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)     

Goldfish brain somatostatin-28 differentially affects dopamine- and pituitary adenylate cyclase-activating polypeptide-induced GH release and Ca(2+) and cAMP signals

Dopamine (DA) and pituitary adenylate cyclase-activating polypeptide (PACAP) stimulate goldfish growth hormone (GH) release via cAMP- and Ca(2+)-dependent pathways while DA also utilizes NO. In this study, identified goldfish somatotropes responded to sequential applications of PACAP and the DA D1 agonist SKF38393 with increased intracellular Ca(2+) levels ([Ca(2+)](i)), indicating that PACAP and DA D1 receptors were present on the same cell. A native goldfish brain somatostatin (gbSS-28) reduced SKF38393-stimulated cAMP production and PACAP- and NO donor-elicited GH and [Ca(2+)](i) increases, but not PACAP-induced cAMP production nor the GH and [Ca(2+)](i) responses to forskolin, 8-bromo-cAMP and SKF38393. gbSS-28 might inhibit PACAP-induced GH release by interfering with PACAP's ability to increase [Ca(2+)](i) in a non-cAMP-dependent manner. However, DA D1 receptor activation bypassed gbSS-28 inhibitory effects on cAMP production and NO actions via unknown mechanisms to maintain a normal [Ca(2+)](i) response leading to unhampered GH release.     

Inhibition of hormonally induced inositol trisphosphate production in Transfected GH4</ sup>C1 cells: A novel role for the D5 subtype of the dopamine receptor

We have previously found that the D5 dopamine receptor couples to a G-protein other than Gsalpha, and could be involved in signaling pathways other than regulation of adenylyl cyclase. To describe interactions of the D5 receptor with cellular effectors, we used GH4C1 cells transfected with cDNA for the human D5 receptor. Thyrotropin-releasing hormone (TRH, 100 nM) stimulated accumulation of inositol phosphates (IPs) fivefold in D5GH4C1 cells. Dopamine (DA, 10 microM) inhibited TRH-stimulated IP values by 29%; at higher concentrations (100 microM), maximal inhibition of 61% was observed. The D5 agonist SKF R-38393 (10 microM) mimicked this effect (28% inhibition). SCH 23390, a D5 antagonist, blocked the inhibition caused by both DA and SKF R-38393. Spiperone, a D2 receptor antagonist, did not block the inhibition. The D2 agonist (+/-)-2-(N-phenylethyl-N-propyl)amino-5-hydroxytetralin (PPHT) did not inhibit TRH-stimulated IP production, nor did it augment the effect of D5 agonists. The DA-mediated suppression of IP levels was not sensitive to pertussis toxin; cholera toxin blocked both TRH stimulation and DA suppression of IP accumulation in response to 100 nM TRH. Neither dibutyryl cAMP nor forskolin lowered IP formation in response to TRH. Phorbol ester decreased TRH-stimulated IP accumulation in D5GH4C1 cells; however, an inhibitor of protein kinase C (PKC) did not block the effect of DA.     

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)     

Excitatory and inhibitory dopaminergic regulation of oxytocin secretion in the lactating rat: evidence for respective mediation by D-1 and D-2 dopamine receptor subtypes.

The present experiments were designed to test whether the previously reported excitatory and inhibitory effects of dopamine (DA) on the secretion of oxytocin (OT) in lactating rats are exerted at different DA receptor subtypes, and to examine whether one or both of these effects might occur at the level of the posterior pituitary. The basal release of OT in nonsuckled, lactating rats was increased after intravenous administration of the D-1 DA agonist SKF 38393, and this effect, as well as the suckling-induced release of OT, was prevented by treatment with the D-1 DA antagonist SCH 23390, suggesting that DA may exert an important stimulatory influence over OT secretion through an action at the D-1 DA receptor subtype. A small stimulation of basal PRL release was also produced by SKF 38393, but blockade of the D-1 DA receptor did not prevent the suckling-induced release of this hormone. Stimulation of the D-2 DA receptor with PPHT had no effect on basal OT release in nonsuckled rats, but this agent, as well as another D-2 DA agonist, bromocriptine, prevented the suckling-induced release of both OT and PRL. The inhibitory effect of D-2 DA receptor stimulation was blocked by the D-2 DA antagonist domperidone, which increased the basal release of both hormones when given alone. These observations confirm previous findings that inhibitory effects of DA on suckling-induced OT release are mediated through activation of the D-2 DA receptor. To test whether either dopaminergic effect occurs at the level of neurosecretory endings in the neurointermediate lobe (NIL), the stalk-NIL was isolated from lactating rats and perifused in vitro. The stalk-NIL junction was electrically stimulated for 4 s, and the effects of selective D-1 DA and D-2 DA agonists and antagonists on the basal and electrically evoked release of OT and vasopressin (VP) was assessed using the two stimulation (S2/S1) paradigm. Electrical stimulation produced marked increases in release of both neural lobe peptides in a Ca(2+)-dependent manner, and the electrically evoked release of OT, but not VP, was enhanced by the opiate antagonist naltrexone (10 microM). Consistent with the in vivo results, SKF-38393 (20 microM) produced a small, but statistically significant, increase in electrically induced OT release, while SCH 23390 (20 microM) was without significant effect. Neither drug affected the basal release of OT or the basal or electrically stimulated release of VP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of dopamine receptors associated with steroid secretion in frog adrenocortical cells.

We investigated the type of receptors involved in the mechanism of action of dopamine on corticosteroid secretion from the frog interrenal (adrenal) gland, using the in-vitro perifusion technique. Exposure of dispersed interrenal cells to 50 microM dopamine for 20 min had a biphasic effect on corticosterone and aldosterone secretion, i.e. a transient stimulation followed by an inhibitory phase. Repeated administration of equimolar pulses of dopamine, given at 150-min intervals, resulted in an enhancement of corticosteroid secretion followed by a subsequent blockade of the stimulatory phase of the response. In contrast, the dopamine-evoked inhibition of corticosteroid release did not show any sensitization or desensitization phenomena. Infusion of repeated pulses of the D1 receptor agonist SKF38393 (32 microM) stimulated corticosteroid release and mimicked the sensitization-desensitization phenomenon induced by dopamine. Repeated administration of the D2 receptor agonist LY171555 (50 microM) resulted in a reproducible inhibition of corticosterone and aldosterone secretion. These results suggested the presence of two different receptors for dopamine, i.e. D1 and D2, on frog adrenocortical cells, responsible respectively for the stimulatory and inhibitory effects of dopamine on steroid secretion. However, bromocriptine (50 microM) and CV205-502 (50 microM), two other D2 receptor agonists, had no effect on corticosteroid release. In addition, several classical D2 receptor antagonists failed to block the effect of dopamine on steroidogenesis. It was also observed that (-)sulpiride, a specific D2 antagonist, did not alter dopamine-induced inhibition of inositol phosphate formation. On the other hand, dopamine and the selective D1 and D2 antagonists SKF38393 and LY171555 did not affect the formation of cyclic AMP by interrenal tissue. Taken together, these data indicate that dopamine directly regulates corticosteroid secretion from frog adrenocortical cells. The effect of dopamine is not coupled to adenylate cyclase activity but is probably mediated through the phosphoinositide-turnover pathway. The pharmacological characteristics of the receptors involved in the mechanism of action of dopamine clearly differ from those of the D1 and D2 subtypes previously described in mammals.     

Dopaminergic regulation of the GT1 gonadotropin-releasing hormone (GnRH) neuronal cell lines: stimulation of GnRH release via D1-receptors positively coupled to adenylate cyclase.

The release of GnRH evoked by dopamine (DA) was studied in the GT1 GnRH neuronal cell lines. Superfusion of GT1-1 cells with DA or the D1-dopaminergic agonist SKF 38393, but not with the D2-dopaminergic agonist bromocriptine, increased 2-fold the amplitude of the spontaneous GnRH pulses. Treatment with DA for 30 min also stimulated GnRH release from static cultures of GT1-7 cells. This effect was mimicked by the selective D1-dopaminergic agonist SKF 38393 and blocked by the D1-dopaminergic antagonist SCH 23390. However, the D2-dopaminergic agonist bromocriptine had no effect, and the stimulation of GnRH release by DA was not blocked by the D2-dopaminergic antagonist spiroperidol. In parallel to the stimulation of GnRH release, DA also rapidly increased (first observed at 120 sec) in a dose-dependent fashion, the intracellular concentration of cAMP in isobutylmethylxanthine-pretreated GT1-7 cells. The pharmacological profile of the increase in cAMP was identical to that for GnRH release. The cAMP responses to DA and norepinephrine were lost after long term treatment with SKF 38393, i.e. heterologous desensitization. GT1 cells also express the mRNA for the dopamine- and cAMP-regulated phospho-protein (mol wt, 32,000; DARPP-32) only seen in cells expressing DA D1-receptors. These results demonstrate a direct stimulatory effect of DA on GnRH release via DA D1-receptors positively coupled to adenylate cyclase in GnRH neuronal cell lines.     

Activation of a D2 receptor increases electrical coupling between retinal horizontal cells by inhibiting dopamine release.

In the fish retina, interplexiform cells release dopamine onto cone-driven horizontal cells. Dopamine decreases the electrical coupling between horizontal cells by activating adenylate cyclase through dopamine D1 receptors. Using intracellular recording, we have studied the effect of dopamine D2 receptor activation on horizontal cell electrical coupling in the intact goldfish retina. Superfusion of the D2 agonist LY171555 (quinpirole; 0.2-10 microM) increased horizontal cell coupling, as indicated by a decrease in responses to centered spots or slits of light. The length constant of the horizontal cell network increased an average of 31%. Although dopamine (0.5-20 microM) uncoupled horizontal cells, lower concentrations (e.g., 0.2 microM) initially uncoupled and then subsequently increased coupling beyond initial control levels. The coupling effect of LY171555 (10 microM) was blocked completely by prior application of the D1 agonist SKF 38393 at saturating (20 microM) or nonsaturating (2.5-5.0 microM) doses. Prior treatment of the retinas with 6-hydroxydopamine, which destroyed dopaminergic neurons, eliminated the coupling effect of LY171555 but not the uncoupling effect of SKF 38393. These results suggest that goldfish horizontal cells contain D1, but not D2, receptors and that dopamine activation of D2 autoreceptors on interplexiform cells inhibits dopamine release onto horizontal cells so that the electrical coupling between horizontal cells increases.     

Involvement of dopamine D1 receptors in the control of growth hormone secretion in the rat

The effects of dopamine on GH release were investigated both in vivo in freely moving intact rats and in rats with a mediobasal hypothalamic lesion, and in vitro in a perifusion system using dispersed male rat pituitary cells kept in primary culture. In vivo, dopamine (5 mg/kg body weight) induced a rapid and very transient increase in plasma GH levels in lesioned but not in intact rats. This increase was markedly inhibited by a prior injection of the D1 antagonist SCH 23390 (0.5 mg/kg) but not of the D2 antagonist domperidone (0.5 mg/kg). The D1 agonist SKF 38393 induced a dose-dependent stimulation of GH release in lesioned rats, and the effect obtained with a dose of 5 mg/kg was abolished by pretreatment with SCH 23390 (0.5 mg/kg). In vitro, dopamine (0.1 mumol/l) and SKF 38393 (0.1 mumol/l) provoked a rapid and reversible release of GH from superfused rat pituitary cells; this effect was markedly inhibited by simultaneous superfusion of SCH 23390 (1 mumol/l). These findings indicate that dopamine can stimulate basal GH release at the pituitary level and that this stimulation is mediated by D1 but not by D2 receptors. They also support the hypothesis that unidentified hypothalamic neurohormones may modulate this effect.     

Pharmacologic evidence that a D2 receptor subtype mediates dopaminergic stimulation of prolactin secretion from the anterior pituitary gland.

The ability of low concentrations of dopamine (DA) to stimulate the secretion of prolactin (PRL) was examined in perifused or monolayer cultures of anterior pituitary cells. In cultures perifused with media containing 100 nM DA, changing the DA concentration to either 1 or 100 pM caused a significant dose-dependent stimulatory PRL secretory response within 6 min when compared to the PRL secretory response to removal of DA altogether. Picomolar concentrations of DA caused a biphasic PRL secretory response. This response is characterized by an immediate increase in the rate of PRL secretion similar to that seen when the cells were treated with 100 nM thyrotropin-releasing hormone followed by a decrease in the rate of PRL secretion to levels comparable to cells receiving media alone. In a monolayer culture system DA, at concentrations between 10 nM and 1.0 pM, caused significant stimulation of PRL secretion relative to media alone. Maximal stimulation occurred at nanomolar concentrations of DA (approximately 60% greater than control). Although the D2 agonists, bromocriptine and 2-(N-phenethyl-N-propyl)-amino-5-hydroxytetralin hydrochloride (PPHT) caused significant (p less than 0.05) inhibition of PRL secretion at nanomolar concentrations and above, neither had stimulatory activity. The D1 agonists, SKF 38393 and SKF 82958, had no effect on PRL secretion when tested at 0.1 pM to 1 microM. These data suggest that DA not only inhibits PRL secretion in vitro, but also stimulates PRL secretion at relatively low concentrations. Stimulation is mediated by a DA receptor which is neither recognized by D2 nor D1 agonists, suggesting a possible third DA receptor subtype.(ABSTRACT TRUNCATED AT 250 WORDS)     

Somatostatin Inhibition of Growth Hormone Release in Goldfish: Possible Targets of Intracellular Mechanisms of Action

Previous studies have demonstrated that growth hormone (GH) release in goldfish is under the stimulatory control of gonadotropin-releasing hormone (GnRH) and dopamine and the inhibitory control of somatostatin (SRIF). GnRH stimulation is mediated through protein kinase C (PKC)- and calcium-dependent mechanisms, whereas dopamine D1 receptor activation increases GH secretion through cyclic (c) AMP-dependent intracellular signal transduction pathways. In this study, the mechanisms of SRIF inhibition on GH secretion were examined using primary cultures of dispersed goldfish pituitary cells in static incubation. Application of 1 μMSRIF inhibited the GH-release responses to 100 nMsalmon GnRH, 100 nMchicken GnRH-II, and 1 μMSKF38393, a D1 agonist. These results indicate that inhibitory action of SRIF on stimulated GH release is direct, at the level of the pituitary cells. Addition of SRIF reduced the GH release responses to two activators of PKC (100 μMdioctanoyl glycerol and 100 nMtetradecanoyl phorbol acetate) and to two ionophores (10 μMA23187 and 10 μMionomycin). Similarly, SRIF abolished the GH responses to an activator of adenylate cyclase (10 μMforskolin), a membrane-permeant cAMP analog (1 mM8-bromo-cAMP), and a voltage-sensitive calcium channel agonist (1 μMBay K 8644). Taken together, these observations indicate that the inhibitory actions of SRIF on D1- and GnRH-stimulated GH release can be exerted at sites distal to cAMP production and PKC activation, respectively. SRIF also exerts its effect at sites distal to calcium mobilization. Since SRIF inhibition was more effective against Bay K 8644-induced response than against ionophore-induced GH response, an inhibitory action at the level of extracellular calcium entry through voltage-sensitive channels is also possible.     

Iso stimulation of GH and cAMP: comparison of beta-adrenergic- to GRF-stimulated GH release and cAMP accumulation in monolayer cultures of anterior pituitary cells in vitro

Growth hormone (GH) release and cAMP content were measured in monolayer cultures of anterior pituitary cells after beta-adrenergic and GH-releasing factor (GRF) receptor activation. Isoproterenol (Iso, ED50-20 nM) was less potent than GRF (ED50-20 pM) in stimulating GH release. Iso caused a rapid stimulation of GH release that was maximal after 15 min and declined thereafter, while GRF caused a more gradual increase in GH secretion that was maximal after 30 min and remained elevated after 3 h. Both Iso- and GRF-stimulated GH release were preceded by an increase in cAMP content in the pituitary cells. Further, the addition of 3-isobutyl-1-methylxanthine (IBMX) to the medium enhanced the GH-stimulatory and cAMP-accumulating effects of both secretagogues. Experiments performed with native catecholamines and synthetic catecholamine agonists and antagonists indicated that the GH-stimulatory effect of Iso was mediated by a mixed population of beta 1-adrenergic and beta 2-adrenergic receptors. Additionally, experiments performed with cultured GH3 tumor cells, found that incubation with GRF, Iso, vasoactive intestinal polypeptide, forskolin, or cholera toxin caused an increase in cAMP content in the cells. However, compared to the responses observed in primary pituitary cultures the GH secretory response to these agents was comparatively small. Together, these studies suggest that a mixed population of beta 1-adrenergic and beta 2-adrenergic receptors may act, at least in part, on somatotrophs in the anterior pituitary to stimulate GH release. Although both GRF and beta 2-adrenergic receptor agents affect GH release through a common second messenger system, their differing pharmacokinetic properties suggest distinct intracellular mechanisms.     

In vitro goldfish growth hormone responses to gonadotropin-releasing hormone: possible roles of extracellular calcium and arachidonic acid metabolism?

Two hours of incubation of primary static cultures of dispersed goldfish pituitary cells with 0.01 nM to 1 microM [Trp7,Leu8]-gonadotropin-releasing hormone (sGnRH) increased growth hormone (GH) secretion in a dose-dependent manner with an ED50 estimate of 0.13 +/- 0.04 nM. Addition of calcium ionophores, 1 to 100 microM A23187 and 5 to 100 microM ionomycin, significantly elevated GH release with ED50s of 0.84 +/- 0.38 and 4.34 +/- 1.02 microM, respectively. Replacement of normal calcium-containing media with calcium-deficient media (prepared without the addition of calcium salts) significantly depressed basal GH secretion, attenuated the A23187- and ionomycin-stimulated GH release, and completely abolished the GH response to sGnRH. Arachidonic acid (AA) at 1 to 50 microM also enhanced GH secretion with an ED50 of 4.72 +/- 1.52 microM. Coincubation with 1 and 10 microM of a lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA), 10 microM of the cyclooxygenase inhibitor, indomethacin, and 10 microM of eicosatetraynoic acid, an enzyme blocker with mixed activities on both the lipoxygenase and cyclooxygenase pathways, did not alter basal, AA-, and sGnRH-induced GH release. However, at 100 microM concentration, NDGA increased AA- and sGnRH-stimulated, as well as basal GH, responses. These results confirm the direct stimulatory action of GnRH on goldfish somatotropes and indicate the importance of extracellular calcium in mediating basal and GnRH-induced GH responses. Although AA stimulates GH secretion, its lipoxygenase and cyclooxygenase metabolites probably do not mediate sGnRH action on somatotropes.     

Beta-adrenergic binding and secretory responses of the anterior pituitary

Our studies demonstrated that beta-adrenergic agonists stimulate the release of GH from rat anterior pituitary (AP) cells in vitro. Concentration-response experiments with beta-adrenergic agonists demonstrated that beta 2-adrenergic receptors mediated this phasic GH release, while having no apparent effect on PRL or LH release. The ACTH response to beta-adrenergic agonists was equivocal. Half-maximal stimulation of GH release occurred at 14 +/- 2 (+/-SE) nM isoproterenol, 160 +/- 30 nM epinephrine, and over 1 microM l-norepinephrine (n = 4). Direct binding studies in membrane particulates of rat AP confirmed receptors of the beta 2-subtype. Iodocyanopindolol binding to beta-adrenergic receptors of rat AP yielded a dissociation constant of 4.6 +/- 0.1 pM and a maximal capacity of 1.9 +/- 0.4 fmol/mg protein (n = 3). In contrast, porcine AP contained beta 1-adrenergic receptors. These results support the hypothesis that the endogenous beta 2-adrenergic agonist l-epinephrine may be a GH-releasing factor of physiological significance in the rat.     

Possible involvement of protein kinase C in gonadotropin and growth hormone release from dispersed goldfish pituitary cells

Static incubation with tumor-promoting 4 beta-phorbol esters, activators of the Ca2(+)- and phospholipid-dependent protein kinase C enzyme (PKC), caused dose-dependent increases in gonadotropin (GTH) and growth hormone (GH) secretion in primary cultures of dispersed goldfish pituitary cells. The estimated half-maximal effective doses (ED50) for stimulating GTH and GH release were 0.35 +/- 0.17 and 0.32 +/- 0.13 nM 12-O-tetradecanoyl phorbol 13 acetate (TPA), 3.71 +/- 1.30 and 1.37 +/- 0.76 nM 4 beta-phorbol 12,13-dibutyrate, 6.90 +/- 4.84 and 1.89 +/- 0.25 nM 4 beta-phorbol 12,13-dibenzoate, and 455 +/- 258 and 311 +/- 136 nM 4 beta-phorbol 12,13-diacetate, respectively. In contrast, treatments with up to 10 microM of the inactive 4 alpha-phorbol 12,13-didecanoate ester did not alter GTH and GH release. Additions of the synthetic diacylglycerol, dioctanoyl glycerol, also enhanced GTH and GH secretion in a dose-dependent manner and with ED50s of 1.73 +/- 0.83 and 1.73 +/- 1.19 microM, respectively. The GTH and GH responses to stimulation by TPA were attenuated by incubation with Ca2(+)-depleted medium containing EGTA or by treatment with the Ca2+ channel blocker verapamil. Coincubation with the PKC inhibitor H7 reduced the GTH and GH responses to TPA. As in previous studies, additions of salmon gonadotropin-releasing hormone (sGnRH) or chicken GnRH-II (cGnRH-II) induced GTH and GH release; these hormone responses to sGnRH and cGnRH-II were also decreased by the addition of H7. These results indicate that activation of PKC may stimulate GTH and GH release in goldfish and suggest that sGnRH and cGnRH-II actions on goldfish pituitary GTH and GH secretion are also mediated, at least partially, by PKC.     

Use of a pituitary cell dispersion method and primary culture system for the studies of gonadotropin-releasing hormone action in the goldfish, Carassius auratus. I. Initial morphological, static, and cell column perifusion studies

Two cell dispersion methods for excised goldfish pituitary glands were tested, and a cultured dispersed cell system based on trypsin enzymatic tissue digestion was developed and characterized. Controlled trypsin/DNase treatment of goldfish pituitary gland yielded dispersed cells of high viability (trypsin blue exclusion test) that responded to gonadotropin (GTH)-releasing hormone (GnRH) challenges with GTH secretion in a time- and dose-dependent manner following overnight culture. Electron microscopy revealed that cell preparations produced by the trypsin dispersion were free of cell debris and nerve terminals. The dispersed pituitary cells also retained distinct morphological and immunological identities. Under static incubation conditions, 2-hr treatments with 0.1 nM to 1 microM [Trp7,Leu8]-GnRH (sGnRH) and [D-Arg6,Pro9-N-ethylamide]-sGnRH (sGnRHa) stimulated GTH release with similar efficacy, but with ED50S of 1.92 +/- 0.48 and 0.19 +/- 0.08 nM, respectively. [His5,Trp7,Tyr8]-GnRH (cGnRH-II) stimulated GTH release in a nonsigmoidal, but dose-dependent manner, and with a higher efficacy than sGnRH. In contrast, sGnRH, sGnRHa, and cGnRH-II were equipotent in inducing growth hormone (GH) secretion in static culture studies and with ED50S of 0.29 +/- 0.13, 0.18 +/- 0.11, and 0.19 +/- 0.17 nM, respectively. When trypsin/DNase-dispersed cells cultured overnight with cytodex beads were tested in a cell column perifusion system, dose-related increase in GTH secretion, as well as GH release, were also observed with 0.5 to 50 nM sGnRH. These results suggest that trypsin-dispersed goldfish pituitary cells can be used effectively to study the actions of GnRH on teleost pituitary either in short-term static incubation or column perifusion studies. Differences in the GTH and GH responses to the two native GnRH forms, sGnRH and cGnRH-II, are also indicated.     

Decreased Cannabinoid CB1 Receptor mRNA Levels and Immunoreactivity in Pituitary Tumors Induced by Prolonged Exposure to Estrogens

Recent studies have demonstrated the occurrence of endocannabinoids and their CB₁ receptor subtype in the anterior pituitary gland, despite previous evidence suggesting their exclusive presence and action in the neuroendocrine hypothalamus. The present study was designed to examine the potential changes in these CB₁ receptors located in the anterior pituitary gland in three different experimental situations, which are known to affect anterior pituitary function: (i) estrogen-induced pituitary tumorization, (ii) presence of ectopic pituitaries, and (iii) chronic treatment with D1 or D2 dopaminergic receptor agonists or antagonists. Results were as follows. Induction of pituitary tumorization by implantation of silastic capsules containing diethylstilbestrol, a synthetic estrogen, produced the expected body weight loss and increase in pituitary weight and plasma prolactin (PRL) levels. In hyperplastic pituitaries, both CB₁ receptor mRNA levels and immunoreactivity decreased significantly. Double labelling studies demonstrated that CB₁ receptors colocalized, in pituitary tumors, with PRL- or luteinizing hormone-containing cells, as they did in normal pituitaries. Plasma PRL levels were also increased in rats bearing ectopic pituitaries implanted under the kidney capsule. As previously reported, this increase was originated by the hormone release from the ectopic gland, because the normotopic pituitary collaborated scarcely to elevate PRL levels since it was hypofunctional due to the activation of peripheral PRL-induced feedback mechanisms. However, in this hypofunctional anterior pituitary, there were not any changes in CB₁ receptor mRNA levels and immunoreactivity. Finally, chronic treatment with SKF38393, a D1 receptor agonist, or bromocriptine, a D2 receptor agonist, or with their corresponding antagonists, SCH 23390 or sulpiride, respectively, did not alter CB₁ receptor mRNA levels and immunoreactivity, although produced the expected changes in plasma PRL levels. In summary, estrogen-induced pituitary tumorization was associated with a marked reduction in CB₁ receptors, despite the fact that these receptors were located, among others, on lactotroph cells which develop hyperplasia during tumor induction. Whether this decrease is associated with the ethiology of pituitary tumor induction and whether their pharmacological activation might affect tumorization process are presently unknown, but this will be subjected of further research.     

DA1 Receptor Mediated Regulation of Na+-H+ Antiport Activity in Rat Renal Cortical Brush Border Membrane Vesicles

Our previous studies indicate that dopamine (DA) plays an important role in regulating renal sodium (Na⁺) metabolism during high Na⁺ intake, and that DA₁ receptors are involved in natriuretic response to acute volume expansion. It has also been shown that in addition to the changes in renal hemodynamics, the natriuretic response produced by exogenously administered DA and DA₁ receptor agonists appears to be due to alterations in renal tubular sodium transport mechanisms. This study was designed to investigate the DA₁ receptor-mediated changes in Na⁺-H⁺ antiport activity in tubular brush border membranes of rat kidney. The Na⁺-H⁺ antiport activity, measured as the amiloride-sensitive Na⁺ influx in BBMV, was inhibited by 37%, 46%, 33%, and 42% by 1 μM DA, SKF 82958, SKF 38393, and fenoldopam respectively. The DA₁ antagonist SCH 23390 increased the antiport activity when given alone, while when administered with an agonist it attenuated the effects of the agonist on the antiporter. DA₂ agonists and antagonists failed to affect the antiport activity. These results indicate that the inhibitory effects of DA and DA receptor agonists on Na⁺-H⁺ antiport activity in renal cortical BBMV were mediated by the DA₁ receptors.     

Evidence that nitric oxide is involved in the regulation of growth hormone secretion in goldfish

Whether nitric oxide (NO) plays a role in regulation of growth hormone (GH) secretion from somatotropes in the pituitary of the goldfish Carassius auratus was investigated. Immunocytochemistry with two antibodies against mammalian NO synthase (NOS) revealed the presence of a NOS-like enzyme in primary cultures of dispersed goldfish pituitary cells, including morphologically identified somatotropes. NO donors S-nitroso-N-acetylpenicillamine and sodium nitroprusside (SNP), as well as a cyclic guanosine monophosphate analogue (dibutyryl guanosine 3':5'-cyclic monophosphate), all significantly increased GH secretion from dispersed goldfish pituitary cells in static culture. Somatostatin abolished the response to SNP, and NOS inhibitors aminoguanadine hemisulfate (AGH) and N-(3-aminomethyl)benzylacetamidine, dihydrochloride (1400W) decreased the GH release response to known neuroendocrine factors stimulatory to GH release (gonadotropin-releasing hormone and a dopamine D1 agonist). AGH and 1400W did not alter basal GH secretion. These data suggest that NO plays a role in mediating the GH response to endogenous neuroendocrine factors in goldfish.     

Regulation of growth hormone and prolactin gene expression and secretion by chimeric somatostatin-dopamine molecules

Dopamine (DA) regulates both prolactin (PRL) secretion and gene expression, whereas somatostatin (SRIF) inhibits GH secretion with unclear effects on GH gene expression. We therefore tested the effects of SRIF analogs and chimeric SRIF/DA compounds BIM 23A760 and BIM 23A761 on GH and PRL secretion and gene expression in primary rat pituitary cultures and pituitary tumor GH(3) and MMQ cells. Chimeric SRIF/DA molecules suppressed GH release with a similar efficacy to SRIF receptor subtype 2 agonists in rat pituitary and GH(3) cells. After 24 h, BIM 23A760 and BIM 23A761 did not exert additive effects on GH secretion, and after 48 h were less effective than the combination of respective mono-receptor agonists in GH(3) cells. Real-time PCR did not reveal changes in GH mRNA levels after treatment with SRIF analogs and SRIF/DA molecules. SRIF/DA compounds suppressed PRL and PRL mRNA in rat pituitary and MMQ cells with a similar efficacy to D(2)-DA receptor agonist. In GH(3) cells, they suppressed PRL and PRL mRNA levels with a similar efficacy to SRIF receptor subtype 2 agonists. SRIF/DA molecules did not exhibit additive effects on PRL secretion and mRNA levels as compared with cotreatment with mono-receptor ligands. The results show that SRIF analogs and SRIF/DA molecules inhibit GH and PRL secretion and suppress PRL but not GH gene expression.