The dopaminergic regulation of gonadotropin-releasing hormone receptor binding in the pituitary of the African catfish, Clarias gariepinus

In several teleost species, including the African catfish, dopamine acts as an endogenous inhibitor of gonadotropin-releasing hormone (GnRH)-stimulated gonadotropin (GTH) release. The present in vivo study was carried out to investigate whether this inhibitory action of dopamine can be explained by an effect on the pituitary GnRH receptors. To that end, sexually mature female catfish were treated with dopamine and the dopamine antagonist pimozide (PIM), respectively. At different time intervals after injection, the pituitaries were collected, and in a GnRH receptor assay the GnRH-binding parameters were determined. The dopamine treatment affected neither GnRH-binding capacity nor affinity. The PIM treatment resulted in a two-fold increase in pituitary GnRH-binding capacity without affecting binding affinity. The time course of this effect coincided with the potentiating effect of PIM of the GTH-releasing activity of a GnRH analog. It is concluded that the stimulatory effect of PIM on the action of GnRH might, in part, be due to an increased pituitary GnRH-binding capacity. Reversely, these results suggest that the endogenous dopaminergic inhibition of GnRH-stimulated GTH release may be mediated, at least in part, through down-regulation of the pituitary GnRH receptors.     

In-vivo evaluation of dopamine receptor-mediated inhibition of gonadotrophin secretion from the pituitary gland of the goldfish, Carassius auratus

Dopamine acts directly on the pituitary to modulate gonadotrophin (GtH) secretion in goldfish (Carassius auratus). In the light of this important role for dopamine in the regulation of goldfish reproduction, this investigation was designed to evaluate the receptor specificity of this dopamine inhibition and to describe the use of domperidone, a specific dopamine D2-receptor antagonist, in the manipulation of pituitary function in goldfish. To investigate the specificity of dopamine inhibition of GtH secretion, selected dopamine receptor antagonists were injected i.p. to block dopamine receptors thereby increasing GtH secretion as reflected by increased serum concentrations of GtH. Serum GtH levels were significantly increased by the active stereoisomer (-)-sulpiride in a dose-related fashion; (+)-sulpiride had no effect. Comparison of dopamine antagonists at low doses indicated that only domperidone and pimozide caused significant increases in serum concentrations of GtH. Dopamine antagonists potentiated the action of a gonadotrophin-releasing hormone analogue (GnRH-A) with an order of potency of domperidone = pimozide greater than metoclopramide = fluphenazine. [3H]Domperidone, injected i.p. with unlabelled domperidone, entered the blood and achieved maximum concentrations 12 h after injection, but did not accumulate in the brain in appreciable amounts. Gonadal 3H radioactivity was usually equal to or in excess of blood radioactivity, while [3H]domperidone was highly concentrated in the pituitary in a time-dependent fashion, with maximal accumulation occurring 24 h after injection. The time-course of pituitary accumulation of [3H]domperidone correlated well with the temporal increase in serum GtH levels in response to i.p. injected domperidone or domperidone plus an analogue of LHRH. Domperidone increased serum concentrations of GtH in a dose-related fashion; an analogue of salmon GnRH (sGnRH-A) increased the sensitivity and magnitude of the serum GtH response to domperidone. Serum concentrations of GtH were increased by sGnRH-A in a dose-related fashion; a low dose of domperidone substantially increased the sensitivity of the serum GtH response to sGnRH-A. These results indicate that dopamine inhibits GtH secretion from the goldfish pituitary by acting through a specific mechanism mediated by a dopamine D2 receptor. Domperidone increased serum concentrations of GtH, potentiated the action of gonadotrophin-releasing hormones and did not pass into the brain after i.p. injection into goldfish. The data also suggest that dopamine and GnRH, although acting through different receptors, influence the effect of each other on GtH release.     

Alterations in pituitary GnRH and dopamine receptors associated with the seasonal variation and regulation of gonadotropin release in the goldfish (Carassius auratus)

Seasonal variations in the serum concentrations of gonadotropin (GtH) and the serum GtH response to intraperitoneal injection of domperidone, a specific dopamine receptor antagonist, were examined in goldfish. In addition, the effects of in vivo treatment of goldfish with a superactive analog of salmon gonadotropin-releasing hormone (sGnRH-A) and domperidone on the binding parameters of pituitary GnRH and dopamine receptors were investigated in goldfish. Serum concentrations of GtH and the maximum GtH response to domperidone increased in correlation with advancing gonadal maturation; values increased from those in sexually regressed fish in January to maximal levels observed in fish in late stages of gonadal recrudescence in March, followed by a decrease with gonadal regression. At all stages, injection of domperidone increased serum concentrations of GtH in a dose-related manner; however, the ED50 of domperidone did not vary significantly over the course of the reproductive cycle. Multiple injections of sGnRH-A caused a progressively increasing and more prolonged serum GtH response; as well, multiple sGnRH-A treatment significantly potentiated the serum GtH response to domperidone without altering the ED50 of domperidone. sGnRH-A treatment caused a significant increase in the number of dopamine/neuroleptic receptors in the goldfish pars distalis, accompanied by a nonsignificant increase in dopamine/neuroleptic receptors in the neurointermediate lobe, and significantly increased the number of high-affinity GnRH receptors in the goldfish pituitary. Treatment with domperidone also significantly increased pituitary high-affinity GnRH receptor numbers. Receptor affinities were not significantly altered by either sGnRH-A or domperidone treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gonadotropin-releasing hormone (GnRH)-binding sites in human breast cancer cell lines and inhibitory effects of GnRH antagonists

GnRH-binding sites have previously been described in human breast tumors, and a GnRH agonist has been shown to inhibit growth of the MCF-7 human breast cancer cell line. We have investigated the presence of GnRH-binding sites in ZR-75-1, MDA-MB-231, Sk Br 3, MDA-MB-157, and MCF-7 human breast cancer cell lines and the effect of GnRH analogs on the incorporation of [3H]thymidine and 14C-labeled amino acids into DNA and protein. Specific GnRH-binding sites were present in membrane preparations of all five human breast carcinoma cell lines. Studies in three cell lines indicated low affinity (Kd, 1.6-3.0 X 10(-6) M) GnRH binding similar to that reported in human placenta and corpus luteum. In contrast, human pituitary GnRH receptors were of high affinity (Kd, 4.8 X 10(-9) M). Breast carcinoma cell GnRH-binding sites also differed from the pituitary receptor in their inability to discriminate between GnRH and superactive analogs. Binding of a [125I]GnRH analog to ZR-75-1 breast cancer cells and pituitary membranes was affected similarly by various cations. GnRH antagonists rapidly inhibited [3H]thymidine incorporation into DNA (within 3 hr), and this effect was reversible. GnRH antagonists also inhibited cell growth, but only after 6 days. GnRH agonists did not alter either thymidine incorporation or growth. The present observations of low affinity GnRH-binding sites in breast cancer cell lines and inhibitory effects of GnRH antagonists point to the possibility of an autocrine regulatory role of GnRH-like peptides in mammary cells.     

Dopamine inhibition of gonadotropin and alpha-melanocyte-stimulating hormone release in vitro from the pituitary of the goldfish (Carassius auratus)

The purpose of the present investigation was to examine the receptor specificity of dopamine inhibition of gonadotropin (GtH) and alpha-melanocyte-stimulating hormone (alpha-MSH) release from the goldfish (Carassius auratus) pituitary in vitro. Pars distalis (PD) and neurointermediate lobe (NIL) fragments of the goldfish pituitary were superfused in vitro under various experimental paradigms; eluate from PD and NIL fragments was analyzed for (GtH) and (alpha-MSH), respectively. Spontaneous GtH release from PD fragments was relatively constant over 6 hr; continuous superfusion with dopamine reversibly inhibited spontaneous GtH release with an estimated ED50 of 10(-4.4) M. Domperidone, a specific D-2 receptor antagonist, reversed the inhibitory action of dopamine and increased spontaneous GtH release. Acute treatment of PD fragments with salmon GnRH (sGnRH) stimulated GtH release; dopamine inhibited GtH release from similarly treated fragments with an ED50 of 10(-7.5) M. The spontaneous release of alpha-MSH from NIL fragments was relatively constant over 6 hr; continuous superfusion with dopamine reversibly inhibited this release with an ED50 of 10(-7.2) M. Acute treatment of NIL fragments with thyrotropin-releasing hormone (TRH) caused acute dose-related increases in alpha-MSH release with an ED50 of 10(-8.2) M; dopamine reversibly inhibited alpha-MSH release from similarly treated fragments with an ED50 of 10(-7.7) M. Both stereoisomers of apomorphine, a dopamine agonist, inhibited GtH release from PD fragments treated with sGnRH; in contrast, alpha-MSH release from NIL fragments treated with TRH was stereospecifically inhibited by (-)-apomorphine, but not by (+)-apomorphine. Domperidone reversed (ED50 = 10(-6.6) M) dopamine (10(-6.3) M) inhibition of GtH release from PD fragments treated with sGnRH. In NIL fragments, the inhibitory action of dopamine (10(-6.3) M) was reversed by domperidone (ED50 = 10(-5.5) M), which restored the acute alpha-MSH release response to TRH. These results suggest the involvement of a low-affinity dopamine/neuroleptic receptor in dopamine inhibition of GtH and alpha-MSH release from the pituitary of the goldfish.     

Pituitary gonadotropin-releasing hormone binding sites in persistent estrous rats

Pituitary gonadotropin-releasing hormone (GnRH) binding sites were studied in persistent estrous rats. For comparison females at different stages of the estrous cycle, ovariectomized animals, and intact males were examined. Persistent estrus was induced by a hypothalamic knife cut, administering testosterone to neonatal rats, or by constant illumination. Rats showing spontaneously such alteration were also used in the experiment. Besides pituitary GnRH-binding sites, the GnRH content of the median eminence and plasma luteinizing hormone, follicle-stimulating hormone, estradiol, and progesterone concentrations were determined. Pituitary GnRH receptors changed during the estrous cycle with the highest concentrations observed on proestrus. In rats in persistent estrus, pituitary GnRH-binding sites varied between proestrous and estrous values of cyclic females. Ovariectomy resulted in a significant increase in receptors. Depending on how the persistent estrous syndrome was induced, very different amounts of GnRH were found in the median eminence of the various groups. Our data indicate that the GnRH-binding sites on pituitary cells of persistent estrous rats are in essence not changed.     

Effect of purified 31K bovine inhibin on the specific binding of gonadotropin-releasing hormone to rat anterior pituitary cells in culture

The mechanism by which inhibin decreases the responsiveness of the pituitary gonadotroph to GnRH in terms of secretion of gonadotropins is largely unknown. We studied the effect of pure 31K bovine inhibin on the specific binding of GnRH to rat anterior pituitary cells in culture using iodinated Buserelin as tracer. Results showed that treatment of cultured anterior pituitary cells from adult male rats with inhibin (0-30 U/ml) for 72 h decreased Buserelin binding in a dose-dependent manner. In the presence of a maximally inhibiting dose of manner. In the presence of a maximally inhibiting dose of inhibin, Buserelin binding decreased progressively with time, reaching a minimum of 42% of the control value after 3 days. Exposure of pituitary cells for 3 days to the inhibin-related peptides transforming growth factor-beta (up to 400 pM) and Müllerian inhibitory substance (up to 100 nM) did not decrease binding of Buserelin, suggesting that the effect was specific to inhibin. Inhibin did not compete with iodinated Buserelin for GnRH-binding sites when they were added to the assay tube simultaneously. In addition, treatment with inhibin halved the number, but did not change the affinity, of GnRH-binding sites and had no effect on either cell number of cell viability. It is concluded that the reduction by inhibin of rat gonadotroph responsiveness to GnRH may be partly related to a decrease in the number of GnRH receptors on the cell surface.     

Evidence that gonadotropin-releasing hormone also functions as a growth hormone-releasing factor in the goldfish

The present study examined the influence of GnRH on the in vivo and in vitro secretion of GH in the goldfish (Carassius auratus). Intraperitoneal injection of several GnRH peptides, including a form native to goldfish, salmon GnRH (sGnRH), elevated circulating GH levels in female goldfish. An analog of mammalian GnRH (mGnRH), [D-Ala6,Pro9-NEt] mGnRH (mGnRH-A), at a dosage of 0.1 microgram/g BW increased serum GH levels for up to 48 h after a single ip injection. Goldfish receiving a series of injections of this dose of mGnRH-A also displayed an increased rate of body growth, indicating that the mGnRH-A-induced increase in the circulating GH level was sufficient to accelerate body growth. In vitro experiments using perifused pituitary fragments found that sGnRH stimulated the secretion of GH from the goldfish pituitary in a potent, dose-dependent, and reversible manner. The time course of response and half-maximally effective dose of sGnRH were very similar for both GH and gonadotropin (GTH) secretion in vitro, suggesting that the mechanism(s) mediating the stimulatory actions of GnRH in the goldfish may be similar for both GH and GTH secretion. However, GnRH-induced GH and GTH secretion from the goldfish pituitary can occur independently of each other, as demonstrated by the finding that somatostatin inhibited the GnRH stimulation of GH secretion in vitro, without influencing the GTH response, whereas the dopamine agonist apomorphine inhibited GnRH-induced GTH secretion in vitro, without influencing the GH response. Furthermore, the dopamine antagonist pimozide did not influence serum GH levels, although pimozide potentiated the stimulatory effect of GnRH on GTH secretion in vivo by blocking the endogenous GTH release inhibitory action of dopamine. Results of the present study suggest that the secretion of GH and GTH in the goldfish are regulated, at least in part, through a common releasing factor, GnRH, whereas somatostatin and dopamine appear to act independently as GH and GTH release inhibitory factors, respectively.     

Characterization of gonadotropin-releasing hormone receptors in cultured rat pituitary cells

The properties of gonadotropin-releasing hormone (GnRH) receptors were analyzed in isolated pituitary cells prepared by enzymatic dispersion with trypsin or collagenase-hyaluronidase. The initial impairment of LH responses to GnRH in isolated cells prepared by both methods was reversed during culture for 2 days in multiwell vessels. However, specific binding sites for GnRH, assayed by equilibration with [125I]iodi0[D-Ser(t-BU)6]des-Gly10-GnRH N-ethylamide (GnRH-A) were demonstrable in collagenase-dispersed cells, both initially and after 2 days in culture. Cellular uptake of GnRH-A was temperature dependent, with rapid and saturable binding to a limited number of specific receptor sites with high affinity for the labeled analog (Ka = 4.0 +/- 0.8 X 10(9) M-1). These sites showed common binding specificity for GnRH-A and the native GnRH peptide, with significantly lower affinity for the natural peptide (Ka = 2.3 X 10(7) M-1). Other protein and peptide hormones, including ovine LH, ovine PRL, hCG, TRH, somatostatin, and angiotensin II (up to 10(-6) M) did not inhibit binding of GnRH-A to its receptors. Cellular binding of GnRH-A was followed by increased cGMP production and LH release within 10 min. The analog was 50 times more potent than native GnRH in stimulating LH release. The Kact values derived for GnRH and GnRH-A were 0.5 and 0.01 nM, respectively, considerably lower than the Kd values of 50 and 0.25 nM derived from receptor binding analysis. These results indicate that GnRH receptors can be identified in isolated pituitary cells, in which peptide binding is followed by increased cGMP production and LH release. The impaired LH responses in acutely dispersed pituitary cells are not due to the loss of receptor sites but to a reversible postreceptor defect. Occupancy of about 20% of the GnRH-binding sites elicits a near-maximal LH response, indicating the nonlinearity of GnRH-receptor coupling to secretory responses in the cultured gonadotroph.     

Dopamine agonist- and antagonist-induced modulation of pituitary gonadotrophin releasing hormone receptors are independent of changes in serum prolactin

The effect of drug-induced hypo- and hyperprolactinaemia on pituitary gonadotrophin releasing hormone receptors (GnRH-R), serum and pituitary gonadotrophins (LH and FSH) and prolactin was investigated in intact adult male and female rats. Hypoprolactinaemia (serum prolactin less than 20% of control values) resulting from dopamine agonist (bromocriptine) infusion (4 mg/kg per day for 7 days) was accompanied by a 40-50% increase in GnRH-R in both male and female animals, though this was not accompanied by any major change in serum or pituitary LH and FSH. Hyperprolactinaemia (serum prolactin greater than ten times control values) induced by the dopamine receptor antagonist metoclopramide (65 mg/kg per day for 7 days) increased GnRH-R between 35 and 45% in both male and female rats without altering serum gonadotrophins. Domperidone (1 mg twice daily for 14 days) also increased GnRH-R by 50% but only in female rats. Both dopamine antagonists significantly increased pituitary prolactin content. Pituitary FSH increased in female rats treated with both metoclopramide and domperidone. The stimulatory effects of bromocriptine and metoclopramide on GnRH-R in male rats were prevented by concurrent treatment with a GnRH antiserum, suggesting that the drug effects were mediated through alteration in endogenous GnRH secretion. Induction of massive (serum prolactin greater than 2000 micrograms/l) hyperprolactinaemia in male and female rats with a transplantable prolactin-secreting pituitary tumour did not reduce GnRH-R concentration, although serum gonadotrophins were suppressed and pituitary gonadotrophin content was increased.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro binding characteristics of [3H]spiperone to the pituitary of the goldfish (Carassius auratus)

Homogenates of the pituitary of the goldfish (Carassius auratus) were incubated with [3H]spiperone under various experimental paradigms to evaluate the binding characteristics of the goldfish pituitary dopamine receptor. Binding was tissue specific as binding of [3H]spiperone to goldfish pituitary was greater than other tissue types examined, and the magnitude of binding was found to be dependent on pituitary (protein) content; also, specific binding was heat labile. Association was rapid and binding was reversible (dissociable) by addition of excess competing ligand (domperidone, a specific dopamine D2 receptor antagonist); the half-life (t1/2) of dissociation was 9.2 min and the estimated dissociation rate constant (k-1) was 7.56 x 10(-2) min-1); as well, the association rate was temperature dependent. Binding was saturable; saturation analysis using [3H]spiperone indicated a single class of binding sites with an estimated dissociation constant (Kd) and capacity of 7.39 +/- 1.23 x 10(-6) M and 31.56 +/- 2.72 x 10(-9) mol/mg protein, respectively. [3H]Spiperone binding was displaceable; displacement analysis using unlabeled domperidone indicated a single class of binding sites with estimated Kd and capacity of 2.94 +/- 0.54 x 10(-6) M and 19.47 +/- 3.12 x 10(-9) mol/mg protein, respectively. Binding was specifically inhibited by various dopamine antagonists and agonists. The density of binding sites differed significantly between regions of the goldfish pituitary; the number of binding sites in the pars distalis and neurointermediate lobes was estimated as 38.89 +/- 2.07 x 10(-9) mol/mg protein vs 109.45 +/- 25.33 x 10(-9) mol/mg protein, respectively; while the Kd's estimated as 3.73 +/- 0.248 x 10(-6) M vs 4.1 +/- 1.21 x 10(-6) M, respectively, were not significantly different. These data agree with previous in vivo and in vitro findings of the biological actions of dopamine agonists and antagonists in modifying gonadotropic hormone release in the goldfish and represent the first demonstration of the existence and binding characteristics of a dopamine/neuroleptic receptor in the pituitary of a nonmammalian vertebrate.     

Dopaminergic modulation of respiratory timing mechanisms in carotid body-denervated dogs

The role of central dopaminergic mechanisms in ventilatory control was investigated by monitoring phrenic motorneuronal output of anesthetized dogs which were vagotomized, paralyzed and artificially ventilated and in which bilateral carotid body denervation had been performed. Intravenous administration of dopamine (DA) had no effect on phrenic output in these dogs. In contrast, apomorphine (APO), a potent dopaminergic agonist, which unlike dopamine, crosses the blood-brain barrier consistently and significantly prolonged inspiratory duration and shortened expiratory duration without altering phrenic amplitude. The dopaminergic antagonist haloperidol produced a frequency-dependent decrease in basal phrenic minute activity and reversed or abolished APO-induced changes in the phrenic profile. These data verify the specificity of APO for dopamine receptors and further suggest that DA receptors in the central nervous system exert a tonic effect on central respiratory control mechanisms. In contrast, domperidone, a dopaminergic antagonist which poorly penetrates the blood-brain barrier did not alter basal phrenic characteristics. Moreover, with the exception of inspiratory duration, domperidone did not antagonize APO-induced alterations in the phrenic profile. We conclude that dopaminergic mechanisms within the brain or spinal cord modulate timing relationships of central respiratory output.     

Dopamine stimulates growth hormone release from the pituitary of goldfish, Carassius auratus, through the dopamine D1 receptors.

Previously, we have demonstrated that ip injection of apomorphine, a nonselective dopamine (DA) agonist, increases serum GH levels in the goldfish, suggesting a possible role of DA in GH regulation. In the present study, the effects of DA on GH release in the goldfish were further characterized using an in vitro perifusion system for pituitary fragments. DA increased GH release in a dose-dependent manner with an ED50 of 0.26 +/- 0.06 microM. SKF38393, a DA D1 agonist, mimicked the GH-releasing effect of DA with an ED50 of 0.41 +/- 0.12 microM. Stereoselectivity consistent with mammalian DA D1 systems was demonstrated for the GH response to SKF38393; only the (+)- but not (-)-enantiomer of SKF38393 induced a dose-dependent GH release. Two other D1 agonists, SKF77434 and SKF82958, were also found to have GH-releasing activity. In contrast, high doses (up to 1 microM) of the DA D2 agonists, bromocriptine and LY171555, did not affect basal GH levels. The receptor specificity for DA-stimulated GH release was further investigated by using D1 and D2 antagonists; the D1 antagonists SCH23390 and SKF83566 completely abolished the GH response to DA or the D1 agonist SKF38393, whereas the D2-specific antagonists domperidone and (-)-sulpiride were not effective in this respect. Taken together, the present study demonstrates that DA is stimulatory to GH release from the pituitary of goldfish, and its action is mediated through receptors resembling the mammalian DA D1 receptors. The apparent similarities of the DA D1 receptor pharmacology between the goldfish and the mammals also indicate that D1 receptor is highly conserved during vertebrate evolution.     

Gonadotropin-releasing hormone binding and activation of enriched population of pituitary gonadotrophs

The properties of GnRH receptors were analyzed in isolated rat pituitary cells by binding studies with the labeled GnRH agonist, [¹²⁵I]iodo-[d-Ser(tBu)⁶]des-Gly¹⁰-GnRH-N-ethylamide. The concentration of GnRH-binding sites in pituitary cells from immature female rats was twice as high as in cells from adult females. Electron-microscopic irnmunocytochemistry revealed twice as many gonadotrophs in the immature rat pituitary, indicating that gonadotrophs from immature and mature female rats contain the same number of binding sites. An enriched population of gonadotrophs prepared from immature female rat pituitaries by velocity sedimentation at unit gravity contained 77% of the total radioimmunoassayable LH and 71% of the total GnRH receptors. A second population of small, poorly granulated gonadotrophs was distributed among the cells of other fractions of the gradient and could be detected only by irnmunocytochemistry. The smaller, possibly immature, gonadotrophs did not contain a measurable number of GnRH receptors. When incubated with the cells recovered from the gradient, GnRH stimulated LH release only in the mature gonadotroph-enriched fraction and not in other regions of the gradient. These results indicate that GnRH interacts preferentially with gonadotrophs in the pituitary. The findings also suggest that functional heterogeneity exists among pituitary gonadotrophs and that GnRH binds to and activates only the mature gonadotrophs which exhibit the highest LH-storage capacity.     

LUTEINIZING HORMONE RESPONSE TO DOMPERIDONE IN HYPERPROLACTINAEMIC WOMEN WITH PITUITARY MICROADENOMAS

Administration of a dopamine (DA) antagonist, domperidone, increased circulating levels of LH in hyperprolactinaemic-amenorrhoeic women with pituitary microadenomas but not in normal women in the early follicular phase of the menstrual cycle. This drug does not readily cross the blood-brain barrier and therefore the site of action of domperidone could be the pituitary gland or the median eminence. As the simultaneous administration of GnRH and domperidone did not increase the GnRH-induced LH release in hyperprolactinaemic women, the site of action of domperidone is likely to be the median eminence.     

Adrenergic and dopaminergic regulation of gonadotropin-releasing hormone release from goldfish preoptic-anterior hypothalamus and pituitary in vitro.

The involvement of adrenergic and dopaminergic receptor subtypes on in vitro release of radioimmunoassayable gonadotropin-releasing hormone (GnRH) from incubated preoptic-anterior hypothalamic (P-AH) slices and pituitary fragments of sexually mature male goldfish was studied. Norepinephrine (NE) produced a dose-related stimulation of GnRH from P-AH slices, but not from pituitary fragments. The effects of some adrenergic receptor agonists (1 microM) on GnRH release from P-AH slices were tested: phenylephrine (alpha 1-agonist) significantly stimulated GnRH release; clonidine (alpha 2-agonist) and isoproterenol (beta-agonist) were ineffective. Incubation of P-AH slices with phentolamine (alpha 1/alpha 2-antagonist) and prazosin (alpha 1-antagonist), at a concentration of 1 microM, inhibited the release of GnRH induced by NE (60 microM); the alpha 2-antagonist yombibin and the beta-antagonist propanolol were ineffective. None of the adrenergic antagonists (1 microM) tested produced significant effects on spontaneous release of GnRH from both tissue preparations. Spontaneous release of GnRH from both P-AH slices and pituitary fragments was reduced by dopamine (DA) in a dose-related manner. The effects of some DA agonists (1 microM) were tested: apomorphine (D1/D2-agonist) and SKF 38398 (D1-agonist), but not bromocriptine and LY-171555 (D2-agonists) significantly reduced spontaneous GnRH release from P-AH slices in vitro. On the other hand, D2-agonists, but not D1-agonists, significantly reduced GnRH release from pituitary fragments. The effects of DA antagonists (1 microM) were also tested: in P-AH slices, addition of SKF-83566 (D1-antagonist) significantly reduced spontaneous GnRH release; pimozide and domperidone (D2-antagonist) were ineffective when tested alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct effects of prolactin and dopamine on the gonadotroph response to GnRH

The intrapituitary mechanisms underlying the inhibitory actions of hyperprolactinaemia on the reproductive axis remain unclear. Previous work on primary pituitary cultures revealed combined suppressive effects of prolactin (PRL) and dopamine on the gonadotrophin response to GnRH. However, whether these effects occur directly at the level of the gonadotroph and are accompanied by changes in gene expression is still unresolved. Here, alphaT(3)-1 and LbetaT2 cells were used to investigate the effects of PRL and dopamine on gonadotrophin synthesis and release in gonadotroph monocultures under basal and GnRH-stimulated conditions. PRL receptor and dopamine receptor mRNA expressions were first determined by RT-PCR in both cell lines. Then, PRL and the dopamine agonist bromocriptine (Br), alone or in combination, were shown to block the maximal alpha-subunit and LHbeta-subunit mRNA responses to a dose-range of GnRH. The LH secretory response was differentially affected by treatments. GnRH dose-dependently stimulated LH release, with a 4-5 fold increase at 10(-8) M GnRH. Unexpectedly, PRL or Br stimulated basal LH release, with PRL, but not Br, enhancing the LH secretory response to GnRH. This effect was, however, completely blocked by Br. These results reveal direct effects of PRL and dopamine at the level of the gonadotroph cell, and interactions between these two hormones in the regulation of gonadotrophin secretion. Moreover, uncoupling between LH synthesis and release in both the basal and the GnRH-stimulated responses to PRL and dopamine was clearly apparent.     

LHRH-PITUITARY PLASMA MEMBRANE BINDING: THE PRESENCE OF SPECIFIC BINDING SITES IN OTHER TISSUES

Two specific binding sites for LHRH are present on plasma membranes prepared from rat and bovine anterior pituitary glands. One site is of high affinity (K = 2X108 1/MOL) and the second is of lower affinity (8-5X105 1/mol) and much greater capacity. Studies on membrane fractions prepared from other tissues showed the presence of a single specific site for LHRH. The kinetics and specificity of this site were similar to those of the lower affinity pituitary receptor. These results indicate that only pituitary membranes possess the higher affinity binding site and suggest that the low affinity site is not of physiological importance in the regulation of gonadotrophin secretion. After dissociation from membranes of non-pituitary tissues 125I-LHRH rebound to pituitary membrane preparations. Thus receptor binding per se does not result in degradation of LHRH and the function of these peripheral receptors remains obscure.     

Effects of porcine follicular fluid, inhibin-A, and activin-A on goldfish gonadotropin release in vitro.

Inhibin and activin are important reproductive regulators in mammalian species and have been demonstrated to be highly conserved in structure. The present study examines the effects of porcine follicular fluid (pFF; a crude inhibin and activin preparation) and purified porcine inhibin-A and activin-A on goldfish gonadotropin-II (GTH-II) release. In studies using primary cultures of dispersed goldfish pituitary cells in static incubation, treatments with pFF, inhibin-A, and activin-A for 10 h caused dose-dependent increase in GTH-II release. In perifusion studies using goldfish pituitary fragments, basal GTH-II release was significantly elevated after 12-h exposure to 500 micrograms/ml pFF. Furthermore, GnRH-induced GTH-II secretion was potentiated by pretreatment with pFF. When pFF was applied in the form of 5-min pulses, a rapid dose-related stimulation of GTH-II was observed. Similarly, challenges with 2-min pulses of 15, 150, and 1500 pM inhibin-A and activin-A stimulated GTH-II release by goldfish pituitary fragments in a rapid and dose-dependent manner. This acute stimulatory action of inhibin on goldfish GTH-II release was completely abolished after pretreatment with specific inhibin antibodies. The acute actions of inhibin and activin on GTH-II release are probably not due to the release of endogenous GnRH from nerve terminals in the pituitary fragments or binding to the GnRH receptors. First, a specific GnRH antagonist did not block the actions of inhibin and activin. Second, dopamine, a potent inhibitor of GnRH-stimulated GTH-II secretion in goldfish, was only partially effective in decreasing inhibin- and activin-induced GTH-II release. Third, the stimulatory effects of inhibin and GnRH on GTH-II release were additive. These lines of evidence also indicate that the mechanisms mediating inhibin and activin stimulation of goldfish GTH-II release may be somewhat different from those of GnRH. These results demonstrate that in contrast with the usual inhibitory effects of inhibin on GTH release in mammals, both inhibin and activin exert long term and acute stimulatory actions on GTH-II release in the goldfish.     

Differential actions of dopamine receptor subtypes on gonadotropin and growth hormone release in vitro in goldfish

Incubation of cultured goldfish pituitary cells with 10 nM to 1 microM apomorphine (APO), a non-selective dopamine agonist, increased growth hormone (GH) release in a dose-dependent manner. GH release was also stimulated in a dose-dependent manner by 0.1 nM to 1 microM salmon gonadotropin (GTH)-releasing hormone (sGnRH), sGnRH analog, and chicken GnRH-II (cGnRH-II). The magnitude of GH responses to 1 microM GnRHs were less than that to 1 microM APO. GH responses to 10 nM to 1 microM APO were not significantly increased by the addition of GnRHs. Static incubations with 0.1 nM to 1 microM of the dopamine D1 agonist SKF38393 did not alter basal GTH release, or the GTH responses to 10 nM sGnRH and cGnRH-II. In contrast, the D1 agonist SKF38393 significantly increased basal GH secretion with maximal stimulation achieved at 100 nM concentration, and GH responses to 10 nM sGnRH and 10 nM cGnRH-II were enhanced by simultaneous applications of SKF38393. Incubation with 1 microM of the D2 agonist LY171555 decreased basal GTH release. Additions of 0.1 nM to 1 microM LY171555 caused dose-dependent decreases in the GTH secretion induced by 10 nM sGnRH and cGnRH-II. In contrast, basal and GnRH-stimulated GH release were not affected by coincubations with LY171555. The D1 antagonist SKF83566 and the D2 antagonist domperidone, at 1 microM concentrations, specifically blocked the D1 agonist SKF38393-stimulated increase in GH release and the D2 agonist LY171555-induced depression of GTH secretion, respectively. In cell column perifusion studies, the D1 agonist SKF38393 at 0.1 nM to 1 microM had no effects on GTH release, but significantly elevated GH secretion rates when applied at 0.1-1 microM concentrations. The GH release induced by 1 microM SKF38393 was significantly reduced by simultaneous perifusion with 1 microM of the D1 antagonist SKF83566. Treatments with SKF38393 and/or SKF83566 did not affect net GTH and GH responses to sGnRH challenges. In contrast, perifusion with 0.1 and 1 microM of the D2 agonist LY171555 depressed basal as well as sGnRH-induced GTH responses. These effects of 1 microM LY171555 were completely blocked by simultaneous applications of 1 microM domperidone, a D2 antagonist. Treatments with these D2 selective drugs did not affect basal and sGnRH-stimulated GH release. These results indicate that in cultured goldfish pituitary cells, activation of dopamine D1- and D2-like receptors specifically stimulates GH release and inhibits both basal and stimulated GTH secretion, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)