In vitro release of gonadotropin-releasing hormone from the brain preoptic-anterior hypothalamic region and pituitary of female goldfish

In vitro release of gonadotropin releasing hormone (GnRH) from slices of the preoptic-anterior hypothalamic (P-AH) region and fragments of the pituitary of goldfish was studied using a static incubation system. Release of GnRH from both tissue preparations was stimulated by depolarizing concentrations of extracellular potassium ions (K+). Other putative secretagogues, calcium ionophore A23187 (1 microM), forskolin (100 microM), and prostaglandin E2 1 microM) also stimulated release of GnRH from both tissue preparations. Omission of Ca2+, or chelating the remaining remaining Ca2+ by EGTA (0.1 mM), abolished the release of GnRH stimulated by high K+ concentrations (60 mM), but did not reduce spontaneous release. Verapamil (1 microM), a voltage-sensitive calcium channel blocker, abolished the release of GnRH stimulated by high K+ or A21387 from both tissue preparations. The GnRH released in vitro from both the P-AH region and pituitary was concentrated by Sep-Pak and then separated by high-performance liquid chromatography. The major peak of the GnRH immunoreactivity was found to coelute with synthetic salmon GnRH [( Trp7,Leu8]-GnRH) and the minor peak with chicken GnRH-II [( Gln8]-GnRH). Dopamine (10 and 100 microM) inhibited GnRH release from both P-AH slices and pituitary fragments, while serotonin (1-100 microM) stimulated release from both. Norepinephrine (10-100 microM) stimulated GnRH release from P-AH slices but not from pituitary fragments. The results demonstrate that the release of GnRH from goldfish P-AH slices and pituitary fragments in vitro in response to various secretagogues and monoamines can be studied using a static incubation system.     

Influences of norepinephrine, and adrenergic agonists and antagonists on gonadotropin secretion from dispersed pituitary cells of goldfish, Carassius auratus.

Static incubations of dispersed goldfish pituitary cells with 1-100 nM norepinephrine (NE) stimulated gonadotropin (GTH) release. Additions of the alpha-agonist phenylephrine, and the alpha 1-agonist 6-fluoronorepinephrine, but not the alpha 2-agonist clonidine, nor the beta-agonist isoproterenol, also enhanced GTH secretion. The GTH responses to 1 nM NE was significantly inhibited by coincubations with 1 microM of the alpha-antagonist phentolamine, the alpha 1-antagonists prazosine and benoxathian, but not the alpha 2-antagonist yohimbine nor the beta-antagonist propranolol. The GTH responses to NE and phenylephrine were also additive to salmon GTH-releasing hormone (sGnRH)-induced GTH release. These results suggests that NE directly stimulates GTH secretion independent of sGnRH receptors via alpha 1-like adrenergic receptors.     

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.     

Synthesis and release of luteinizing hormone in vitro by rat anterior pituitary cells: effects of gallopamil hydrochloride (D600) and pimozide

We compared the role of Ca2+ in regulating GnRH-induced LH synthesis and release from cultured rat pituitary cells. LH synthesis and release were measured after a 4-h treatment of cells with gallopamil hydrochloride (D600; 1 and 100 microM), a Ca2+ channel blocker, or pimozide (0.5 and 5.0 microM), a calmodulin inhibitor, with or without 1 nM GnRH. LH translation and glycosylation were monitored by measuring incorporation of [14C]alanine and [3H]glucosamine, respectively, into total (cell and medium) immunoprecipitable LH. GnRH significantly (P less than 0.01) increased total [3H]LH (glycosylation), but had no effect on total [14C]LH (translation). D600 significantly (P less than 0.01) depressed (1 microM) and completely blocked (100 microM) GnRH-induced LH glycosylation and release of [3H]LH, [14C]LH, and immunoreactive LH. D600 (100 microM) also reduced (P less than 0.05) total basal synthesis of [14C]LH. Neither dose of D600 altered uptake of [3H]glucosamine, but 100 microM D600 significantly (P less than 0.01) depressed its incorporation into total protein. D600 (100 microM) significantly (P less than 0.01) depressed [14C]alanine uptake and incorporation into total protein. Pimozide significantly (P less than 0.01) reduced, in a dose-related manner, GnRH-induced LH glycosylation, and release of immunoreactive LH, [3H]LH, and [14C]LH. Pimozide did not alter LH translation or uptake of radiolabeled precursors or their incorporation into total protein. These results demonstrate that D600 and pimozide inhibit both GnRH-induced LH glycosylation and release. Thus, the actions of GnRH on LH glycosylation and release are both mediated by similar Ca2+-dependent pathways.     

Influence of dopamine on the altered release of prolactin, luteinizing hormone, and follicle-stimulating hormone induced by interleukin-2 in vitro.

Interleukin-2 (IL-2) alters the release of anterior pituitary hormones at femtomolar concentrations from hemipituitaries incubated in vitro. This cytokine significantly lowered luteinizing hormone (LH) and follicle-stimulating hormone (FSH) and stimulated prolactin (PRL) release, thus demonstrating a reciprocal action of the lymphokine on lactotrophs and gonadotrophs. Since dopamine (DA) is a powerful inhibitor of PRL release, in the present experiments were evaluated possible dose dependent effects of DA on IL-2-induced alterations of the release of PRL, LH, and FSH. Hemipituitaries were incubated with varying concentrations of DA, a combination of IL-2 plus DA, or a combination of haloperidol (1 x 10(-5) M) with DA for 1 h, followed subsequently by incubation with medium containing only high potassium (K+) to study the effects on depolarization-induced hormone release. DA induced a dose-related, significant lowering of the basal PRL release with a minimal effective dose (MED) of less than 19 nM. The depolarization-induced PRL release was also inhibited, but the MED was 100-fold higher than the MED to inhibit basal PRL release. DA at much higher concentrations (30, 60, and 90 microM) significantly reduced pituitary PRL content. The addition of 0.187, 3.75, 15, or 60 microM DA to IL-2-induced PRL release. IL-2 (10(-15) M) produced a significant decrease in LH and FSH release. The combination of 3.75 or 15 microM DA plus IL-2 failed to alter the IL-2 suppressed LH release, whereas the addition of 0.187 microM DA to IL-2 blocked its suppressive influence, and 60 microM DA added to Il-2 produced an additive inhibitory effect. Thus, the interaction of IL-2 and DA is biphasic on LH release. The significant reduction of FSH release induced by IL-2 was blocked in the presence of 0.187, 3.75, 15, or 60 microM DA. DA alone at relatively high concentrations of 30, 60, and 90 microM suppressed basal LH and FSH release. The effects of DA on PRL, LH, and FSH at all doses tested were blocked by the DA receptor blocker, haloperidol which by itself at the concentration tested (1 x 10(-5) M) had no effect. Thus, the actions of DA at all concentrations tested appear to be mediated via DA receptors. In conclusion, DA was capable of blocking the stimulatory action of IL-2 on PRL release and its inhibitory action on FSH release by a DA receptor mediated action.(ABSTRACT TRUNCATED AT 400 WORDS)     

The alpha-1-adrenergic neuronal system is involved in the pulsatile release of luteinizing hormone-releasing hormone in the ovariectomized female rhesus monkey.

It has been postulated that catecholamines are integral to the control of LHRH release. In the present study, the roles of adrenergic and dopaminergic receptors in the control of pulsatile LHRH release are examined. The effects of prazosin (an alpha 1-antagonist), rauwolscine (an alpha 2-antagonist), propranolol (a beta-antagonist), haloperidol (a dopamine antagonist), SCH23390 (a D1 antagonist), and LY163502 (a D2 agonist), on in vivo LHRH release in the stalk-median eminence were tested in ovariectomized female rhesus monkeys using push-pull perfusion. Prazosin caused a significant suppression of the LHRH release. This was primarily due to a significant suppression of LHRH pulse amplitude, but not pulse frequency, i.e. the interpulse interval was not affected by the administration of prazosin. In contrast to prazosin, none of the other adrenergic or dopaminergic drugs had significant effects on LHRH release. We conclude from these results that (1) the stimulatory effects of norepinephrine (NE) and/or epinephrine on pulsatile LHRH release are mediated by alpha 1-adrenergic receptors but not alpha 2- or beta-adrenergic receptors, and (2) dopaminergic receptors do not appear to be involved in pulsatile LHRH release in ovariectomized rhesus monkeys.     

Serotonin release mechanisms in bovine pineal gland: stimulation by norepinephrine and dopamine

An assessment of serotonin (5HT) release was made in bovine pineal gland. Bovine pineal fragments took up [3H]5HT by a Na+-dependent process exhibiting two apparent Km, i.e. a high affinity uptake system (Km = 220 nM) and a low affinity uptake system (Km = 197 microM). A significant release of [3H]5HT was elicited by increasing K+ concentrations in the medium (20-80 mM). Exposure of bovine pineal fragments to varying doses of catecholaminergic agonists indicated that a significant [3H]5HT release was elicited at the following threshold concentrations: 10(-6) M norepinephrine (NE), 10(-7) M dopamine (DA), 10(-6) phenylephrine and 10(-6) M isoproterenol. By employing specific receptor agonists and antagonists, the 5HT release activity of adrenergic agonists was found to be mediated by alpha 1-adrenoceptors, while that of DA by D2-dopaminergic receptors. 5HT release elicited by NE or DA, as well as that by 30 mM K+, was Ca2+-dependent. Both NE and DA increase 45Ca2+ uptake in a dispersed cell preparation of bovine pineal glands. As in the case of 5HT release, the effect of NE and DA on calcium uptake was mediated by alpha 1-adrenoceptors and D2-dopaminergic receptors, respectively. These results indicate that both NE and DA control 5HT release in bovine pineal gland.     

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.     

Sepsis causes presynaptic histamine H3 and alpha2-adrenergic dysfunction in canine myocardium

OBJECTIVE: Histamine H3 receptors and alpha2-adrenoceptors are presynaptic receptors that modulate norepinephrine (NE) release from sympathetic nerves innervating the cardiovascular system. We previously showed that cardiac H3 receptors are activated in sepsis, and that this activation leads to a decrease in the adrenergic response (AR) [J. Appl. Physiol. 85 (1998) 1693-1701] H3-receptors and alpha2-receptors appear to be coupled to GTP binding regulatory proteins (G) that modulate transmitter release by reducing calcium current into the nerve terminals through neuronal calcium channels. There may also be interaction between H3-receptors and alpha2-receptors on AR that may occur either at the receptor or a more downstream level. METHODS: In the present study, we examined the effect of septic plasma on AR in a canine ventricular preparation in which field stimulation was used to produce AR. We determined whether there was interaction between H(3)-receptors and alpha2-adrenoceptors and tested whether H3 activation would attenuate the alpha2-agonist and alpha2-antagonist effects of clonidine and yohimbine, respectively. We also determined whether the mechanism by which septic plasma decreases the adrenergic response involves inactivation of an inhibitory G protein and used pertussis toxin (PTX) to assess this effect. RESULTS: We found that septic plasma attenuated AR produced by field stimulation, and that this decrease was mediated by a PTX sensitive inhibitory G protein. H3 activation also attenuated the alpha2-agonist and alpha2-antagonist effects on adrenergic activation as compared with nonseptic plasma. CONCLUSION: We conclude that presynaptic sympathetic dysfunction may contribute to cardiovascular collapse in sepsis.     

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.     

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.     

Modulation of noradrenergic transmission by neuropeptide Y and presynaptic alpha 2-adrenergic receptors in the hypothalamus of spontaneously hypertensive rats.

Neuropeptide Y (NPY) has a wide and specific distribution in the central nervous system, and is colocalized with catecholamines in specific neuronal systems. In this study, in order to investigate the regulatory mechanisms of NPY and presynaptic alpha 2-adrenergic receptors on central noradrenergic transmission in hypertension, we have examined the effects of NPY and the alpha 2-agonist, UK 14,304, on (3H)-noradrenaline (NA) release from hypothalamic slices of spontaneously hypertensive rats (SHR). Electrical stimulation (1 Hz)-evoked (3H)-NA release was significantly greater in the hypothalamic slices of SHR than in those of Wistar Kyoto rats (WKY). NPY and the alpha 2-agonist, UK 14,304, inhibited the stimulation-evoked (3H)-NA release in a dose-related manner. The inhibitory effects of NPY and UK 14,304 on NA release were significantly attenuated in SHR compared with WKY. These results suggest that NPY and alpha 2-adrenoceptors might be involved in the regulation of central sympathetic nervous activity in hypertension.     

Comparison of vascular responses of isolated, perfused simian and canine coronary arteries to adrenergic agonists

The vascular responses of the isolated, perfused simian left circumflex coronary arteries to adrenergic agonists were compared to those of canine coronary arteries in isolated, perfused preparations. Norepinephrine and epinephrine produced only vasoconstriction in monkey arteries in contrast to vasoconstrictor, vasodilator and biphasic responses of canine coronary arteries. Isoproterenol induced a dose-dependent vasodilatation. Salbutamol, a selective beta 2-agonist, produced either a slight vasodilatation or no response. Phenylephrine, a selective alpha 1-agonist, usually caused a marked vasoconstriction in a dose-related manner in both preparations. Xylazine and clonidine, selective alpha 2-agonists, caused a slight vasoconstriction in low doses, but these two agonists relaxed both preparations in high doses. These observations suggest that adrenoceptor subtypes may be mainly alpha-1 and beta-1 in the large coronary arteries of monkeys and dogs and that the alpha-adrenoceptor is predominant in simian large coronary arteries.     

Opioid-receptor-mediated inhibition of [3H]dopamine but not [3H]noradrenaline release from rat mediobasal hypothalamus slices.

The modulation of the electrically evoked release of [3H]dopamine (DA) and [3H]noradrenaline (NA) by opioid receptor activation was examined in superfused slices of rat mediobasal hypothalamus (MBH). [3H]DA release was inhibited (maximally by 30-35%) by both the selective kappa-agonist U 50,488 (1 nM to 1 microM) and the selective mu-agonist DAGO (0.01-1 microM) but not by the delta-selective agonist DPDPE (1 microM). Naloxone partly antagonized the inhibitory effect of U 50,488 and completely that of DAGO, whereas the selective kappa-antagonist norbinaltorphimine (nor-BNI) only antagonized the inhibition caused by U 50,488. The dopamine D2 receptor agonist quinpirole as well as the alpha 2-adrenoceptor agonist oxymetazoline both decreased (by 25-30%) the evoked overflow of [3H]DA. The evoked release of [3H]NA was not modulated by any of the opioid agonists nor by quinpirole. However, the alpha 2-adrenoceptor agonist oxymetazoline inhibited the release of [3H]NA by 30-40%. Activation of alpha 2-adrenoceptors by oxymetazoline prevented the inhibitory effect of U 50,488, but not DAGO, on evoked [3H]DA release, whereas the selective kappa-antagonist nor-BNI antagonized the inhibition by oxymetazoline of [3H]DA, but not [3H]NA, release. In conclusion, activation of both kappa- and mu-opioid receptors results in an inhibition of evoked DA release from MBH slices but does not modulate NA release. Therefore, several of the reported effects of opioids on hormone secretion may be an (indirect) consequence of a reduction of DA release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dopamine stimulates release of thyrotrophin-releasing hormone from perfused intact rat hypothalamus via hypothalamic D2-receptors

We have studied the effect of dopamine together with agonist and antagonist drugs of different specificities on the release of TRH from the perfused, intact hypothalamus of the adult rat in vitro. Dopamine produced a dose-related stimulatory effect on TRH release with maximal effect being achieved at 1 mumol/l (increase over basal, 118 +/- 16.5 (S.E.M.) fmol TRH; P less than 0.001 vs basal). This effect was mimicked by the specific D2-agonist drugs bromocriptine (0.1 mumol/l) and LY 171555 (0.1 mumol/l) (increase over basal values, 137.5 +/- 13.75 fmol and 158.6 +/- 10.7 fmol respectively; P less than 0.001 vs basal), but not by the D1-agonist SKF 38393A. The stimulatory effect of dopamine (1 mumol/l) was blocked in a stereospecific manner by the active (D) but not by the inactive (L) isomers of the dopamine antagonist butaclamol. Similar blockade was achieved with the specific D2-antagonist domperidone (0.01 mumol/l) whereas the D1-antagonist SCH 23390 was only effective when used at a concentration 100 times greater. Lower concentrations (0.01 mumol/l) of this D1-antagonist did not block the stimulatory effect of dopamine. High-performance liquid chromatography characterization of the material secreted within the hypothalamus showed one single peak of immunoreactive material which coeluted with synthetic TRH. These data suggest that dopamine exerts a stimulatory role in the control of hypothalamic TRH release by acting at specific D2-receptors.     

Mechanism of action of dopamine on the in vitro release of gonadotropin-releasing hormone

Controversy exists on whether dopamine (DA) stimulates or inhibits GnRH secretion and whether its effects are mediated via alpha-adrenergic receptors or dopaminergic receptors. As a means to examine this conflict, we have utilized an in vitro superfusion system to study the effects of DA, norepinephrine (NE), phentolamine (alpha-antagonist), pimozide (DA antagonist), and two DA agonists (apomorphine and bromocryptine) on GnRH release from isolated mediobasal hypothalami from adult male rats. In this dynamic system, graded concentrations of both NE and DA (2.0 nM to 2.0 microM) led to a dose-dependent increase in GnRH output during the 10 min interval that followed each pulse dose of NE (P less than 0.02) or DA (P less than 0.05). The DA-induced GnRH release was reproducible, consistent, and significant over five successive pulses (20 microM) at 30-min intervals (P less than 0.02). Coinfusion of phentolamine (20 microM) prevented the DA (20 microM) induced release of GnRH (P less than 0.03), but pimozide (20 microM) had no significant effect on DA-induced GnRH release (P greater than 0.3). The two DA agonists, apomorphine and bromocryptine, at doses up to 2.0 microM and 200 nM, respectively, had no significant effect on GnRH release. To determine whether DA was causing a direct stimulation of alpha-adrenergic receptors or being enzymatically converted to NE which could then stimulate alpha-receptors to induce GnRH release, rats were injected with sodium diethyldithiocarbamate (DDC) (550 mg/kg BW) ip, 1 h before death. DDC blocks the enzymatic conversion of DA to NE, and this was reflected by a 37% decrease in hypothalamic NE efflux during the superfusion. However, pulses of DA, even in the presence of DDC, were associated with a marked dose-dependent increase in hypothalamic NE efflux, and DDC failed to prevent the subsequent stimulation of GnRH release. We conclude that the apparent DA-induced release of GnRH is most probably attributable to DA-induced release of hypothalamic NE which, in turn, acts through alpha-adrenergic receptors on peptidergic neurons to stimulate GnRH release.     

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.     

Gonadotropin-releasing hormone neurosecretion in the human hypothalamus: in vitro regulation by dopamine

An in vitro perifusion system was used to investigate GnRH release from adult human hypothalami in response to dopamine (DA) and the DA receptor antagonist haloperidol (HAL). Administration of a 1-microM pulse of DA consistently elicited a mean +/- SE 218 +/- 59% increase (P less than 0.05; n = 5) in GnRH release, whereas 1 microM HAL had no effect. Administration of 1 microM DA during three perifusions in which 1 microM HAL was added to the medium failed to alter basal GnRH release. In contrast, DA did evoke an acute 98 +/- 39% increase (P less than 0.06) in GnRH release during three matching perifusions with medium containing the alpha-adrenergic antagonist phentolamine. These studies demonstrate that DA can stimulate in vitro release of GnRH from the adult human hypothalamus by a DA receptor-mediated mechanism.     

Involvement of cyclic adenosine monophosphate in the stimulation of gonadotropin secretion from the pituitary of the teleost fish, tilapia.

The present study examines the involvement of cAMP in the transduction of the short-term effect of gonadotropin-releasing hormone (GnRH) on gonadotropin release in the teleost fish, tilapia. A 5 min pulse of dibutyryl cyclic AMP (dbcAMP; 0.03-3 mM) or forskolin (0.1-10 microM) resulted in dose-dependent surges in tilapia gonadotropin (taGTH) secretion from the perifused pituitary. The initial increase in taGTH in response to dbcAMP (3 mM) occurred within 6 min. The concentration of cAMP in the effluent medium increased about 20-fold after a pulse of [D-Ala6,Pro9-NEt]-luteinizing hormone-releasing hormone (LHRH) (GnRHa; 100 nM). To rule out the possibility that the observed effects were due to stimulation by endogenous GnRH release from intact nerve terminals present in the fragments, further experiments were performed in primary cultures of dispersed pituitary cells. Exposure (30 min) of the cells to forskolin (0.01-1.0 microM) resulted in a dose-dependent increase in taGTH release similar to that achieved by GnRHa (1 pM to 10 nM). Also 8-bromo cAMP (0.01-1.0 mM) evoked a dose-related increase in taGTH release. A 3-fold increase in the release occurred in the presence of isobutylmethylxanthine (IBMX) (0.2 mM), similar to that obtained by GnRHa (1.0 nM) in the absence of IBMX. However, when combined, the increase in taGTH release was 16-fold. Moreover, exposure of the cultured cells to GnRHa (0.1 or 10 nM, 60 min) resulted in a dose-related elevation of intracellular cAMP levels and taGTH release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of topically applied adrenergic agents on cochlear blood flow.

This study was designed to assess the role of adrenergic receptors in the control of cochlear blood flow. Laser Doppler flowmetry was used to determine the effects of adrenergic drugs topically applied to the round window membrane of the cochlea. The relative influence of the various receptor types (alpha 1, alpha 2, beta 1, and beta 2) was examined by a selection of agonists and antagonists. The agonists norepinephrine and epinephrine, which have mixed alpha- and beta-receptor effects, and phenylephrine, a strong alpha 1-agonist, all induced a dose-dependent reduction in cochlear blood flow. The agonists isoproterenol (beta-active), salbutamol (alpha 2-active) had no effect on cochlear blood flow. Of the antagonists, when tested alone, only the selective alpha 1-antagonist prazosin had a direct effect on cochlear blood flow, demonstrating an increase in cochlear blood flow. The selective alpha 2-antagonist idazoxan, the beta-antagonist propranolol, and the unselective alpha-antagonist phentolamine had no effect on cochlear blood flow. Interaction studies of agonists and antagonists were performed to specifically define the receptor subclasses responsible for the cochlear blood flow increases with norepinephrine and epinephrine. The results are consistent with the presence of an alpha 1-adrenergic sympathetic control of cochlear blood flow.