interconnected and recently we have reported that 5-HT(1) and 5-HT(2) receptor agonists blunted (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-stimulated GH secretion in prepubertal rats. The present experiments were carried out to analyze the effects of activation of different 5-hydroxytryptamine (5-HT) receptor subtypes on gonadotropin secretion and their role in the N-methyl-d-aspartate (NMDA)-stimulated LH release. DESIGN AND METHODS: We analyzed the gonadotropin secretion after manipulation of serotoninergic and aminoacidergic systems and their interactions in 5-, 16- and 23-day-old male and female rats. To this end, serum LH and FSH concentrations were measured in rats treated with 5-hydroxytryptophan methyl ester (5-HTP) (a precursor of 5-HT synthesis) plus Fluoxetine (Fx, a blocker of 5-HT reuptake), d,l-p-chlorophenyl-alanine methyl ester (PCPA, a blocker of 5-HT synthesis), R-(+)-8-hydroxydipropylaminotetralin hydrobromide (8-OH-DPAT, an agonist of 5-HT(1A) receptors), (+/-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI) and alpha-methyl-5-hydroxytryptamine (alpha-Me-5-HT, agonists of 5-HT(2) receptors), and 1-Phenylbiguanide (1-PHE an agonist of 5-HT(3) receptors). In addition, the effects of 8-OH-DPAT and DOI on NMDA-stimulated LH secretion were analyzed. RESULTS: Neither the activation nor blockade of the serotoninergic system modified LH secretion. Basal gonadotropin secretion remained unchanged in 23-day-old male and female rats after activation of 5-HT(1A), 5-HT(2) and 5-HT(3) receptors. The stimulatory effect of NMDA on LH secretion was blocked in both sexes after activation of the serotoninergic system, through specific 5-HT(1) and 5-HT(2) receptor agonists. CONCLUSIONS: Activation of serotoninergic receptors decreased the stimulatory effect of NMDA on LH secretion in prepubertal male and female rats.     

Appearance of a ventricular 5-HT4 receptor-mediated inotropic response to serotonin in heart failure

BACKGROUND: Current pharmacological treatment of congestive heart failure (CHF) addresses changes in neurohumoral stimulation or cardiac responsiveness to such stimulation. Yet, undiscovered neurohumoral changes, adaptive or maladaptive, may occur in CHF and suggest novel pharmacological treatment. Serotonin [5-hydroxytryptamine (5-HT)] enhances contractility and causes arrhythmias through 5-HT(4) receptors in human atrium and ventricle but not through rat ventricular 5-HT(4) receptors. OBJECTIVE: We investigated whether CHF could induce ventricular responsiveness to serotonin. METHODS: Postinfarction CHF was induced in male Wistar rats by coronary artery ligation. Contractility was measured in left ventricular papillary muscles 6 weeks after infarction. Messenger RNA was quantified by RT-PCR and cAMP by RIA. RESULTS: Serotonin caused positive inotropic (-logEC(50)=7.5) and lusitropic effects in CHF but not Sham papillary muscles. The inotropic effect of 10 muM serotonin in CHF (31.3+/-2.2%) was of similar size as the effect of 10 muM isoproterenol (34.0+/-1.7%). The effects of serotonin were antagonised by GR113808 (0.5-5 nM), consistent with mediation through 5-HT(4) receptors. This was further supported by positive inotropic effects of the 5-HT(4)-selective partial agonist RS67506. Carbachol blunted the serotonin responses and serotonin increased ventricular and cardiomyocyte cAMP, consistent with coupling to G(s) and adenylyl cyclase. Quantitative RT-PCR revealed fourfold increased 5-HT(4(b)) mRNA expression in CHF vs. Sham ventricles. CONCLUSION: Functional ventricular 5-HT(4) receptors are induced by myocardial infarction and CHF of the rat heart. We propose that they are a model for ventricular 5-HT(4) receptors of human failing heart and may play a pathophysiological role in heart failure.     

Production and metabolism of serotonin (5-HT) by the human adrenal cortex: paracrine stimulation of aldosterone secretion by 5-HT

In the human adrenal cortex, serotonin (5-HT) is contained in mast-like cells, and we have shown that 5-HT stimulates aldosterone secretion, suggesting that 5-HT may control glomerulosa cells through a paracrine mechanism. Concurrently, the presence of 5-hydroxyindolacetic acid in human adrenocortical extracts indicates that 5-HT may be metabolized after local release by mast cells. The aim of the present study was to investigate in vitro the production and metabolism of 5-HT by the human adrenal cortex. Perifused adrenal slices released spontaneously detectable amounts of 5-HT (0.74 +/- 0.38 fmol/mg wet tissue.min). The mast cell-depleting drug compound 48/80 induced a burst of 5-HT secretion followed by a gradual increase in aldosterone production. Administration of the specific 5-HT(4) receptor antagonist GR 113808 (10(-6) M) did not affect compound 48/80-induced 5-HT release but abolished the stimulatory effect of compound 48/80 on aldosterone secretion, indicating that 5-HT released locally is responsible for a paracrine control of steroidogenesis. Incubation of cells from the human adrenal cortex with 5-HT (10(-5) M) provoked the formation of the 5-HT metabolite 5-hydroxytryptophol. The type A monoamine oxidase (MAO) inhibitor clorgyline (10(-6) M) suppressed the metabolism of 5-HT into 5-hydroxytryptophol. Immunocytochemical staining of cultured cells revealed the presence of a subpopulation of MAO-A-positive cells. Double labeling with an antiserum against chromogranin A showed that MAO-A was actually contained in chromaffin cells. Similarly, immunohistochemical staining of adrenal slices showed that MAO-A was expressed in chromaffin cells located both in the medulla and in intracortical rays. In conclusion, the present study shows that, in the human adrenal cortex, 5-HT, released by mast-cells, may stimulate aldosterone secretion in a paracrine manner. Our data also indicate that 5-HT is metabolized by MAO-A located in intracortical chromaffin cells.     

Beta-adrenergic stimulation of adenosine-3',5'-monophosphate (c-AMP) accumulation and of prolactin and growth hormone secretion in rat anterior pituitary cell cultures

The beta-adrenergic agonist 1-isoproterenol (ISO) (10(-9)-10(-7) M) provoked a prompt and profound increase of intracellular c-AMP accumulation in monolayer cultures of rat anterior pituitary. In superfused reaggregate cell cultures ISO also stimulated c-AMP outflow. The effect was blocked by propranolol and the highly selective beta 2-receptor blocker ICI 118.551. Epinephrine (E), norepinephrine (NE) and the highly selective beta 2-agonist zinterol (ZIN) also stimulated c-AMP accumulation. The order of potency ZIN greater than or equal to ISO greater than E much greater than NE together with the high potency of ICI 118.551 suggests the beta-effect is mainly through the beta 2-receptor subtype. The same concentrations of ISO strongly stimulated growth hormone (GH) release and, as previously shown, prolactin (PRL) release from superfused reaggregate cell cultures, but not luteinizing hormone or thyroid stimulating hormone release. Stimulation of PRL and GH release from these cultures was also induced by the adenylyl cyclase activator forskolin. Since interference of beta-adrenergic effects on contaminating fibroblasts or endothelial cells could be reasonably excluded, the present data suggest that beta-adrenergic stimulation of c-AMP accumulation in anterior pituitary cells elicits GH and PRL release.     

Selective blockade of serotonin 5-HT2A receptor increases coronary blood flow via augmented cardiac nitric oxide release through 5-HT1B receptor in hypoperfused canine hearts

Serotonin (5-hydroxytryptamine [5-HT]), which induces vasoconstriction via 5-HT2A receptors in smooth muscle cells and vasodilation through activating nitric oxide (NO) synthase (NOS) via 5-HT1B receptors in endothelial cells, possesses divergent effects on regulating vascular tone. These facts lead us to consider that sarpogrelate, a 5-HT2A receptor blocker, may increase coronary blood flow (CBF) via either attenuation of vasoconstriction through 5-HT2A receptor blockade or augmentation of vasodilation by relative stimulation of NOS through 5-HT1B receptor and we tested this hypothesis in ischemic canine hearts. In open chest dogs, coronary perfusion pressure was reduced so that CBF was decreased to 33% of the baseline and kept constant. Thereafter, sarpogrelate was infused selectively into the left anterior descending artery with and without either an inhibitor of NOS (NG-nitro-L-arginine methyl ester (L-NAME)) or a 5-HT1B receptor antagonist (GR55562). An intracoronary administration of sarpogrelate increased CBF (34.0 +/- 4.0 to 44.5 +/- 4.4 ml/100 g/min, P < 0.05), along with the cardiac NOx release (3.2 +/- 0.6 to 6.8 +/- 1.2 nmol/ml, P < 0.05). The increases in both CBF and NOx by sarpogrelate were completely blunted by the co-administration of either L-NAME or GR55562. Interestingly, sarpogrelate increased the cardiac serotonin release (-4.8 +/- 3.2 vs. 22.1 +/- 1.5 ng/ml, P < 0.05, respectively) in the hypoperfused heart. Immunohistochemical analysis showed that sarpogrelate induced serotonin production in ischemic cardiac myocytes. These results suggest that sarpogrelate increases CBF via augmented cardiac NO production through 5-HT1B receptor activation along with the blockade of 5-HT2A receptors. The increase in cardiac release of serotonin may increase NO production in the ischemic heart.     

Direct effects of sex steroids on prolactin release at the anterior pituitary level: interactions with dopamine, thyrotropin-releasing hormone, and isobutylmethylxanthine

When present alone for 4 or 8 days, 5 alpha-dihydrotestosterone (DHT) or the pure progestin R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione) inhibits spontaneous PRL release by 33--50% in rat anterior pituitary cells in primary culture. This inhibitory effect of DHT and R5020 can only be partially reversed by 17 beta-estradiol (E alpha). DHT and R5020 inhibit spontaneous PRL release in E2-primed cells at ED50 values of 0.5 and 3 nM, respectively. While E2 diminishes by 30--60% the maximal inhibitory effect of dopamine on PRL release and increases by 10-fold the ED50 value of dopamine action, DHT and R5020 can prevent by 30--60% the action of E2 and thus increase the potency of dopamine to inhibit PRL release. The inhibitory action of DHT and R5020 as well as the stimulatory action of E2 on spontaneous PRL release are similarly expressed on TRH- and 3-isobutyl-1-methylxanthine-induced PRL release, thus suggesting that at least part of the highly effective modulatory effects of sex steroids are exerted at a step after cAMP formation.     

Effect of ionotropic and metabotropic glutamate agonists and D-aspartate on prolactin release from anterior pituitary cells.

Although the presence of ionotropic (iGluRs) and metabotropic (mGluRs) glutamate receptors has been demonstrated in the anterior pituitary, recent reports on the direct effect of glutamate on prolactin (PRL) secretion by anterior pituitary cells have presented contradictory results. Hence, the aim of this study was to determine the effect of ionotropic (iGluRs) and metabotropic (mGluRs) glutamate receptor agonists on prolactin (PRL) release. In addition, since D-Aspartate (D-Asp) is found in the pituitary and is involved in neuroendocrine regulation, we also studied the direct action of D-Asp on PRL secretion. Finally, since the posterior pituitary participates in the regulation of PRL secretion, we examined the influence of the posterior pituitary on the effects of NMDA and D-Asp on PRL release. Glutamate (1000 microM) increased PRL secretion from cultured anterior pituitary cells. Both NMDA (100 microM) and kainate (100 microM) increased PRL secretion and these effects were blocked by a specific NMDA receptor antagonist. AMPA did not modify PRL release in these cultures. The group I and II mGluR agonist, trans-ACPD (1000 microM), and a specific group II mGluR agonist, L-CCG-I (100-1000 microM), inhibited whereas specific group I and III mGluR agonists, 3-HPG and L-AP4 respectively, had no effect on PRL release. Finally, D-Asp (100-1000 microM) stimulated PRL secretion and this effect was reduced by a NMDA receptor antagonist. When anterior pituitary cells were cultured in the presence of posterior pituitary cells, NMDA did not modify PRL or GABA release, while D-Asp increased PRL secretion and decreased GABA release in these cocultures. In conclusion, our results show that L-glutamate has a differential direct effect on PRL release: it exerts a stimulatory action via iGluRs and an inhibitory effect via mGluRs. D-Asp could directly stimulate PRL release through NMDA receptors. D-Asp may also stimulate PRL release by decreasing GABA release from the posterior pituitary.     

The functional state of the dopamine receptor in the anterior pituitary is in the high affinity form

In order to determine whether the high affinity state or the low affinity state of the dopamine receptor mediated the inhibition of release of PRL by dopamine agonists, a large number of dopaminergic agonists (n = 31) and antagonists (n = 24) were tested for their potencies to inhibit the binding of [3H] spiperone to porcine anterior pituitary tissue, and for their potencies to affect the release of PRL from rat anterior pituitary cells in culture. All agonists (except bromocriptine, ergocryptine, and dihydroergocryptine) inhibited [3H]spiperone binding in two phases: one phase occurred at nanomolar or subnanomolar concentrations (representing the high affinity state of the dopamine receptor) and the other phase occurred at much higher concentrations of agonist (the low affinity state of the dopamine receptor). The dissociation constants (K) for each drug at each state were derived by computer, with the program LIGAND. It was observed that the agonist K values for the high affinity state were virtually identical with those agonist concentrations inhibiting PRL release; the K values for the low affinity state were about 2 orders higher. These data suggest that the high affinity state of the D2 dopamine receptor is the functional state which mediates the inhibition of PRL release.     

D2 Dopamine receptor subtype mediates the inhibitory effect of dopamine on TRH-induced prolactin release from the bullfrog pituitary

Dopamine receptors in mammals are known to consist of two D1-like receptors (D1 and D5) and three D2-like receptors (D2, D3 and D4). The aim of this study was to determine the dopamine receptor subtype that mediates the inhibitory action of dopamine on the release of prolactin (PRL) from the amphibian pituitary. Distal lobes of the bullfrog (Rana catesbeiana) were perifused and the amount of PRL released in the effluent medium was measured by means of a homologous enzyme-immunoassay. TRH stimulated the release of PRL from perifused pituitaries. Dopamine suppressed TRH-induced elevation of PRL release. Quinpirole (a D2 receptor agonist) also suppressed the stimulatory effect of TRH on the release of PRL, whereas SKF-38393 (a D1 receptor agonist) exhibited no such an effect. The inhibitory action of dopamine on TRH-induced PRL release from the pituitary was nullified by the addition of L-741,626 (a selective D2 receptor antagonist) to the medium, but not by the addition of SCH-23390 (a selective D1 receptor antagonist). These data indicate that the inhibitory effect of dopamine on TRH-evoked PRL release from the bullfrog pituitary gland is mediated through D2 dopamine receptors.