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)     

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.     

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.     

Relationship between cyclic AMP-stimulated and native gonadotropin-releasing hormone-stimulated gonadotropin release in the goldfish.

The relationship between drugs elevating intracellular cAMP levels and gonadotropin (GTH)-releasing hormone (GnRH) in the stimulation of GTH secretion in the goldfish was investigated using dispersed goldfish pituitary cells in primary culture. In static incubation experiments, activation of adenylyl cyclase by forskolin and the inhibition of cAMP phosphodiesterase by 3 isobutyl-1-methylxanthine (IBMX) increased cAMP release and stimulated GTH secretion. The addition of membrane permeant cAMP analogs, 8-bromoadenosine 3':5'-cyclic monophosphate (8Br-cAMP), and dibutyryl cAMP also increased GTH release, suggesting that elevation of cAMP levels can induce GTH secretion. In the goldfish, dopamine is a physiological inhibitor of GTH release. Application of the dopamine agonist apomorphine decreased the GTH responses to forskolin, 8Br-cAMP, and salmon GTH-releasing hormone (sGnRH). The ability of agents that elevate cAMP levels to mimic GnRH action on GTH release suggests that cAMP may mediate GnRH-stimulated GTH secretion in the goldfish; however, this possibility was not substantiated by results from further experiments. In 2-hr static incubation studies, the GTH responses to sGnRH and chicken GnRH-II (cGnRH-II) were enhanced by coincubations with forskolin, IBMX, and 8Br-cAMP. The magnitudes of these enhancements were at least additive, if not synergistic. The levels of cAMP released into the media were unaffected by treatment with sGnRH and cGnRH-II, either in the absence or in the presence of IBMX. Replacement of normal testing media with Ca(2+)-deficient media (without Ca2+ salts and in the presence of 0.1 mM EGTA) decreased sGnRH and cGnRH-II stimulation of GTH release but did not affect forskolin and 8Br-cAMP actions. These results indicate that sGnRH and cGnRH-II stimulation of short term (less than or equal to 2-h) GTH release in the goldfish is not mediated by cAMP. The kinetics of the interactions between sGnRH, forskolin, and IBMX were also investigated in cell column perifusion studies. Applications of 5-min pulses of forskolin and IBMX stimulated rapid increases in GTH release; the latencies of these responses were similar to that observed with sGnRH. The simultaneous applications of sGnRH with either forskolin or IBMX resulted in GTH responses that were of greater magnitude and longer duration than those in response to sGnRH alone. These results together indicate that elevation of cAMP levels can potentiate the GTH response to the native GnRHs by increasing the magnitude of the acute GTH release and by prolonging the duration of GnRH action; however, cAMP does not appear to be involved directly in mediating GnRH stimulation of GTH release.(ABSTRACT TRUNCATED AT 400 WORDS)     

Alpha 1-adrenoreceptors on intact rat anterior pituitary cells: correlation with adrenergic stimulation of thyrotropin secretion

An in vitro study of the alpha-adrenergic control of TSH secretion was carried out on rat anterior pituitary cells in monolayer culture. The ability of adrenergic agonists and antagonists to alter TSH release from the cells was determined. With the use of parallel cell cultures under the same conditions, alpha-adrenergic binding sites were measured and characterized with [3H]dihydroergocryptine (DHE) as the radioligand. Epinephrine (E) and norepinephrine (NE) released TSH over a 2-h period in a dose-dependent and stereospecific manner (ED50 = 1 and 700 nM for the (-) and (+/-) stereoisomers of E; 7 and 600 nM for the active and inactive stereoisomers of NE respectively). Maximum release was 3- to 4-fold greater than basal secretion for both isomers of E but less (2- to 3-fold) for the isomers of NE. Phenylephrine, an alpha 1-agonist, elicited a 2- to 3-fold increase in TSH secretion (ED50 = 13 nM). Clonidine, an alpha 2-agonist, produced only slight stimulation at concentrations greater than 10(-6) M, and isoproterenol was ineffective. Prazosin, an alpha 2-antagonist (IC50 = 0.12 nM), was 500-fold more effective than yohimbine, an alpha 2-antagonist (IC50 = 60 nM), in reversing the TSH stimulation induced by 10(-7) M E. With [3H]DHE and prazosin as competing ligands, alpha-adrenergic receptors could be quantified independently of dopamine receptors present upon the same mixed cell preparations. The kinetics of specific radioligand binding to the cells were rapid (k1 = 1.75 X 10(-7) M-1 min-1, k2 = 0.131 min-1), equilibrium being reached within 15 min at 22 C. Adsorption isotherms and Scatchard analysis revealed a single population of binding sites with high affinity (kd = 7.2 nM) and low capacity (3 fmol/10(5) cells). Competition by adrenergic agonists for [3H]DHE binding was stereospecific. The rank order of potency against binding was identical with that determined functionally against TSH secretion (Ki for prazosin, 0.7 nM greater than thymoxamine, 2.7 nM greater than (-) E, 7 nM greater than phentolamine, 8 nM greater than (-) NE, 11.5 nM greater than phenylephrine, 100 nM greater than yohimbine, 300 nM greater than clonidine, 4500 nM greater than (+/-) E, 5000 nM greater than (+/-) NE, 7000 nM greater than isoproterenol, 3 X 10(5) nM), and typical of binding to an alpha 1-adrenoreceptor. It is concluded that TSH can be specifically released from rat anterior pituitary cells in monolayer culture by the direct effects of adrenergic agonists and that the stimulation is mediated via a single high affinity population of alpha 1-adrenergic receptors.     

Use of a pituitary cell dispersion method and primary culture system for the studies of gonadotropin-releasing hormone action in the goldfish, Carassius auratus. II. Extracellular calcium dependence and dopaminergic inhibition of gonadotropin responses

Primary static cultures of dispersed goldfish pituitary cells obtained by controlled trypsinization released gonadotropin (GTH) in response to 2-hr stimulations of 0.1 nM to 1 microM [Trp7,Leu8]-gonadotropin-releasing hormone (sGnRH), [D-Arg6,Pro9-N-ethylamide]-sGnRH (sGnRHa), and [His5,Trp7,Tyr8]-GnRH (cGnRH-II) in a dose-dependent manner. Coincubation with 10 to 1000 nM of a dopamine agonist, apomorphine, dose dependently reduced the GTH response to increasing concentrations of sGnRH. Apomorphine at 1 microM completely abolished the dose-dependent GTH response to sGnRHa and cGnRH-II, but only partially inhibited the GTH-releasing action of high concentrations of sGnRH. Addition of calcium ionophores, 1 to 100 microM A23187 and 10 to 100 microM ionomycin, significantly increased GTH release. The ED50S of the GTH response to A23187 and ionomycin were 0.88 +/- 0.15 and 13.67 +/- 2.76 microM, respectively. Incubation with Ca2(+)-deficient media (media prepared without the addition of Ca2+ salts) did not significantly affect basal GTH release, but severely decreased the hormone response to increasing concentrations of sGnRH, A23187, and ionomycin. These results confirm the direct inhibitory dopaminergic influence on GTH release in goldfish and further suggest that extracellular Ca2+ plays a role in mediating GnRH action on gonadotropes in fish.     

Interactions between Signaling Pathways in Mediating GnRH-Stimulated GTH Release from Goldfish Pituitary Cells: Protein Kinase C, but Not Cyclic AMP Is an Important Mediator of GnRH-Stimulated Gonadotropin Secretion in Goldfish

In the goldfish, it has been proposed that gonadotropin (GTH) release induced by GTH-releasing hormone (GnRH) involves Ca²⁺entry through voltage-sensitive Ca²⁺channels (VSCC), protein kinase C (PKC) activation, and arachidonic acid (AA) metabolism, but not cyclic AMP (cAMP) action. However, cAMP appears to mediate GnRH action in other teleosts. In this study, the relative importance of PKC and cAMP in mediating GnRH action in goldfish was studied using primary cultures of dispersed pituitary cells. Consistent with an involvement of PKC in GnRH action, the GTH responses to the PKC activating tetradecanoyl phorbol acetate (TPA), salmon (s)GnRH, and chicken (c)GnRH-II were inhibited by two selective PKC inhibitors, calphostin C, and staurosporine. Furthermore, GTH release responses induced by sGnRH or cGnRH-II were not additive to responses stimulated by the PKC-activating diglyceride DiC8, in either long-term static incubation or acute perifusion experiments. In static incubation studies, the GTH responses to sGnRH and DiC8 were potentiated by the VSCC agonist Bay K 8644, suggesting that VSCC participates in both PKC and GnRH action. Concentrations of K⁺< 100 mMdid not elicit GTH secretion when tested alone, but were effective in stimulating GTH release in the presence of subthreshold doses of DiC8 or TPA. This suggests that minimal activation of PKC greatly enhances the effectiveness of Ca²⁺influx to increase GTH secretion. Taken together, these results indicate that PKC is an important mediator of GnRH-induced, VSCC-dependent GTH release. In contrast to the involvement of PKC, cAMP-dependent mechanisms showed no evidence of direct participation in GnRH-induced GTH release in goldfish. In static incubation studies, the GTH responses to sGnRH and cGnRH-II were not affected by H89, a cAMP-dependent protein kinase (PKA) inhibitor. Furthermore, the GTH release stimulated by cAMP was additive to the response to sGnRH, cGnRH-II, DiC8, TPA, or AA. However, compared to the response to forskolin or TPA alone, combinations of forskolin and TPA resulted in a potentiated increase in GTH release. The acute GTH response to forskolin was also enhanced by DiC8. Thus, cAMP-dependent mechanisms may constitute an independent pathway that interacts positively with GnRH-dependent mechanisms in the regulation of GTH release.     

Synergistic effects of salmon gonadotropin-releasing hormone and estradiol-17beta on gonadotropin subunit gene expression and release in masu salmon pituitary cells in vitro

Effects of salmon gonadotropin-releasing hormone (sGnRH) and estradiol-17beta (E2) on gene expression and release of gonadotropins (GTHs) were examined in masu salmon (Oncorhynchus masou) using primary pituitary cell cultures at three reproductive stages, initiation of sexual maturation in May, pre-spawning in July, and spawning in September. Amounts of GTH subunit mRNAs were determined by real-time polymerase chain reaction, and levels of GTH released in the medium were determined by RIA. In control cells, the amounts of three GTH subunit mRNAs (alpha2, FSHbeta, and LHbeta) peaked in July prior to spawning. FSH release spontaneously increased with gonadal maturation and peaked in September, whereas LH release remained low until July and extensively increased in September. Addition of E2 to the culture extensively increased the amounts of LHbeta mRNA in May and July in both sexes. It also increased the alpha2 mRNA in July in the females. In contrast, sGnRH alone did not have any significant effects on the amounts of three GTH subunit mRNAs at all stages, except for the elevation of alpha2 and FSHbeta mRNAs in July in the females. Nevertheless, synergistic effects by sGnRH and E2 were evident for all three GTH subunit mRNAs. In May, sGnRH in combination with E2 synergistically increased the amounts of LHbeta mRNA in the males and alpha2 mRNA in the females. However, in July the combination suppressed the amounts of alpha2 and FSHbeta mRNAs in the females. sGnRH alone stimulated LH release at all stages in both sexes, and the release was synergistically enhanced by E2. Synergistic stimulation of FSH release was also observed in May and July in both sexes. These results indicate that a functional interaction of sGnRH with E2 is differently involved in synthesis and release of GTH. The synergistic interaction modulates GTH synthesis differentially, depending on subunit, stage, and gender, whereas it potentiates the activity of GnRH to release GTH in any situation.     

Differences in extracellular calcium involvement mediating the secretion of gonadotropin and growth hormone stimulated by two closely related endogenous GnRH peptides in goldfish pituitary cells.

Two endogenous gonadotropin-releasing hormone (GnRH) peptides, salmon GnRH (sGnRH) and chicken GnRH II (cGnRH II), stimulate gonadotropin (GtH) and growth hormone (GH) secretion in the goldfish. The extracellular calcium (e-Ca2+) dependence of the GtH and GH response to the two GnRH peptides were compared using static incubations of dispersed goldfish pituitary cells. Incubation with Ca(2+)-depleted medium (without the addition of Ca2+ salts and in the presence of EGTA) did not alter basal GtH secretion, but reduced the GtH response to sGnRH, and abolished the cGnRH II-induced GtH release. Blockade of e-Ca2+ entry by low concentrations of CoCl2 had no effect on basal GtH secretion but reduced cGnRH II and sGnRH stimulated GtH release when applied at 0.1 and 0.5 mM concentrations, respectively. In general, treatments with voltage-sensitive Ca2+ channel (VSCC) antagonists, verapamil, nifedipine and nicardipine, did not alter basal GtH release but attenuated GnRH-stimulated GtH responses. cGnRH II-induced GtH release was decreased by 10 nM verapamil and 1 nM nifedipine, whereas the reduction of GtH responses to sGnRH required 100 times higher concentrations of these VSCC antagonists. cGnRH II but not sGnRH stimulation of GtH secretion was also abolished by 10 microM nicardipine. In contrast to GtH release, exposure to Ca(2+)-depleted medium reduced basal GH release and abolished the GH responses to both GnRH peptides. sGnRH and cGnRH II-stimulated GH responses were both abolished by 0.1 mM CoCl2, decreased by 1 nM verapamil, and reduced by 10 nM nicardipine. Addition of 0.1 and 10 microM nifedipine inhibited the GH responses to sGnRH and cGnRH II, respectively. Basal GH release was not affected by the VSCC antagonists tested. Results from this study indicate that entry of e-Ca2+, in part through VSCC, is involved in GnRH stimulation of GtH and GH release from goldfish gonadotropes and somatotropes; however, the e-Ca2+ dependence of the GtH and GH responses to the two endogenous GnRHs differ. The stimulatory effects of cGnRH II on GtH secretion is more dependent on and sensitive to e-Ca2+ than sGnRH. Whereas the sensitivity of GH responses to manipulations of e-Ca2+ availability is, in most instances, similar for both GnRH peptides. These results further suggest that basal secretion of GH is more sensitive to e-Ca2+ than basal GtH release; however, VSCC are not involved in the maintenance of basal release of either hormone.     

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)     

Stimulatory effect of beta-adrenergic agonists on ileal L cell secretion and modulation by alpha-adrenergic activation.

Postprandial release of peptide YY (PYY) and glucagon-like peptide-1 (GLP-1) from L cells results from both nutrient transit in the ileal lumen and neural drive of endocrine cells. The adrenosympathetic system and its effectors have been shown to induce secretion of L cells in vivo or in vitro. Because these transmitters act through three receptors, beta, alpha1, alpha2, coupled to different intracellular pathways, we evaluated the responses of L cells to specific agonists, using the model of isolated vascularly perfused rat ileum. General stimulation of adrenergic receptors with epinephrine (10(-7) M) induced significant GLP-1 and PYY secretions (94+/-38 and 257+/-59 fmol/8 min respectively) which were abolished upon propranolol (10(-7) M) pretreatment and strongly decreased upon infusion with 10(-8) M prazosin. Blockade of alpha2-receptors with idazoxan (10(-8) M) did not alter epinephrine-induced peptide secretion. The beta-adrenergic agonist isoproterenol (10(-6) M) infused for 30 min induced a transient release of GLP-1 and PYY (integrated release over the 8 min of the peak secretion: 38+/-16 and 214+/-69 fmol for GLP-1 and PYY respectively, P<0.05). Because terbutaline but not dobutamine or BRL 37,344 (10(-5) M) induced significant GLP-1 and PYY secretions (135+/-30 and 305+/-39 fmol/8 min respectively), isoproterenol-induced secretions are suggested to result mainly from stimulation of the beta2-isoreceptor type. In contrast, the alpha1-agonist phenylephrine (10(-7) M) did not stimulate peptide release. When co-infused with 10(-6) M or 10(-7) M isoproterenol, 10(-7) M phenylephrine raised GLP-1 release to 174+/-53 and 108+/-28 fmol/8 min respectively (vs 38+/-16 and 35+/-10 fmol/8 min for isoproterenol alone, P<0.05) whereas PYY secretion was not significantly increased. Clonidine (10(-7) M), an alpha2-agonist, induced a moderate and delayed increase of GLP-1 and PYY but abolished the isoproterenol-induced peptide secretion. Our results showed that general stimulation of adrenergic receptors stimulates the secretory activity of ileal endocrine L cells. The net peptide secretion results from the activation of the beta2-isoreceptor type. Additionally, GLP-1 and PYY secretions are positively modulated by alpha1-receptor stimulation and inhibited by alpha2-receptor activation upon beta-receptor occupation.     

Norepinephrine stimulates immunoreactive (ir) atrial natriuretic peptide (ANP) secretion and pro-ANP mRNA expression from rat hypothalamic neurons in culture: effects of alpha 2-adrenoceptors.

Although ANP or its smaller congeners are produced and secreted from rat hypothalami, the role or roles of neurotransmitter(s) in regulating their release and production from the neurons remains unclear. We report here that norepinephrine or epinephrine (NE/EPI) facilitates irANP secretion and pro-ANP mRNA expression in long term cultures of rat hypothalamic neurons through their effects on alpha 2-adrenoceptors. Hypothalami of 3 day-old Sprague-Dawley rats were removed and digested with collagenase. The dispersed cells were plated on poly-D-lysine coated culture dishes (10(6) cells/well) in Hepes buffered Dulbecco's Modified Eagle Medium supplemented with 8% fetal calf serum. Six days after plating, media were replenished with serum free media and the cultures incubated for 4 more days with vehicle or various doses of NE, EPI, alpha- or beta-adrenoceptor agonists in the presence of absence of antagonists. Culture media were then extracted with C18 Sep-pak and the levels of irANP determined by a well characterised RIA for ANP. NE or EPI treatment significantly increased irANP secretion from the cultures in a dose related manner with ED50 and Emax of approximately 0.2 microM and 1 microM respectively. The stimulation effect of NE was blocked by yohimbine (alpha 2-antagonist), but not prazosin (alpha 1-antagonist) or propranolol (beta-antagonist). Clonidine (alpha 2-agonist), but not phenylephrine (alpha 1-agonist) or isoprenaline (beta-agonist) mimicked the effects of NE or EPI. At the concentration of 0.1 microM, clonidine increased irANP release approximately 3 fold above that of control values (34.7 +/- 3.3; mean +/- SE, n = 4). These changes were accompanied by corresponding increments in the abundance of pro-ANP mRNA in the cultures as examined by a colorimetric Northern blot analysis. Our results indicate that NE or EPI, acting through its alpha 2-adrenoceptors, may modulate the function of ANP neurons in rat hypothalami by regulating the secretion and production of the neuropeptide at the genomic level.     

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

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

Desensitization to native molecular forms of gonadotropin-releasing hormone in the goldfish pituitary: dependence on pulse frequency and concentration.

Homologous desensitization of gonadotropin-releasing hormone (GnRH) was investigated using goldfish pituitary fragments in vitro. The two native GnRH peptides, sGnRH [( Trp7, Leu8]-GnRH) and cGnRH-II [( His5, Trp7, Tyr8]-GnRH) were administered either continuously or in pulsatile fashion at different frequencies and concentrations. Continuous treatment (60 min) with either sGnRH or cGnRH-II at 10(-7), 10(-8), and 10(-9) M resulted in desensitization of goldfish pituitary in a biphasic fashion, characterized by an initial rapid peak of GTH release (phase 1), followed by a lower sustained release of GTH remaining at a stable concentration above the basal level (phase 2). Pititary fragments were then washed for 60 min and further treated continuously (60 min) with the same concentrations of sGnRH or cGnRH-II (second treatment). Total sGnRH- or cGnRH-II-induced GTH release during the second treatment period was significantly lower than that observed during the initial treatment period, depending upon the concentration of the peptides. The second phase of GTH release was more pronounced at lower concentrations compared to that observed following 10(-7) M treatment, especially for sGnRH. Pulsatile treatment with either sGnRH or cGnRH-II (2-min pulses of 10(-7), 10(-8), and 10(-9) M given every 20 min) resulted in significant desensitization of the pituitary GTH release. Reduction of pulse frequency to 2 min treatment every 60 min resulted in a lower degree of desensitization; little or no desensitization was observed following treatment with 10(-8) and 10(-9) M cGnRH-II or 10(-9) M sGnRH. A further reduction in frequency to 2-min pulses of sGnRH or cGnRH-II (10(-7) or 10(-8) M) given every 90 min did not result in desensitization of the pituitary GTH release. In summary, the present study demonstrates that GnRH-induced desensitization is dependent on both pulse frequency and concentration in the goldfish pituitary. These findings support the hypothesis that pulsatile secretion of the native GnRH peptides may be essential for maintenance of normal pituitary GTH release in goldfish.     

Participation of arachidonic acid metabolism in gonadotropin-releasing hormone stimulation of goldfish gonadotropin release

Two intraperitoneal injections of a mammalian gonadotropin-releasing hormone (GnRH) analog, [D-Ala6, Pro9-N-ethylamide]-GnRH (mGnRHa; 0.1 micrograms/g), at 12-hr intervals increased serum gonadotropin (GTH) levels in sexually mature and sexually regressed female goldfish 2 and 6 hr after the second injection. This serum GTH response was decreased by the coinjection of a lipoxygenase enzyme inhibitor, nordihydroguaiaretic acid (NDGA: 0.1 to 10 micrograms/g) at the time of the second mGnRHa application. In static cultures of dispersed goldfish pituitary cells, 1-100 microM arachidonic acid (AA) and 0.1-1000 nM [Trp7, Leu8]-GnRH (salmon GnRH, sGnRH) and [D-Arg6, Pro9-N-ethylamide]-sGnRH (sGnRHa) caused dose-dependent increases in GTH release. Additions of 1-40 microM NDGA reduced the sGnRH-stimulated GTH release in a dose-dependent manner, and completely inhibited the GTH response to increasing concentrations of AA. NDGA 40 microM also decreased the elevated GTH levels induced by sGnRHa treatment. Exposure to 10 microM 5,8,11,14-eicosatetraynoic acid, an inhibitor with mixed action on lipoxygenase and cyclooxygenase enzymes, reduced the dose-dependent GTH response to sGnRH and AA. In contrast, coincubation with another cyclooxygenase blocker, indomethacin, at 10 microM, did not alter AA and sGnRH-induced GTH release. These results provide in vivo and in vitro evidence for the participation of AA metabolism in mediating GnRH-stimulated GTH release in the goldfish. The importance of AA metabolism through the lipoxygenase pathway is also indicated.     

Noradrenaline and prostaglandin E2 stimulate LH-RH release from rat median eminence through distinct 1-alpha-adrenergic and PGE2 receptors

Noradrenaline (NA) and prostaglandin (PG) E2 produced a dose-related stimulation of luteinizing hormone releasing hormone (LH-RH) release from incubated median eminence of adult male rats, with ED50 values of 6.10(-7) and 8.10(-8) M, respectively. The effects of some adrenoceptor agonists (10(-5) M) on LH-RH release were tested: only phenylephrine (alpha 1-agonist) stimulated LH-RH release; clonidine (alpha 2 greater than alpha 1-agonist) and isoproterenol (beta-agonist) were ineffective. Adrenoceptor antagonists (10(-6) M) were also tested: prazosin (alpha 1-antagonist) and phentolamine (alpha 1/alpha 2-antagonist) almost completely suppressed the enhanced release of LH-RH induced by NA. In contrast, neither yohimbine (alpha 2-antagonist) nor propranolol (beta-antagonist) altered this effect of NA. When tested alone, no significant effect was obtained on basal LH-RH release with any of the antagonists tested. Moreover, at concentrations that blocked the stimulation produced by NA, the adrenoceptor antagonists did not alter the effect of PGE2. Among seven PGs tested at 10(-6) M, only PGE2, PGE1, PGA2, and 16,16-dimethyl PGE2 significantly enhanced LH-RH secretion. 8-iso PGE2 weakly stimulated LH-RH secretion, whereas PGF2 alpha and PGD2 were ineffective. A direct correlation existed between the potency of these compounds to modify LH-RH secretion and to inhibit specific [3H]-PGE2 binding to hypothalamic membranes. In conclusion, these results suggest that the stimulation of LH-RH from median eminence induced by NA and PGE2 involves the activation of an alpha 1-adrenergic receptor and a PGE2 receptor, respectively.     

Interactions of estradiol with gonadotropin-releasing hormone and thyrotropin-releasing hormone in the control of growth hormone secretion in the goldfish.

The effects of testosterone (T) and estradiol (E2) on serum growth hormone (GH) concentrations were investigated throughout the seasonal reproductive cycle of the female goldfish. Gonad-intact female goldfish were implanted intraperitoneally for 5 days with silastic pellets containing no steroid (blank), T(100 micrograms/g) or E2 (25-100 micrograms/g). In blank-implanted females, seasonal variations in serum GH were evident; maximal serum GH levels were found in spring while minimal GH levels were found in summer and early autumn. Implantation of E2-containing silastic capsules stimulated increases (2-4 times control) in serum GH levels throughout the reproductive cycle. Implantation of T did not affect serum GH at any time of the year. One possible mechanism by which E2 could exert its effects may be through alteration of pituitary sensitivity to GH-releasing factors. The decapeptide salmon gonadotropin-releasing hormone (sGnRH) is found in the brain and pituitary of goldfish and stimulates gonadotropin (GTH) and GH secretion. In contrast, thyrotropin-releasing hormone (TRH) stimulates GH, but not GTH, release from pars distalis fragments obtained from sexually regressed (ED50 = 5.7 +/- 3.8 nM; August) or sexually mature (ED50 = 0.53 +/- 0.28 nM; March) fish; in vivo E2 treatment resulted in a 3-fold increase in the in vitro GH response to TRH. Furthermore, E2 treatment increased sGnRH-stimulated GH release by approximately 4-fold. These results demonstrate that E2 but not T stimulates GH secretion throughout the reproductive cycle of female goldfish. Furthermore, sGnRH and TRH stimulate GH release in a teleost, and these stimulatory responses are enhanced by physiological levels of E2.     

Differential effects of central adrenoceptor agonists on luteinizing hormone release

This study examined the alterations in episodic luteinizing hormone (LH) release in response to third ventricle infusions of various alpha- and beta-adrenoceptor agonists in ovariectomized (OVX) rats as well as the effects of steroid priming with 50 micrograms estradiol benzoate (EB) and 25 mg progesterone (P) on the LH responses to these agonists. Unanesthetized rats with indwelling atrial cannulae were bled at 10-min intervals for 0.5-1.5 h prior to infusion and up to 1.5 h following infusion of equimolar amounts (0.06 or 0.3 mumol in 2 microliters saline adjusted to pH 5.5 and infused slowly over a 2-min period) of norepinephrine (NE), phenylephrine (Phen, alpha 1-agonist), isoproterenol (Iso, beta-agonist) or clonidine (Clon, alpha 2-agonist). In unprimed OVX rats, 0.06 mumol NE induced a significant lengthening (by approximately 121%) of the episodic interval between the peak LH levels and caused a decrease in mean blood LH levels of approximately 24%, which began almost immediately and lasted for approximately 1 h after infusion. When administered in the same manner and dosage, both alpha- and beta-adrenergic agonists were similarly effective in suppressing pulsatile LH release in OVX unprimed rats, with the following rank order being apparent: Clon greater than NE congruent to Phen greater than Iso. However, in OVX-EBP-primed rats, while 0.06 mumol NE significantly stimulated LH release, none of the other adrenoceptor agonists administered at this dosage was effective in altering the low nonpulsatile levels of blood LH characteristic of the steroid-primed animal. Nevertheless, at a concentration 5 times higher (0.3 mumol) Clon and Phen did induce LH surges while Iso, even at this higher dose, was not stimulatory to LH release. These results suggest that the inhibitory action of NE on LH secretion in OVX rats may be exerted via activation of both alpha- and beta-adrenoceptors, whereas primarily alpha-adrenoceptors are responsible for mediating the NE-induced stimulation of LH release in OVX steroid-primed animals.     

Effects of catecholamine-depleting agents and receptor blockers on basal and angiotensin II- or norepinephrine-stimulated luteinizing hormone release in female rats

Depletion of hypothalamic norepinephrine (NE) and epinephrine by administration of diethyldithiocarbamate abolished the stimulatory effects of intraventricular (IVT) angiotensin II (AII) on LH release in ovariectomized rats pretreated with estradiol and progesterone. The increase in blood LH produced by IVT NE or iv LHRH was unaffected in these drug-treated animals. Selective depletion of hypothalamic epinephrine by the administration of LY134046 (8,9-dichloro-2,3,4,5-tetrahydro-1H-2-benzazepine hydrochloride) potentiated the effect of AII on LH secretion. Blockade of alpha 1-, alpha 2-, or beta-adrenergic receptors resulted in a transient increase in basal LH levels. The stimulation of LH secretion induced by IVT AII or NE was unaffected by alpha 1-receptor blockade with prazosin, but was abolished by alpha 2-receptor blockade with yohimbine. beta-Receptor blockade with propranolol potentiated both NE- and AII-induced LH release. AII receptor blockade with IVT saralasin prevented the LH rise due to AII without modifying that due to NE. Taken together, these data suggest that IVT AII stimulates LH release in ovariectomized rats treated with estradiol and progesterone by releasing endogenous NE, which, in turn, acts on facilitatory alpha 2-receptors to affect LH secretion, presumably by increasing the secretion of LHRH. Exogenous NE also acts at this receptor. beta-Receptors provide inhibitory tone to this facilitatory system, and blockade of this receptor subtype results in potentiated LH responses to both AII and NE.     

Alpha 2-adrenergic potentiation of adenosine-stimulating effect on glucagon secretion

Previous studies from our laboratory showed 1) that adenosine (1.65 microM), a substance released by tissues in energy-deficient states, stimulated glucagon secretion by activation of A2 purinergic receptors, and 2) that this effect was potentiated by a low substimulating concentration of epinephrine through activation of alpha-adrenergic receptors. The present work was undertaken to assess the subtype of alpha-adrenergic receptor involved in this potentiation. Therefore, we used adrenergic blockers and agonist drugs more specific for alpha 1- or alpha 2-adrenergic receptors. The potentiating effect of epinephrine (0.01 microM) on glucagon secretion induced by adenosine (1.65 microM) was not prevented by an alpha 1-adrenergic blocker, prazosine (6 microM), but was suppressed by an alpha 2-adrenergic blocker, yohimbine (0.6 microM). The implication of alpha 2-adrenergic receptors in the potentiating effect was confirmed by the use of selective alpha 1- or alpha 2-adrenergic agonist drugs. Indeed, clonidine (0.01 microM), an alpha 2-agonist, ineffective per se, potentiated, whereas phenylephrine (0.01 microM), an alpha 1-agonist, had no effect on glucagon secretion induced by adenosine. We conclude that the potentiation by epinephrine of adenosine-induced glucagon secretion is mediated by alpha 2-adrenergic receptor activation. A potentiation between the effects of A2 purinergic and alpha 2-adrenergic agonists may be of physiological relevance in stressful energy-deficient states, when an increase in glucagon secretion is necessary.