Endothelin-induced biphasic response of lactotrophs cultured under different conditions.

Endothelin (ET), a recently discovered vasoconstrictor peptide, is widely distributed in different tissues including brain and pituitary. Although evidence regarding the role of ET in neuroendocrine processes is still fragmentary, it appears that the release of several anterior pituitary (AP) hormones can be modulated by peptides of the ET family. In the present study, we compared the effects of ET-1 and ET-3 on the release of PRL from AP cells cultured in serum-free (SFM) or serum-containing (SCM) medium. AP obtained from adult male rats were enzymatically dispersed, and the cells were plated in either SFM or SCM. After 4 days, cells were incubated with ET-1 or ET-3 for designated periods of time, and PRL levels in the incubation media were measured by RIA. When AP cells maintained in SCM were exposed to ET for 20 min, both peptides inhibited PRL release, with ET-1 being a more potent inhibitor than ET-3. In contrast, a biphasic response was observed in cultures grown in SFM: low concentrations of ET peptides inhibited the secretion of PRL, while high concentrations caused a significant stimulation. Further characterization of the effects of ET-1 revealed that the stimulatory phase was relatively short (15-30 min) and was followed by inhibition of PRL release. The addition of either horse serum or fetal bovine serum to SFM on the day of plating resulted in a dose-dependent reduction in the ET-induced stimulation of lactotrophs. These findings indicate that the presence of serum in culture medium alters the expression of cell properties underlying inhibitory and stimulatory responses to ET in terms of PRL secretion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of the pituitary effects of the mammalian endothelins: vasoactive intestinal contractor (endothelin-beta, rat endothelin-2) is a potent inhibitor of prolactin secretion.

Direct pituitary effects of vasoactive intestinal contractor (VIC), which has been described recently to be the rat form of endothelin-2 (ET-2), were compared to those previously reported for rat ET-1, rat ET-3, and human ET-2. In static incubations of cultured dispersed anterior pituitary cells, the minimum effective dose of VIC necessary to inhibit PRL release after 1-h incubation was 1 pM, and the maximum effective dose was 1 nM. Similar inhibition was observed with human ET-2. The minimum effective inhibitory dose of ET-1 was also 1 pM; however, that of ET-3 was 0.1 nM. PRL release inhibition by VIC was not mediated via the D2-dopamine receptor and was not prevented by calcium channel blockade with 100 nM nifedipine. The inhibitory effect of VIC was not present in cells treated with 100 nM staurosporine, a dose that inhibits protein kinase-C activity. Time-course studies revealed a transient stimulation of PRL release with higher doses of VIC (10 and 100 nM), which occurred within the first 15 min of incubation and was unaffected by calcium channel blockade or inhibition of protein kinase-C activity. No stimulation of PRL release was observed with doses of VIC lower than 10 nM. Instead, we observed the maintenance of the inhibitory effect for 4 h of incubation. GH release was not significantly affected by doses of VIC ranging from 10(-13)-10(-7) M; however, the release of LH was slightly, yet significantly, stimulated by 10 and 100 nM VIC. This release was prevented by pretreatment with nifedipine, but unaffected by protein kinase-C inactivation. A physiological role for VIC (rat ET-2) in the control of lactotroph function is suggested by its effectiveness at picomolar doses and its long-lasting action.     

Regulatory role of galanin in control of hypothalamic-anterior pituitary function.

The role of the neuropeptide galanin in the regulation of anterior pituitary function was studied in vivo in conscious male rats and in vitro with cultured anterior pituitary cells. Galanin (50-200 ng; 15-60 pmol) injected into the third cerebral ventricle of rats produced highly significant, dose-related increases of plasma growth hormone (GH) concentrations, whereas galanin increased prolactin (PRL) and decreased thyroid-stimulating hormone (TSH) levels only at the highest dose (60 pmol) tested. Intravenous galanin failed to alter PRL and TSH levels in these rats. In contrast with the results with intraventricular injection of the peptide, intravenous injection of 30 or 300 pmol of galanin produced small, brief, dose-related increases in plasma GH. The response to the 300-pmol dose was less than that induced by a factor-of-20-lower intraventricular dose, which establishes a central action of galanin. Galanin in concentrations ranging from 1 nM to 1 microM failed to alter significantly GH, PRL, or TSH release from dispersed anterior pituitary cells. It also failed to alter GH secretion in response to 100 nM GH-releasing hormone; however, at this dose galanin did potentiate the effect of 100 nM TSH-releasing hormone on TSH and PRL release. Thus, the effects of third-ventricular injection of the peptide are mediated by the hypothalamus. To determine the physiological significance of galanin in control of pituitary hormone release, highly specific antiserum against galanin was injected intraventricularly. Third-ventricular injection of 3 microliter of galanin antiserum resulted in a dramatic decrease in plasma GH values as compared with those of normal rabbit serum-injected controls within 15 min, which persisted until the end of the experiment (5 hr postinjection). Galanin antiserum did not decrease plasma PRL or TSH levels at any time period after its third-ventricular injection; however, a transient increase of plasma TSH levels occurred after 30 and 60 min in comparison with TSH levels in normal rabbit serum-injected controls. Since there was no effect of the antiserum on plasma PRL and only a transient elevation in TSH, galanin may not be physiologically significant enough during resting conditions to alter PRL and TSH release in the male rat. The results of the experiments with galanin antiserum indicate that endogenous galanin has a tonic action within the hypothalamus to stimulate GH release. The rapidity of onset of the effects of galanin and the antiserum directed against it suggest that it acts to stimulate release of GH-releasing hormone from periventricular structures, which then stimulates the release of GH.     

Bombesin stimulates prolactin and growth hormone release by pituitary cells in culture

Bombesin (BBS) has been previously shown to stimulate the secretion of PRL and GH in steroid-primed rats. To determine whether these effects were mediated by the central nervous system or were due to direct action on the pituitary gland, we studied the interaction of BBS with GH4C1 cells, a clonal strain of rat pituitary cells which synthesizes and secretes PRL and GH. The addition of 100 nM BBS to GH4C1 cells for 60 min increased PRL release to 140 +/- 3% of the control value (mean +/- SE) and GH release to 133 +/- 5% of the control value. Stimulation of hormone secretion was observed within 15 min of treatment with 100 nM BBS and continued for at least 2 h. Half-maximal stimulation of PRL release occurred with 0.5 nM BBS, and a maximal effect was observed with 10 nM peptide. The BBS analogs ranatensin, litorin, and [Tyr4]BBS, each at a concentration of 100 nM, caused the same stimulation of PRL release as maximal concentrations of BBS itself. BBS stimulated hormone release selectively in two of five different clonal pituitary cell strains examined. Pretreatment of GH4C1 cells with 1 nM estradiol and/or 100 nM insulin resulted in more powerful stimulation of PRL release by both TRH and BBS. When epidermal growth factor and vasoactive intestinal peptide were added simultaneously with BBS, PRL release was greater than in the presence of either peptide alone. In contrast, the stimulatory effects of TRH and BBS were not additive. Somatostatin inhibited both basal and stimulated PRL release. Thus, low concentrations of BBS can directly stimulate PRL and GH release by a clonal pituitary cell strain in culture. These results suggest that BBS may stimulate PRL and GH secretion in vivo by direct action on the pituitary gland.     

The rat posterior pituitary contains a potent prolactin-releasing factor: studies with perifused anterior pituitary cells

We previously reported that removal of the posterior pituitary abolished the suckling-induced rise in plasma PRL. This suggested that the posterior pituitary contains a PRL-releasing factor (PRF). Using perifused anterior pituitary cells, the objectives of this study were 1) to examine whether the posterior pituitary contains PRF activity as compared to the medial basal hypothalamus (MBH), and 2) to determine to what extent substances known to be present in the posterior pituitary and/or MBH contribute to this activity. Anterior pituitary cells, attached to Cytodex beads, were perifused with medium 199. Tissues were extracted with acid, lyophilized, and reconstituted in medium 199. Tissue extracts and synthetic compounds were introduced to the cells in short pulses. Fractions were collected and analyzed for PRL, LH, and GH by RIA. Posterior pituitary extracts contained a potent substance(s) which stimulated PRL release in a concentration-dependent manner, but did not alter LH secretion. As little as 1% of the extract increased PRL release. In contrast, the MBH extract contained significantly less PRF activity but was capable of stimulating and inhibiting LH and GH release, respectively. Cerebellar extracts did not alter PRL secretion. Of more than 25 neuroactive substances tested in the perifusion system, oxytocin, TRH, and angiotensin II (A II) appeared as likely candidates for PRF. Therefore, the specific receptor antagonists d(CH2)5Tyr(Me) ornithine vasotocin (for oxytocin), chlordiazepoxide (for TRH), or saralasin (for A II) were infused together with the posterior pituitary extract. These antagonists completely abolished the PRL-releasing activities of their respective peptides but failed to reduce the PRF activity of the posterior pituitary. In contrast, PRF activity in the MBH was nearly eliminated by the TRH antagonist. Conclusions: 1) The rat posterior pituitary contains a potent PRF capable of inducing a rapid, hormone-specific, concentration-dependent stimulation of PRL release from perifused anterior pituitary cells. 2) The MBH contains significantly less PRF activity, which is largely attributable to TRH. 3) Although the chemical identity of PRF is yet unknown, the PRF activity in the posterior pituitary is not accounted for by oxytocin, TRH, or A II.     

Dihydropyridine-sensitive calcium channel activity related to prolactin, growth hormone, and luteinizing hormone release from anterior pituitary cells in culture: interactions with somatostatin, dopamine, and estrogens

In the present work, we determined the activity of voltage-dependent dihydropyridine (DHP)-sensitive Ca2+ channels related to PRL, GH, and LH secretion in primary cultures of pituitary cells from male or female rats. We investigated their modulation by 17 beta-estradiol (E2) and their involvement in dopamine (DA) and somatostatin (SRIF) inhibition of PRL and GH release. BAY-K-8644 (BAYK), a DHP agonist which increases the opening time of already activated channels, stimulated PRL and GH secretion in a dose-dependent manner. The effect was more pronounced on PRL than on GH release. BAYK-evoked hormone secretion was further amplified by simultaneous application of K+ (30 or 56 mM) to the cell cultures; in parallel, BAYK-induced 45Ca uptake by the cells was potentiated in the presence of depolarizing stimuli. In contrast, BAYK was unable to stimulate LH secretion from male pituitary cells, but it potentiated LHRH- as well as K+-induced LH release; it had only a weak effect on LH secretion from female cell cultures. Basal and BAYK-induced pituitary hormone release were blocked by the Ca2+ channel antagonist nitrendipine. Under no condition did BAYK affect the hydrolysis of phosphoinositides or cAMP formation. Pretreatment of female pituitary cell cultures with E2 (10(-9) M) for 72 h enhanced LH and PRL responses to BAYK, but was ineffective on GH secretion. DA (10(-7) M) inhibited basal and BAYK-induced PRL release from male or female pituitary cells treated or not treated with E2 (10(-9) M). SRIF (10(-9) and 10(-8) M) reversed BAYK-evoked GH release to the same extent in cell cultures derived from male or female animals. It was ineffective on BAYK-induced PRL secretion in the absence of E2, but antagonized it after E2 pretreatment. The effect was dependent upon the time of steroid treatment and was specific, since 17 alpha-estradiol was inactive. In addition, DA and SRIF decreased the 45Ca uptake induced by the calcium agonist. These data demonstrate that DHP-sensitive voltage-dependent calcium channels of the L type present on different pituitary cells are not equally susceptible to BAYK activation under steady state basal conditions, indicating that their spontaneous activity and/or distribution vary according to the cell type; their activity is modulated by sex steroids. In addition, these data suggest that Ca2+ channels represent a possible site of DA and SRIF inhibition of PRL and GH release, respectively, by gating calcium entry into the corresponding cells.     

A possible role of prostacyclin to stimulate prolactin and growth hormone release by hypothalamic and pituitary actions, respectively

Prostacyclin (PGI2) (1-5 micrograms in 3 microliters 0.05 M Tris/HCl buffer, pH 7.5) and its stable metabolite, 6-oxo-PGF1 alpha, were microinjected into the third ventricle of ovariectomized rats, and plasma FSH, GH, PRL, and TSH levels were measured by RIA. Control animals received 3 microliters buffer. Injection of 5 micrograms PGI2 dramatically elevated plasma PRL values (4- to 5-fold) at 5 and 15 min, whereas the same dose of 6-oxo-PGF1 alpha produced a significant but smaller (2-fold) stimulatory effect. A delayed increase (1.5-fold) in plasma GH occurred after intraventricular PGI2 at 30 and 60 min. 6-Oxo-PGF1 alpha failed to alter GH levels. There were no alterations in plasma FSH and TSH after intraventricular injection of PGI2. Dispersed, overnight cultured cells from anterior pituitaries of ovariectomized rats were tested with 10(-4)-10(-7) M PGI2 and its metabolite. After 15 min of incubation, 3 X 10(-5) PGI2 produced a highly significant elevation in GH release (P less than 0.001), whereas there was no alteration in PRL levels. Only pharmacological doses of 6-oxo-PGF1 alpha (10(-4) M) stimulated GH release. There was no alteration in PRL release by the cultured cells even in the presence of 10(-4) PGI2. These results suggest that PGI2 stimulates PRL release by a hypothalamic action either to increase the release of PRL-releasing factor, or to decrease release of PRL-inhibiting factor, or by both mechanisms. The delayed stimulatory effect of PGI2 on the release of GH may be exerted via an effect on the anterior lobe itself, since PGI2 was effective in stimulating GH release by the incubated pituitary cells.     

The effects of endothelins on the secretion of prolactin, luteinizing hormone, and follicle-stimulating hormone are mediated by different guanine nucleotide-binding proteins

Different bacterial toxins capable of modifying specific alpha-subunits of G-proteins were used to characterize the guanine nucleotide-binding protein (G-protein) dependency of the effects of endothelins (ETs) on PRL, LH, and FSH secretion. Primary cultures of anterior pituitary cells obtained from female rats were preincubated for 24 h with 20 ng/ml pertussis toxin (PTX) or 2 micrograms/ml cholera toxin (CTX) before challenge with ETs. Both ET-1 and ET-3 elicited a concentration-dependent inhibition of PRL secretion and stimulated the release of LH and FSH secretion on pituitary cells not treated with toxins. Based on the calculated ration of the half-maximal effective concentrations (EC50) of ET-1 and ET-3, ET-1 showed 7800, 20, and 14 times greater potency than ET-3 on PRL, LH, and FSH secretion, respectively. PTX, a selective inhibitor of Gi and several other G proteins, increased the basal secretion of PRL and completely eliminated the responsiveness of lactotroph cells to ET-1 and ET-3. Pretreatment with PTX caused a markedly different effect on LH and FSH secretion: while basal LH release was slightly increased, FSH secretion was markedly depressed by PTX. Moreover, while ET-induced LH secretion was enhanced by PTX, the effectiveness of ETs on FSH release was completely abolished. CTX, known as an activator of Gs proteins, decreased the basal secretory activity of lactotrophs but did not influence the ET-induced decrease of PRL release. CTX pretreatment (like PTX before) elicited a strikingly different effect on LH and FSH: while basal LH secretion was enhanced, basal FSH secretion was markedly inhibited by CTX. Moreover, while the effectiveness of ETs on LH secretion was not changed significantly, the stimulatory effect of ETs on FSH secretion was diminished after CTX pretreatment. Thus, the inhibition of PRL secretion by ETs requires a PTX-sensitive G protein while the ET-induced stimulation of FSH secretion involves both PTX- and CTX-sensitive elements. The fact that pretreatments with PTX or CTX influenced basal secretion of PRL, LH, and FSH suggests that PTX- and/or CTX-sensitive G proteins are directly involved in the process of exocytosis. Additionally, these findings might indicate an active paracrine/autocrine regulation of pituitary cells in culture that are impaired or enhanced by the bacterial toxins employed. Though the broad substrate specificity of PTX and CTX and the multiplicity of G protein families did not allow us to identify the specific G protein(s) involved, these data reveal the diversity of ET-induced intracellular signaling mechanisms in lactotrophs and gonadotrophs.     

The role of histamine in the neuroendocrine regulation of pituitary hormone secretion

The neurotransmitter histamine participates in the neuroendocrine regulation of pituitary hormone secretion by an indirect action at a hypothalamic level where histaminergic neurons are abundant. The effect of histamine is caused by activation of postsynaptic H1- or H2-receptors. Histamine stimulates the secretion of ACTH, beta-endorphin (mediated by CRH and AVP), alpha-MSH (mediated by dopamine and peripheral catecholamines), and PRL (mediated by dopamine, serotonin and AVP), and participates in the stress-induced release of these hormones and possibly in the suckling- and estrogen-induced PRL release. The release of GH and TSH is predominantly inhibited by histamine; however, uncertainty exists regarding its role and the hypothalamic factors involved. Histamine increases the secretion of LH in females (mediated by GnRH), and may be involved in the mediation of the estrogen-induced LH surge. AVP and oxytocin are stimulated by histamine, probably by an effect in the supraoptic and paraventricular nuclei of the hypothalamus.     

The role of calcium in LH release from the pituitary by phorbol ester

GnRH stimulates LH release from pituitary cells, and this process is calcium dependent. On the other hand, phorbol ester, 12-0-tetradecanoylphorbol-13-acetate (TPA), a potent activator of calcium- and phospholipid-dependent protein kinase (protein kinase C), stimulates luteinizing hormone (LH) release from rat pituitary cells. To investigate the involvement of the calcium dependent process in LH release by TPA, the effects of calcium channel antagonists, verapamil and nifedipine, on TPA-mediated LH release were compared with those of a GnRH superagonist, [D-Ala6] des-Gly10-GnRH N-ethylamide (GnRHa) in cultured pituitary cells. Furthermore, pituitary cells saturated with 45Ca2+ were stimulated by GnRHa or TPA and calcium mobilization after the stimuli were monitored. The pituitary cells from adult male rats were dispersed by trypsin and cultured for 3 days. Cultured pituitary cells were incubated with GnRHa or TPA in the presence of increasing concentrations of verapamil or nifedipine for 3hrs, and LH released into medium was measured by RIA for rat LH. For 45Ca2+ experiment, 3 day-cultured pituitary cells were saturated with 45Ca2+ (10(6) cells/1 microCi/100 microliters) and incubated with secretagogues for the indicated times. Incubations were terminated by filtration, and the radioactivity on the filter was measured by a beta-counter. LH release was stimulated by 0.1 nM TPA, and the maximum response at 10 nM TPA was 50% of the LH response to GnRHa. A23187 also stimulated LH release in relatively high concentrations (10(-5)-10(-4) M), and no additive stimulatory effect was observed when a half-maximal dose of TPA (10(-9) M) was added with increasing concentrations of A23187. Verapamil partially inhibited both GnRHa- and TPA-stimulated LH release, and a similar inhibitory effect on LH release was observed when nifedipine was incubated with GnRHa or TPA, although high concentrations (10(-5)-10(-4) M) of nifedipine stimulated LH release induced by GnRHa and TPA. GnRHa and TPA stimulated 45Ca2+ influx into the cells, and its peak was observed 15 and 30 seconds after stimulation, respectively, while GnRH antagonist did not mobilize 45Ca2+ until 120 seconds after stimulation. These results suggest that TPA-stimulated LH release from pituitary cells involves a calcium dependent process as does GnRH-stimulated LH release.     

Atrial natriuretic factor inhibits luteinizing hormone secretion in the rat: evidence for a hypothalamic site of action

The presence of atrial natriuretic factor (ANF) immunoreactivity and receptors for ANF in the median eminence, hypothalamus, and anterior pituitary gland suggests a role for the peptide in the hypothalamic control of anterior pituitary function. In conscious ovariectomized female rats, transient elevation of plasma levels of ANF by volume loading, a stimulus known to release endogenous ANF from the heart, or by bolus iv injection of 0.1, 1.0, or 10 micrograms synthetic ANF failed to result in altered circulating levels of LH or GH. Constant iv infusion of ANF for 30 min, such that 2- to 3-fold elevations in plasma ANF were detected by RIA resulted, however, in significant inhibition of LH release in ovariectomized female rats (0.05 and 0.1 micrograms ANF/kg.min) and orchidectomized male rats (0.1 microgram ANF/kg.min). It was unlikely that this effect was exerted at the level of the anterior pituitary, since ANF failed to alter basal or LHRH-stimulated LH release from cultured anterior pituitary cells in vitro and since iv infusion of 0.1 microgram ANF/kg.min failed to alter pituitary responsiveness in vivo to a 10-ng bolus injection of LHRH. Significant inhibition of LH secretion was also observed after third cerebroventricular injection of 1.0 or 2.0 nmol ANF. As with iv infusion, central administration of ANF failed to significantly alter GH secretion. LHRH release from median eminence explants incubated in vitro in the presence of dopamine (60 or 120 microM) was inhibited by 10(-7) M ANF, suggesting a median eminence site of action of the peptide. Finally, an opiate involvement in the mechanism of ANF's action was suggested, since naloxone (0.5 mg, iv, followed by a 60-min infusion of an additional 1 mg) completely blocked the ability of ANF (0.1 or 0.5 microgram/kg.min, infused over the last 30 min of naloxone administration) to inhibit LH release. These data suggest that ANF can act centrally to alter the hypothalamic control of gonadotropin secretion, possibly by interacting with central dopaminergic and peptidergic systems. They further suggest actions of ANF within the brain unrelated to its previously described effects on fluid and electrolyte homeostasis.     

Role of Neurokinin B and Dynorphin A in pituitary gonadotroph and somatolactotroph cell lines

The role of Neurokinin B (NKB) and Dynorphin A (Dyn) in the regulation of the hypothalamic pituitary axis is an important area of recent investigation. These peptides are critical for the rhythmic release of GnRH, which subsequently stimulates the secretion of the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The present study utilized the gonadotroph cell line LβT2 and the somatolactotroph GH3 cell line to examine the possible role of these peptides in pituitary hormone secretion. The NKB receptor (NK3R) and the Dyn receptor (the κ-opiate receptor (KOR)) were both detected in LβT2 cells and GH3 cells. NKB, by itself, failed to increase gonadotropin LHβ and FSHβ promoter activities and did not modulate the effects of GnRH on gonadotropin promoter activity. In GH3 cells, NKB significantly increased TRH-induced PRL promoter activity although NKB alone did not have an effect on basal PRL promoter activity. Dyn had no effect on gonadotropin promoters alone or in combination with GnRH stimulation. PRL promoters stimulated by TRH were not significantly changed by Dyn. TRH-induced PRL promoter activity was further increased in the presence of higher concentrations of NKB, whereas Dyn did not have a significant effect on the PRL promoter even at a high concentration. In addition, TRH-induced ERK (Extracelluar signal-regulated kinase) activation was enhanced in the presence of NKB. Our current study demonstrated that NKB had a stimulatory effect on PRL expression in a PRL-producing cell, but had no effect on gonadotropin secretion from a gonadotroph cell line.     

The impact of euglycemia and hyperglycemia on stimulated pituitary hormone release in insulin-dependent diabetics

To study the influence of different blood glucose (BG) concentrations on the release of pituitary hormones, the effect of the simultaneous iv administration of LRH (200 micrograms), TRH (400 micrograms), and arginine (30 g/30 min) upon the serum concentrations of LH, FSH, TSH, PRL, and GH was determined in six male insulin-dependent diabetics. BG concentration was clamped by feedback control and an automated glucose-controlled insulin infusion system at euglycemic (BG 4-5 mmol/liter) or hyperglycemic (BG, 14-18 mmol/liter) levels. Increments in serum concentrations of LH, FSH, TSH, and PRL were similar in the euglycemic and hyperglycemic steady states, whereas the GH response to arginine was suppressed during the hyperglycemic clamp (P less than 0.01). Omission of exogenous insulin during hyperglycemia did not modify the observed hormonal responses. Thus, the release of LH, FSH, TSH, and PRL in response to adequate acute stimuli at the pituitary level is not modulated by hyperglycemia in insulin-dependent diabetes, while arginine-induced GH release is suppressed. Since the effect of arginine on GH is most likely mediated by an action on the hypothalamus, the data suggest that elevated glucose concentrations may exert their modulatory influence on GH secretion at the hypothalamic rather than at the pituitary level.     

Calcitonin inhibits basal and thyrotropin-releasing hormone-induced release of prolactin from anterior pituitary cells: evidence for a selective action exerted proximal to secretagogue-induced increases in cytosolic Ca2+

Salmon calcitonin (sCT)-like peptide is present in the central nervous system and pituitary gland of the rat, and this peptide inhibits basal and TRH-stimulated PRL release from cultured rat anterior pituitary (AP) cells. The present studies were designed to examine further the inhibitory actions of sCT on basal and TRH-stimulated PRL release and investigated 1) the temporal dynamics of the responses, 2) the effects of sCT on PRL release induced by other secretogogues, and particularly those acting via elevations of cytosolic Ca2+, and 3) the selectivity of sCT action on basal and stimulated AP hormone release. The inhibition of basal PRL release by sCT (0.1-10 nM) was dose-dependent and was characterized by a rapid onset with a gradual recovery to normal rates of release after the period of sCT inhibition. The inhibitory effect of sCT on basal PRL release was reversed by treatment with either the Ca2+ ionophore A23187 or with the phorbol ester, phorbol myristate acetate (PMA). sCT infusion did not affect the basal release of GH, TSH, FSH, or LH by perifused AP cells. When administered in short pulses, TRH, at concentrations from 1-100 nM, elicited a dose-dependent increase in PRL release. When coadministered with short 10 nM TRH, sCT (1-100 nM) inhibited TRH-induced PRL release in a dose-dependent manner, with a maximal inhibition of 78% at a concentration of 10 nM, and an ED50 concentration of approximately 3 nM. During longer (30 min) pulses of TRH (100 nM), PRL release increased sharply over 4-fold within 2 min, followed within 12 min by a rapid decline to a level 1.5-2-fold higher than basal, and this level was maintained for the remainder of the stimulation period. sCT pretreatment inhibited the overall PRL response to TRH. In contrast to its inhibition of TRH-induced PRL release, sCT failed to prevent the stimulation of PRL release by either ionophore A23187, PMA, vasoactive intestinal peptide, or forskolin. In addition, sCT failed to block TRH-induced TSH release or GnRH-induced LH release.(ABSTRACT TRUNCATED AT 400 WORDS)     

Divergent influences of calcium ions on releasing factor-stimulated anterior pituitary hormone secretion in normal man

To investigate the influence of calcium ions on the secretion of anterior pituitary hormones in response to stimulation by exogenous hypothalmic releasing factors in man, we measured serum concentrations of pituitary hormones serially during a continuous infusion of combined TRH (2 micrograms/min) and GnRH (1 microgram/min), with concomitant iv saline or calcium administration. Compared to saline, calcium administration was associated with a significant increase in GnRH-TRH-stimulated LH and FSH release and a corresponding rise in serum testosterone concentrations. The effect of calcium ions on gonadotropin secretion was specific, because releasing factor-stimulated secretion of TSH and PRL was suppressed by hypercalcemia. Serum concentrations of GH were not significantly altered under these conditions. In summary, the present results provide the first in vivo evidence that acute infusion of calcium ions augments GnRH-TRH-stimulated secretion of LH and FSH, with an accompanying increase in serum testosterone levels. In contrast, hypercalcemia did not alter serum GH concentrations, and it suppressed GnRH-TRH-stimulated release of PRL and TSH. We conclude that calcium ions can selectively influence releasing factor-stimulated secretion of certain anterior pituitary hormones in man.     

Endothelin ETA receptors mediate the signaling and secretory actions of endothelins in pituitary gonadotrophs.

Specific receptors for endothelin (ET), localized by autoradiographic studies with [125I]ET in frozen sections of the rat pituitary gland, were abundant in the adenohypophysis, but not in the neurohypophysis. Specific binding of [125I]ET-1 and [125I]ET-3 was also demonstrable in 3-day-old primary cultures of anterior pituitary cells. The binding of [125I]ET-1 to its receptors was time and temperature dependent and was followed by rapid internalization of the receptor-ligand complex. Binding of [125I]ET-1 and [125I]ET-3 to pituitary tissues and cells was more effectively displaced by ET-1 and ET-2 than by ET-3. In cultured pituitary cells, ET-1 caused a rapid increase in polyphosphoinositide hydrolysis, and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] production, with a prompt rise in the cytoplasmic calcium concentration ([Ca2+]i) and LH secretion. The Ins(1,4,5)P3 response to 100 nM ET-1 was transient, with a spike at 10 sec followed by an exponential decrease toward the low steady state level. Ins(1,3,4)P3 and inositol bisphosphate (InsP2) increased more slowly, reaching peak values 30-40 sec after stimulation. The kinetics of the [Ca2+]i response to ET-1 were similar to those of the Ins(1,4,5)P3 response and more rapid than those of the Ins(1,3,4)P3 and InsP2 responses. In perifused cells, ET-stimulated increases in LH release showed the same biphasic patterns as the Ins(1,4,5)P3 and [Ca2+]i responses. ET-1 was more potent than ET-3 in stimulating [Ca2+]i and LH responses, consistent with its higher affinity for the pituitary ET receptors. The initial activation of Ca2+ signaling and LH exocytosis by ETs was followed by prolonged refractoriness to both ET-1 and ET-3. The development of desensitization occurred more rapidly in ET-1- than ET-3-stimulated cells and correlated temporally with endocytosis of the receptor-ligand complex. These findings indicate that stimulation of gonadotropin release by ETs occurs via activation of ETA-type receptors, which are coupled to polyphosphoinositide hydrolysis and [Ca2+]i mobilization, and undergo rapid internalization and profound desensitization.     

Paradoxical signal transduction mechanism of endothelins and sarafotoxins in cultured pituitary cells: stimulation of phosphoinositide turnover and inhibition of prolactin release.

Endothelins (ET-1, ET-2, ET-3 and vasoactive intestinal contractor, VIC) and sarafotoxins (SRTX-b and SRTX-c) appear to bind with high affinity to a homogeneous class of binding sites in cultured rat pituitary cells. All of these ligands seem to interact with the same receptor (ETA-R), except for SRTX-c which apparently binds to a separate receptor. Binding was followed by phosphodiesteric cleavage of phosphoinositides, resulting in the formation of inositol phosphates. No consistent effect on basal or gonadotropin-releasing hormone (GnRH)-induced release of luteinizing hormone (LH) was exerted by ET or SRTX during 2 h of static incubation. On the other hand, both groups of vasoactive peptides inhibited basal and thyrotropin-releasing hormone (TRH)-induced prolactin secretion. Surprisingly, activation of phosphoinositide turnover by TRH in pituitary mammotrophs led to stimulation of prolactin secretion, whereas activation of the same pathway by ET or SRTX resulted in inhibition of prolactin secretion. ET and SRTX stimulated inositol phosphate formation in GH3 cell line and in the gonadotroph-like cell line alpha T-3 (which is capable of producing the alpha subunit of the gonadotrophins), indicating that the peptides interact with both pituitary mammotrophs and gonadotrophs. The very low concentrations (nM range) needed to stimulate phosphoinositide turnover and to inhibit prolactin secretion, as well as the recent finding that ETs are present in the hypothalamo-pituitary axis suggest that ET might participate in the neuroendocrine modulation of pituitary functions. One such possibility is that ETs might be members of the prolactin inhibiting factors (PIFs) family.     

Platelet-activating factor stimulates prolactin release from dispersed rat anterior pituitary cells in vitro

The biologically active phospholipid (platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) stimulated PRL release from dispersed rat anterior pituitary cells in culture. PAF-induced PRL release was dose dependent, with threshold stimulation at 1 nM and maximal stimulation at 100 nM. Stimulation occurred as early as 1 min of incubation and persisted for 2 h. The action of PAF on PRL release is consistent with a receptor-mediated mechanism based on the observations that the action of PAF is blocked by dopamine agonists and the PAF receptor antagonists L 652731 and SRI 63072. The structural analogs 1-O-alkyl-2-oleoyl-sn-glycero-3-phosphocholine and 1-O-alkyl-2-acetyl-sn-glycero-3-phosphoethanolamine, which lack the biological activity of PAF, are not able to stimulate PRL release over the dose range 0.2-2 microM. In addition, the PAF precursor lyso PAF and diacyl-sn-glycero-3-phosphocholine (phosphatidylcholine) were ineffective in stimulating PRL release. PAF induced the secretion of PRL and GH but not that of LH or TSH from hemipituitaries in short term incubations. PAF did not effect PRL release from GH3 cells. In conclusion, these data indicate that PAF stimulates PRL release from primary cultures of rat anterior pituitary cells in a dose-related, rapid, and specific manner.     

Endothelin-3 immunoreactivity in gonadotrophs of the human anterior pituitary.

Endothelin (ET), originally discovered in vascular endothelial cells, has also been demonstrated in nonvascular tissues. The present study was undertaken to elucidate the presence of ET in the human pituitary. The avidin-biotin complex method with antiserum to ET-1 (and ET-2) or ET-3 was used to identify ET in human pituitaries obtained by autopsy. ET-3 immunoreactivity was found in the cytoplasm of large ovoid cells of the anterior pituitary. Using the double staining method, the cells containing ET-3 immunoreactivity were differentiated from cells containing ACTH, TSH beta, GH, PRL, and protein S-100. By staining with anti-LH beta antiserum in adjacent sections and using the double staining method, the cells were identified as gonadotrophs. No staining was observed in the posterior pituitary. In addition, no ET-1 (and ET-2) immunoreactivity was detected. The specific localization of ET-3 immunoreactivity in the gonadotrophs of the human pituitary suggests a possible role of ET-3 in the regulation of anterior pituitary function.