Inhibitory effect of beta-endorphin on gonadotropin-releasing hormone and thyrotropin-releasing hormone releasing activity in cultured rat anterior pituitary cells

To study the effect of human beta-endorphin (beta h-End) on pituitary response to gonadotropin-releasing hormone (LH-RH) and thyrotropin-releasing hormone (TRH) in vitro, we used dispersed rat pituitary cells. When beta h-End (10(-7) M) was simultaneously added along with LH-RH, its stimulatory effect was blocked and naloxone (NAL, 10(-5) M) did not reverse the beta h-End inhibitory effect. NAL alone elicited an increase in LH release, but in the presence of both stimulants (LH-RH and NAL), LH secretion was lower than that observed with LH-RH alone. TRH stimulatory activity of TSH and PRL secretion was blunted by the presence of beta h-End (10(-7) M) and was not reversed by NAL (10(-5) and 10(-3) M). These data suggest that beta h-End directly blocks the LH, TSH- and PRL-secreting activity of both LH-RH and TRH at the pituitary level. This beta h-End effect is not reversed by the specific opiate receptor blocker NAL.     

Stimulatory effect of thyrotrophin-releasing hormone (TRH) on the release of luteinizing hormone (LH) from rat anterior pituitary cells in vitro

The effect of thyrotrophin-releasing hormone (TRH, 10(-7) M) on luteinizing hormone (LH) release from rat anterior pituitary cells was examined using organ and primary cell culture. The addition of TRH to the culture medium resulted in a slightly enhanced release of LH from the cultured pituitary tissues. However, the amount of LH release stimulated by TRH was not greater than that produced by luteinizing hormone-releasing hormone (LH-RH, 10(-7) M). Actinomycin D (2 X 10(-5) M) and cycloheximide (10(-4) M) had an inhibitory effect on the action of TRH on LH release. The inability of TRH to elicit gonadotrophin release from the anterior pituitary glands in vivo may partly be due to physiological inhibition of its action by other hypothalamic factor(s).     

Effect of naloxone and beta-casomorphin on the hypothalamic-pituitary-luteinizing hormone axis in vitro.

The effect of naloxone and beta-casomorphin on luteinizing hormone (LH) release from pituitary cell aggregates, obtained by three-dimensional culture, with or without mediobasal hypothalamic fragments was studied in vitro. Short-term naloxone perifusion at a concentration of 10(-5)M did not modify either basal or LHRH-stimulated LH release from the pituitary cell aggregates. In contrast, a 12-min naloxone perifusion at the same concentration caused an increase in LH release in the mediobasal hypothalamic-pituitary cell aggregate axis. This increase was rapid (12-16 min after time pulse), marked [up to 10 times (p less than 0.004) the initial base line], short (return to the base line secretion 32-40 min after the beginning of the time pulse) and dose-dependent, with a rise greater than 1000% at a concentration of 10(-4) (p less than 0.006). The same effect was observed when a second pulse was applied 48 min after the first one. LH release induced by naloxone was antagonized 56 +/- 2% (p less than 0.03) by beta-casomorphin (an exogenous opiate) at a concentration of 10(-5) M. beta-casomorphin alone did not modify LH basal secretion, but inhibited 25.1 +/- 2.4% (p less than 0.008) LH release enhanced by LHRH. These results indicate that naloxone, an opiate antagonist, markedly increases LH release via a mu-type opioid receptor mechanism at the hypothalamic level only, during short-term exposure.     

The role of protein kinase C in LH release

To investigate the postreceptor mechanism, especially the role of protein kinase C (C-kinase), in luteinizing hormone (LH) release from anterior pituitary cells, dispersed rat anterior pituitary cells were stimulated with luteinizing hormone-releasing hormone (LH-RH), [D-Ser(tBu)]6 des-Gly-NH2(10) ethylamide (Buserelin), 12-0-tetradecanoyl phorbol-13-acetate (TPA) and trifluoperazine (TFP) and the LH released into the medium was determined by radioimmunoassay. LH released by combined stimulation with TPA and either LH-RH or Buserelin was significantly less than that released by LH-RH or Buserelin alone (LH-RH: p less than 0.05; Buserelin: p less than 0.01). It is thought that this paradoxical phenomenon occurred due to desensitization accompanied by down-regulation of LH-RH receptors induced by TPA. This hypothesis was supported by the finding indicating that the binding capacity of LH-RH receptors decreased in a time-course manner during incubation with TPA. The amount of LH released by combined stimulation with TPA and TFP was significantly greater than with TPA alone (P less than 0.01). This suggests that TFP has dual actions, i.e., facilitating and inhibiting LH release.     

An increase of pituitary 3', 5' cyclic adenosine monophosphate produced by estradiol benzoate in vitro: possible implication of this increase in the secretion of luteinizing hormone.

In an attempt to study the site and mechanism of action of estrogen in producing positive feedback control, porcine anterior pituitary slices were incubated in vitro in the presence of estradiol benzoate (EB). EB elevated pituitary cyclic AMP concentration within 5 min and augmented pituitary release of luteinizing hormone (LH). The magnitude of increase of cyclic AMP and LH release was related to the doses of EB used. Also, luteinizing hormone releasing hormone (LH-RH) elevated pituitary cyclic AMP concentration and stimulated pituitary release of LH. The magnitude of increase of cyclic AMP and LH release was inversely related to the doses of LH-RH used. EB and LH-RH were additive in increasing cyclic AMP. Progesterone and clomiphene citrate interfered with an increase of pituitary cyclic AMP produced by EB, but did not significantly affect the basal level of pituitary cyclic AMP. Testosterone propionate, human chorionic gonadotropin and hexestrol were without effect on either basal or stimulated level of pituitary cyclic AMP. Since cyclic AMP and dibutyryl cyclic AMP (DBC) stimulated LH release, it is suggested that EB directly stimulates the release of LH by augmenting cyclic AMP synthesis in the anterior pituitary.     

Rapid release of substance P and LH-RH from synaptosomes prepared from the medial basal hypothalamus and substantia nigra

We investigated Ca2+-dependent, depolarization-induced release of substance P (SP) and LH-RH from medial basal hypothalamic (MBH) and substantia nigra (SN) synaptosomes prepared from male rat brain. Depolarization of MBH synaptosomes evoked significant release of SP from 10.0 +/- 0.1 (5 mM K+) to 28.0 +/- 2.4 (75 mM K+) pg released/10 seconds. Fractional release was 1.0% and 2.7% respectively. In contrast, LH-RH was not released by depolarization of MBH synaptosomes: 11.6 +/- 0.9 (5 mM K+) to 11.0 +/- 0.7 (75 mM K+) pg released/10 seconds. Fractional release was 1.1 and 1.0% respectively. Depolarization-induced LH-RH release also did not occur in the presence of 10(-4) or 10(-6) M norepinephrine, 10(-7) M 12-O-tetradecanoylphorbol-13-acetate (TPA, PMA), 10(-5) M forskolin or in female rats. The inability of depolarizing concentrations of K+ to stimulate LH-RH release in physiological buffers remains an enigma. Significant depolarization-induced SP release was seen from MBH and SN synaptosomes at 20, 15, 10, 5 and only 1 second of release. Despite comparable basal release of SP from MBH and SN synaptosomes, the rate and magnitude of evoked release were much more pronounced in SN synaptosomes. The initial rate (0-1 second) of SP release was 4.5-fold greater from SN than from MBH synaptosomes [krel = 0.027(-1) (SN), krel = 0.006(-1) (MBH)]. The magnitude of SP release from SN synaptosomes was 2- to 3-fold greater at any given time interval compared with release from MBH synaptosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of atrial natriuretic peptide on the release of beta-endorphin from rat hypothalamo-neurohypophysial complex

The aim of this study was to determine whether atrial natriuretic peptide (ANP) alters beta-endorphin (beta-END) secretion from rat intermediate pituitary and whether this effect is a direct action on the intermediate pituitary or an indirect one mediated by hypothalamic factor(s). We studied the release of beta-END from rat neuro-intermediate lobes of the pituitary (NIL) and from the hypothalamo-neurohypophysial complex (HNC), which consists of the hypothalamus, pituitary stalk, intermediate and posterior lobes of the pituitary, by means of an in vitro perifusion system. NIL and HNC were prepared from male Wistar rats and individually perifused for 30 min with perifusion medium followed by 20 min perifusion with medium containing alpha-rat ANP and/or dopamine (DA). Samples of perifusion medium were collected every 5 min and subjected to RIA for beta-END. The basal release of beta-END from NIL was 180% of that from HNC (p less than 0.01), which provides further support for the presence of hypothalamic factors that inhibit beta-END release from the intermediate pituitary. The perifusion of HNC with ANP at 10(-7) and 10(-6) M increased the beta-END concentration by 25 and 50%, respectively (p less than 0.01). In contrast, ANP (10(-8) to 10(-6) M) had no effect on beta-END release from NIL. The inhibitory effect of DA (10(6) M) on beta-END release from NIL and HNC (51% and 50% of the basal release, respectively, p less than 0.01) was confirmed. However, this inhibitory effect was not reversed by ANP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of metal cations on pituitary hormone secretion in vitro

Increased body burdens of metal cations are known to affect adversely reproductive function in several species. The effects of these metals on gonadal function are well documented. In contrast, little is known about their possible direct effects on pituitary hormone release. The purpose of this study was to determine, in vitro, the effects of nickel, cadmium, and zinc (50 μM) on both baseline and potassium chloride (KCl)-stimulated pituitary luteinizing hormone (LH), prolactin (Prl), and thyroid-stimulating hormone (TSH) release. Anterior pituitary fragments from adult male Long-Evans rats were evaluated using a continuous-flow perifusion system. Baseline and stimulated LH releases were unaffected by nickel and zinc; however, cadmium caused an increase in baseline LH secretion. Baseline Prl release was decreased by zinc, while cadmium resulted in increased release of this hormone. Stimulated Prl release was lower during exposure to zinc but unaltered by nickel and cadmium. Following exposure to zinc, a rebound in stimulated release was noted for all three hormones measured. These results showed that the metal cations tested did have a direct effect on pituitary hormone release at a dose lower than those reported to alter testicular function in vitro. Furthermore, the changes in pituitary hormone secretion varied depending upon the metal and hormone being evaluated.     

Mechanism of LH release in cultured rat pituitary cells

To investigate the mechanisms of the synthesis and the release of gonadotropin, rat anterior pituitary cells were stimulated in vitro with luteinizing hormone releasing hormone (LH-RH), [D-Ser(tBu)]6 des-Gly-NH2(10) ethylamide (Buserelin) and 12-0-tetradecanoyl phorbol-13-acetate (TPA), and then the LH and LH-beta subunit released into the medium were determined by radioimmunoassay. Buserelin showed its biological activity at a much lower concentration than LH-RH, but both of them caused the release of LH and LH-beta subunit in a dose-dependent manner. Furthermore, intracellular LH synthesis from LH-beta subunit by stimulation with LH-RH or Buserelin was also found. After inducing various degrees of desensitization by stimulation with LH-RH or Buserelin in a dose-dependent manner (the first stimulation), pituitary cells were stimulated with a fixed dose of TPA (the second stimulation) and the released LH was assayed. LH was released almost constantly by the second stimulation, regardless of the dose used for the first stimulation. These results suggest that the C-kinase pathway was unaffected by the desensitization induced with LH-RH or Buserelin.     

In vitro and in vivo effects of ghrelin on luteinizing hormone and growth hormone release in goldfish

We studied the in vitro and in vivo effects of octanoylated goldfish ghrelin peptides (gGRL-19 and gGRL-12) on luteinizing hormone (LH) and growth hormone (GH) release in goldfish. gGRL-19 and gGRL-12 at picomolar doses stimulated LH and GH release from dispersed goldfish pituitary cells in perifusion and static incubation. Incubation of pituitary cells for 2 h with 10 nM gGRL-12 and 1 or 10 nM gGRL-19 increased LH-beta mRNA expression, whereas only 10 nM gGRL-19 increased GH mRNA expression. Somatostatin-14 abolished the stimulatory effects of ghrelin on GH release from dispersed pituitary cells in perifusion and static culture. The GH secretagogue receptor antagonist d-Lys(3)-GHRP-6 inhibited the ghrelin-induced LH release, whereas no effects were found on stimulation of GH release by ghrelin. Intracerebroventricular injection of 1 ng/g body wt of gGRL-19 or intraperitoneal injection of 100 ng/g body wt of gGRL-19 increased serum LH levels at 60 min after injection, whereas significant increases in GH levels were found at 15 and 30 min after these treatments. Our results indicate that, in addition to its potent stimulatory actions on GH release, goldfish ghrelin peptides have the novel function of stimulating LH release in goldfish.     

Interactions between neuropeptide Y, luteinizing hormone-releasing hormone and estradiol in the control of luteinizing hormone release from cultured ovine pituitary cells

This study used pituitary cells in culture firstly to test the hypothesis that NPY may augment the pituitary LH response to LHRH and secondly to determine whether this interaction is dependent on the presence of estradiol. LHRH (10(-10)-10(-6) M) caused a significant increase in LH secretion from dispersed ovine pituitary cells maintained in culture for six days, a response which was enhanced when cells were pretreated for three days with 4 x 10(-11) M estradiol. NPY 10(-10)-10(-6) M) had no effect on basal LH release from ovine pituitary cells maintained either in the presence or absence of estradiol. NPY (10(-10) and 10(-8) M) also had no effect on LHRH-stimulated LH release either in the presence or absence of estradiol. These results substantiate previous observations that physiologically relevant concentrations of estradiol enhance the LH response to LHRH in cultured ovine pituitary cells. However, in contrast to experiments carried out using rat pituitary cells in culture, the present data provide no evidence to support the hypothesis that NPY alone interacts with LHRH in the control of LH secretion from the ovine pituitary gland.     

Comparison between the biological effects of LH-RH and buserelin on the induction of LH release from hemi-pituitary glands of female rats in vitro

The biological effects of LH-RH and the agonist [D-Ser(But)6-des Gly10]-LH-RH(1-9)-ethylamide (buserelin) were compared during 8 h of incubation with female rat hemi-pituitary glands. Similar dose-response relationships were found for LH-RH and buserelin as concerns the release of luteinizing hormone (LH) by pituitary glands from intact and ovariectomized rats. Also the LH secretion patterns from glands of intact rats were similar: an initial low response was followed by a fast increase (priming effect) after which the response declined again (desensitization). In a subsequent experiment pituitary glands from ovariectomized rats were first exposed to LH-RH or buserelin for 4 h and then further incubated in medium only. After discontinuation of the stimuli the rate of LH release decreased in all cases, but this decrease was significantly greater when the glands had been exposed to LH-RH. Short-term (1/2, 1 or 2 h) exposures to LH-RH or buserelin followed by an intervening period (1 1/2, 1 or 0 h, respectively) of incubation in medium only resulted in an almost similar, significant increase in the subsequent protein synthesis-independent LH response to LH-RH (priming effect). Only preincubation with LH-RH for 2 h was significantly more effective. The results demonstrate equal intrinsic activities for LH-RH and buserelin. Differences in the biopotencies for LH-RH and buserelin in vivo and in vitro may occur only after discontinuation of the external stimuli.     

Indomethacin and gonadotrophin release from rat pituitaries.

The influence of low (5 mcM) and high (200 mcM) concentrations of indomethacin on gonadotropin release from rat pituitaries was studied in vitro. Low concentrations significantly (p less than .05) reduced the release of luteinizing hormone (LH) in comparison with controls, whereas high concentrations significantly (p less than .05) increased the rate of release. The release of follicle stimulating hormone (FSH) was not affected. When pituitary tissue was stimulated by LH-releasing hormone (LH-RH), the high concentration of indomethacin significantly (p less than .05) increased LH release and produced a nearly significant (p less than .01) increase in FSH. The low concentration was without effect. The effect of the addition of prostaglandins (PGs) alone and in combination with indomethacin was also investigated. PGE-1 significantly (p less than .05) increased the release of LH. However, there was no significant (p greater than .1) interaction between the 2 drugs. The effects on the release of FSH were similar. The addition of PGE-2 or PGF-2alpha slightly increased the release of LH and FSH (p greater than .1). It is suggested that high concentrations of indomethacin probably do not inhibit PG synthesis, but may inhibit cyclic nucleotide phosphodiesterase.     

Effects of hypothalamic-releasing hormones on LH, FSH, and prolactin in pituitary monolayer cultures.

Four-day-old pituitary monolayer cultures were incubated with various hypothalamic releasing hormones. Rat hypothalamic extract stimulated the release of LH, FSH, and PRL by these cultures in a dose-related fashion. Synthetic LH-RH stimulated the release of LH and FSH but not of PRL. Synthetic TRH increased the release of PRL but had no effect on LH or FSH. At 10(-8) M, somatostatin did not affect any of the three adenohypophyseal hormones. Incubation with DBcAMP or theophylline also stimulated PRL release without any detectable effect on LH and FSH release. These data suggest the involvement of cyclic AMP--adenylate cyclase system in the mechanism of PRL release, but their involvement in gonadotropin release requires further studies.     

Effect of serotonin on the basal and gonadotrophin-releasing hormone-induced release of luteinizing hormone from rat pituitary glands in vitro

The effect of serotonin (5-HT) on the basal and gonadotrophin-releasing hormone (GnRH)-stimulated release of luteinizing hormone (LH) was studied in rat adenohypophysis in vitro. Anterior pituitary glands from ovariectomized rats were incubated for 1h in the presence of different doses of 5-HT (0.01 to 3 mumol/l). Serotonin added to the culture medium slightly dimished the basal release of LH and markedly inhibited the release of LH induced by GnRH. Responsiveness to GnRH (3 nmol/l) was significantly reduced, in a dose-dependent manner, by the simultaneous treatment of glands with 5-HT. Maximal inhibition to 65% of the response obtained with GnRH alone, was attained with 1 mumol/l 5-HT. The EC50 value was estimated to be about 1.9 X 10(-7) M. The inhibitory effect of 5-HT was evident within 30 min of incubation. Furthermore, 5-HT appear to exert a short-lasting action, since the rate of basal and GnRH-induced release of LH was reduced during the first hour of incubation, but after 2h the suppressive effects of 5-HT were no longer apparent. Methysergide, a serotonin receptor blocking agent, partially antagonized the inhibitory effect of 5-HT on LH release, either basal or GnRH-stimulated. This suggests that a receptor-mediated component may be involved in the mechanism of 5-HT action. The present results indicate that 5-HT can affect the release of LH by acting directly at the pituitary gland level.     

Release of hypothalamic neuropeptide Y and effects of exogenous NPY on the release of hypothalamic GnRH and pituitary gonadotropins in intact and ovariectomized does in vitro

The effect of NPY on the hypothalamic release of GnRH and pituitary release of gonadotropins was examined in intact and ovariectomized (OVEX) rabbits in a superfusion system. Exposure of mediobasal hypothalami (MBH) from intact rabbits to NPY (8 X 10(-8) M) resulted in a sustained stimulation of GnRH secretion into the medium. The same dose of NPY had no effect on MBH-GnRH release from OVEX rabbits. NPY also produced a sustained stimulation of LH and FSH release by pituitary fragments from intact rabbits, but NPY caused only a transient release of these hormones by pituitaries from OVEX does. Media samples from MBH superfusions were also measured for NPY concentrations. NPY was released episodically into the medium. The amplitude and frequency of NPY pulses in intact and OVEX rabbits did not differ; nor were mean levels of NPY significantly affected by castration. These results suggest that NPY has direct effects on both the hypothalamus and pituitary to modulate the the activities of GnRH neurons and gonadotropes. The pattern of GnRH and gonadotropin response to NPY exposure is determined by ovarian factors.     

Action of luteinizing hormone-releasing hormone and synthesis of prostaglandin in the pituitary gland

The possibility that prostaglandin E₂ (PGE₂) may play a role in luteinizing hormone (LH) release was examined using an model. Addition of luteinizing hormone-releasing hormone (LH-RH) to the culture medium stimulated cyclic AMP accumulation and LH-release by incubated hemipituitaries, but did not affect the level of PGE₂ or prostaglandin synthetase activity in the gland. Aspirin and indomethacin reduced both prostaglandin synthetase activity and PGE₂ content in the pituitary, but did not impair the stimulatory action of LH-RH on either cyclic AMP accumulation or LH-release. Flufenamic acid on its own caused LH-release, but the drug abolished the effect of LH-RH on cyclic AMP accumulation. The mechanism of this action of flufenamic acid is not understood.It is concluded that the stimulatory action of LH-RH on pituitary cyclic AMP production and LH release is not mediated by prostaglandins.     

Effect of pre-incubation with different concentrations of LH-RH on subsequent LH release caused by supramaximally active amounts of LH-RH; role of LH-RH-induced protein synthesis.

Anterior pituitary glands from intact diestrous female rats were incubated for two consecutive periods of 3 hours. During the first period various submaximally active amounts of luteinizing hormone-releasing hormone (LH-RH) were added to the media, whereas during the second period a supramaximally active concentration of LH-RH was present. When during the second incubation period protein synthesis was inhibited by cycloheximide, the amount of luteinizing hormone (LH) released during that period was positively correlated to the concentration of LH present during the first incubation period. This relationship was not seen when cycloheximide was absent, or when cycloheximide was present throughout both periods. Total LH was not affected by LH-RH; thus no effect of LH-RH on LH synthesis was observed. It is concluded that the amounts of protein synthesized by the pituitary glands in response to the different amounts of LH-RH during the first incubation period can constitute a limiting factor for the response to the supramaximally active amount of LH-RH added during the second incubation period.     

Further evidence that peptide histidine isoleucine (PHI) may function as a prolactin releasing factor in rats

Intraventricular administration of peptide histidine isoleucine (PHI) (200 ng, 1, 5 and 10 micrograms/rat) resulted in a significant and dose-related increase in plasma prolactin (PRL) levels in urethane-anesthetized rats and in conscious rats with intraatrial and intraventricular catheters. Intravenous injection of PHI (10 micrograms/rat) also raised plasma PRL levels in these animals. In in vitro studies, PRL release from superfused rat anterior pituitary cells was stimulated by PHI (10(-9), 10(-8) and 10(-7) M) in a dose-related manner. The stimulating effect of PHI (10(-7)M) on PRL release in vitro was as potent as that of vasoactive intestinal polypeptide (VIP) (10(-7) M) and was observed even in the presence of dopamine (10(-7) M). These results suggest that PHI plays a stimulating role in regulating PRL secretion by acting, at least in part, directly on the pituitary in the rat.     

The effects of gonadectomy and oestradiol-17 beta on pituitary responsiveness to LH-RH in the adult male marmoset (Callithrix jacchus).

Exogenous luteinizing hormone-releasing hormone (LH-RH) administered in a wide range of doses (0.2-25 micrograms) to intact male marmoset monkeys induced a marked increased in plasma luteinizing hormone (LH) concentrations. Maximum LH concentrations achieved after injection of LH-RH occurred progressively later as the dosage increased. Bilateral orchidectomy sigificantly enhanced pituitary responsiveness to a standard dose (2.0 microgram) of LH-RH, whereas the introduction of oestradiol-17 beta implants effectively inhibited the responses. LH-RH-induced LH release after gonadectomy (with and without oestradiol-17 beta treatment) was similar in males and females. The use of marmosets for appropriate investigation into the physiological role of LH-RH in controlling LH secretion in primates is proposed.