Norepinephrine regulation of growth hormone release from goldfish pituitary cells. I. Involvement of alpha2 adrenoreceptor and interactions with dopamine and salmon gonadotropin-releasing hormone

Adrenergic regulation of growth hormone (GH) release in the goldfish was examined in vitro using dispersed goldfish pituitary cells under column perifusion. Norepinephrine and epinephrine suppressed basal GH release from goldfish pituitary cells in a reversible and dose-dependent manner. At high doses, a transient rebound of GH release was observed after termination of norepinephrine and epinephrine treatment. In this study, the dose-dependence of adrenergic inhibition on basal GH release was mimicked by the alpha2 agonists clonidine and UK14304. Basal GH secretion, however, was not affected by the beta agonist isoproterenol and alpha1 agonist methoxamine. In addition, the inhibitory actions of norepinephrine and clonidine on basal GH release were blocked by the alpha2 antagonists yohimbine and RX821002. The beta antagonist propranolol and alpha1 antagonists prasozin and benoxathian were not effective in this respect. Salmon gonadotropin-releasing hormone (sGnRH) and dopamine, two known GH-releasing factors in fish, stimulated GH release from goldfish pituitary cells and their GH-releasing actions were inhibited by simultaneous treatment with norepinephrine. Furthermore, the GH rebound after norepinephrine treatment was significantly enhanced by prior exposure to sGnRH and this effect was not observed with dopamine treatment. These results, taken together, suggest that in the goldfish adrenergic input at the pituitary level inhibit basal GH release through activation of alpha2 adrenoreceptors. This alpha2 inhibitory influence may interact with dopaminergic and GnRH input to regulate GH secretion from goldfish pituitary cells. The 'post-inhibition' GH rebound after NE treatment and its sensitivity to sGnRH potentiation may also represent a novel mechanism for GH regulation in fish.     

PACAP Stimulation of Gonadotropin-II Secretion in Goldfish Pituitary Cells: Mechanisms of Action and Interaction with Gonadotropin Releasing Hormone Signalling

Pituitary adenylate cyclase-activating polypeptide (PACAP) has recently been shown to be a hypophysiotropic factor in the goldfish. In this study, we examined the mechanisms of PACAP action on goldfish maturational gonadotropin (GTH-II) release using primary cultures of pituitary cells. The GTH-II response to mammalian PACAP1-38 (mPACAP) was inhibited by a PACAP receptor antagonist suggesting a receptor-mediated action. Addition of either an adenylate cyclase inhibitor or a protein kinase A (PKA) inhibitor reduced the mPACAP-induced GTH-II release. In addition, when GTH-II release was already stimulated by either forskolin or 8-bromo-cAMP (8Br-cAMP), mPACAP did not further increase GTH-II secretion. These results strongly implicated the involvement of an adenylate cyclase/cAMP/PKA pathway in PACAP-stimulated GTH-II release. Although mPACAP induced a rise in intracellular Ca2+ level in identified gonadotropes, results with voltage-sensitive Ca2+ channel inhibitors indicated that the GTH-II responses to mPACAP, forskolin and 8Br-cAMP did not depend upon Ca2+ entry through these channels. Two protein kinase C (PKC) inhibitors did not affect mPACAP-elicited GTH-II release, and mPACAP further increased GTH-II secretion in the presence of PKC activators. These results indicate that PKC-dependent elements are not essential for the stimulatory action of mPACAP in gonadotropes. Interestingly, while GTH-II responses to a stimulatory concentration of mPACAP were additive to responses elicited by maximal effective concentrations of two endogenous gonadotropin releasing hormones (GnRHs), a subthreshold concentration of mPACAP potentiated GnRH and PKC activator stimulation of GTH-II secretion. Similarly, submaximal concentrations of forskolin potentiated the GTH-II response to the PKC activator, tetradecanoyl phorbol acetate. These data suggest that PACAP and its cAMP-dependent signalling mechanisms provide an alternate stimulatory input to goldfish gonadotropes and may influence the effectiveness of the major neuroendocrine control exerted by the PKC-dependent GnRH signalling pathway.     

Somatostatin-14 actions on dopamine- and pituitary adenylate cyclase-activating polypeptide-evoked Ca2+ signals and growth hormone secretion

Using single-cell Ca(2+) imaging and a growth hormone (GH) radioimmunassay, we investigated somatostatin-14 (SS(14)) inhibition of cAMP-dependent, stimulated GH secretion from primary cultures of dispersed goldfish pituitary cells. The dopamine-D1 receptor agonist SKF-38393, and the hypothalamic neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) both elevated intracellular Ca(2+) concentration ([Ca(2+)](i)) and stimulated GH release. When increases in [Ca(2+)](i) were prevented by intracellular loading of BAPTA, a Ca(2+) chelator, SKF-38393- and PACAP-stimulated GH release were inhibited, suggesting that these Ca(2+) signals are required for stimulated GH release. SS(14) inhibited SKF-38393- and PACAP-stimulated GH release, but did not prevent these Ca(2+) signals. Kinetic analysis revealed that SS(14) lowered the maximum amplitude of the SKF-38393- and PACAP-evoked Ca(2+) responses, but had no effect on other aspects of the Ca(2+) signal. We then examined the ability of SS(14) to act subsequent to dopamine-D1 or PACAP receptor activation using the adenylate cyclase activator forskolin, or the membrane permeant cAMP analogue 8Br-cAMP. Forskolin and 8Br-cAMP both increased [Ca(2+)](i) and GH secretion and, as expected, SS(14) inhibited the resultant GH release. Although SS(14) significantly increased the time to maximum amplitude of the forskolin-evoked Ca(2+) signals, it had no detectable effect on any of the kinetic parameters used to describe the Ca(2+) signals evoked by 8Br-cAMP. Taken together, these results establish that SS(14) has the ability to suppress Ca(2+)-dependent exocytosis by acting distal to elevations in [Ca(2+)](i). Furthermore, it appears likely that the cellular mechanisms underlying the observed effects of SS(14) on Ca(2+) signalling are upstream of cAMP and may be unrelated to those responsible for inhibiting GH release.     

Extracellular Sodium Dependence of GnRH-Stimulated Growth Hormone Release in Goldfish Pituitary Cells

In goldfish, gonadotropin-releasing hormone (GnRH) stimulation of growth hormone (GH) release has been shown to involve extracellular Ca²⁺ entry through voltage-sensitive Ca²⁺ channels and the activation of protein kinase C (PKC). In this study, the possible involvement of extracellular Na⁺ in mediating the GH response to GnRH was examined using dispersed pituitary cells. Perifusion with Na⁺-depleted medium reversibly reduced the acute GH response to 5-min pulses of either 10 nM salmon (s)GnRH or 10 nM chicken (c)GnRH-II. Similarly, replacement of normal medium with Na⁺-depleted medium attenuated the long-term GH release response to sGnRH and cGnRH-II under static incubation conditions. These results suggest that GnRH-induced GH release requires the presence of extracellular Na⁺. Treatment with 5-min pulses of the Na⁺-channel agonist veratridine (10 μM) increased GH release in an extracellular Ca²⁺-dependent manner, presumably due to activation of voltage-sensitive Ca²⁺ channels resulting from the depolarizing effect of increased Na⁺ influx. On the other hand, Na⁺ entry through tetrodotoxin (TTX)-sensitive, voltage-dependent Na⁺ channels is not involved in GnRH-induced GH release. Application of 250 nM TTX, which abolished the voltage-sensitive Na⁺ currents in identified goldfish somatotropes, did not affect the acute GH responses to 5-min pulses of sGnRH and cGnRH-II. The possible participation of Na⁺/H⁺ antiport in mediating the extracellular Na⁺-dependent GnRH action on GH release was then examined. In static incubation experiments, sGnRH- and cGnRH-II-induced GH secretion were reduced by inhibitors of the Na⁺/H⁺ antiport, amiloride and dimethylamiloride (DMA). Likewise, the GH response to the PKC activator, tetradecanoyl phorbol acetate, was attenuated by treatment with Na⁺-depleted medium, amiloride, and DMA. The inhibitory actions of amiloride and DMA were selective as these drugs did not affect the GH response elicited by the Ca²⁺ ionophore ionomycin and the voltage-sensitive Ca²⁺ channel agonist, Bay K 8644. Taken together, these results indicate that extracellular Na⁺ and the Na⁺/H⁺ exchanger are involved in the mediation of GnRH-stimulated GH release in goldfish. Furthermore, this dependence on Na⁺ and Na⁺/H⁺ antiport probably occurs distal to the activation of PKC by GnRH.     

Differential involvement of phosphoinositide 3-kinase in gonadotrophin-releasing hormone actions in gonadotrophs and somatotrophs of goldfish, Carassius auratus

In goldfish, two endogenous gonadotrophin-releasing hormones (GnRHs) [salmon (s)GnRH and chicken (c)GnRH-II] control maturational gonadotrophin-II [lutenising hormone (LH)] and growth hormone (GH) secretion via Ca(2+)-dependent intracellular signalling pathways. We investigated the involvement of phosphoinositide 3-kinase (PI3K) in GnRH-evoked LH and GH release and associated intracellular Ca(2+) increases ([Ca(2+)](i) ) in goldfish gonadotrophs and somatotrophs. Immunoreactive PI3K p85α, the predominant regulatory subunit for class IA PI3Ks, was detected in goldfish pituitary tissue extracts and both endogenous GnRH isoforms increased phosphorylation of PI3K p85α in excised pituitary fragments. sGnRH- and cGnRH-II-elicited LH release responses from primary cultures of pituitary cells and [Ca(2+)](i) increases in identified gonadotrophs were significantly reduced in the presence of PI3K inhibitors wortmannin (100 nm) and LY294002 (10 μm). Unexpectedly, wortmannin and LY294002 inhibited GnRH-evoked GH release but only attenuated the [Ca(2+)](i) response in identified somatotrophs to cGnRH-II, and not sGnRH. On the other hand, Ca(2+) ionophore-evoked LH and GH secretion remained unaltered in the presence of the PI3K inhibitors, suggesting that general decreases in the releasable hormone pool or sensitivity to [Ca(2+)](i) changes did not underlie the ability of wortmannin and LY294002 to reduce the actions of GnRH. These results provide the first evidence for the presence and involvement of PI3K in GnRH-induced LH and GH release in any primary pituitary cell system. In gonadotrophs, the inhibitory action of PI3K on both sGnRH and cGnRH-II involves the attenuation of their evoked [Ca(2+)](i); in contrast, GnRH isoform-specific effects occur in somatotrophs.     

Modulation of Gonadotropin II Release by K+ Channel Blockers in Goldfish Gonadotropes: A Novel Stimulatory Action of 4-Aminopyridine

The effects of K+ channel blockers on basal gonadotropin II (GTH-II) release were examined in cultured goldfish gonadotropes. Tetraethylammonium (TEA) inhibited basal GTH-II release, whereas 4-aminopyridine (4-AP) increased basal release, although both K+ channel blockers generated increases in [Ca2+]i. Other K+ channel blockers had no significant effect on GTH-II release. We examined whether Ca2+ entry that arises from blockade of K+ channels by 4-AP mediates the secretory response. Secretion evoked by 4-AP was slightly reduced by TEA but was unaffected by reducing Ca2+ entry using either an inhibitor of Ca2+ channels, verapamil, or nominally Ca2+-free medium. In contrast, the Ca2+ signal evoked by 4-AP was largely blocked by Ca2+-free medium, as predicted by its inhibitory action on K+ channels. Together, these data suggest that the hormone release response to 4-AP is independent of entry of extracellular Ca2+. Finally, the mechanism of hormone release evoked by 4-AP appeared to be independent of mechanism(s) evoked by caffeine since 4-AP did not affect caffeine-evoked release and caffeine did not affect 4-AP evoked release. That both 4-AP and TEA generated Ca2+ signals but affected hormone release in either an extracellular Ca2+ independent (4-AP) or inhibitory (TEA) manner suggests that Ca2+ entry is linked to GTH-II secretion in a highly nonlinear fashion.     

Agonist-specific and sexual stage-dependent inhibition of gonadotropin-releasing hormone-stimulated gonadotropin and growth hormone release by ryanodine: relationship to sexual stage-dependent caffeine-sensitive hormone release

Differential utilization of intracellular Ca2+ stores with specific functional characteristics could be a potential mechanism for coupling various stimuli to specific cellular responses. In the goldfish pituitary, both gonadotropes and somatotropes possess multiple intracellular Ca2+ stores that are differentially coupled to agonist-evoked exocytosis. We investigated the role of ryanodine receptor/Ca2+-release channels (RyR) in basal and gonadotropin-releasing hormone (GnRH)-evoked hormone secretion from cultured gonadotropes and somatotropes using radioimmunoassay for gonadotropin (GTH-II) and growth hormone (GH). As is the case in vivo, the basal and evoked secretion of both hormones varied with seasonal reproductive status. GnRH-stimulated hormone release was three-fold higher in cells from sexually mature animals compared to those in a sexually regressed state. Nanomolar doses of ryanodine evoked significant GTH-II and GH secretion, suggesting that ryanodine-sensitive Ca2+ stores can couple to exocytosis in both cell types. In gonadotropes, 10 microM ryanodine abolished cGnRH-II-evoked GTH-II release in both sexually mature and sexually regressed fish, while sGnRH signalling was mediated by ryanodine-sensitive Ca2+ stores in cells from sexually regressed fish only. Ryanodine-sensitive Ca2+ stores in somatotropes were only involved in cGnRH-II-stimulated GH release during gonadal regression. In contrast, sGnRH-stimulated, but not cGnRH-II-stimulated, GH release was significantly reduced by 1 microM xestospongin C. Although hormone release stimulated by mobilizing caffeine-sensitive Ca2+ pools was also markedly seasonal, it was largely independent of ryanodine-sensitive Ca2+ stores. Ryanodine-sensitive Ca2+ stores in both cell types are not active downstream of ionomycin, BayK 8644, protein kinase C or cyclic adenosine monophosphate signalling pathways, suggesting difference from a classical Ca2+-induced Ca2+ release system. Ours study is the first to suggest that RyR2 may be involved in the seasonal plasticity of pituitary function, which may be related to cyclic changes observed in reproduction and growth.     

Opiate- and alpha 2-adrenoceptor-induced hyperpolarizations of locus ceruleus neurons in brain slices: reversal by cyclic adenosine 3':5'-monophosphate analogues

The purpose of this study was to investigate the ionic and second messenger mechanisms underlying the hyperpolarizations induced by the selective alpha 2-adrenoceptor agonist clonidine and the opiate agonist morphine in the locus ceruleus. Intracellular recordings were carried out in rat brain slices, and drugs at known concentrations were administered in the perfusate. The cyclic adenosine 3':5'-monophosphate (cAMP) analogues 8-bromo-cAMP and dibutyryl cAMP, while not altering basal activity, reversed the hyperpolarizations induced by clonidine or morphine. In contrast, administration of the parent compound adenosine failed to affect these responses. These results are consistent with previous biochemical studies suggesting that alpha 2-adrenergic and opiate agonists might signal their actions by reducing intracellular cAMP levels. Under manual voltage clamp, both clonidine and morphine elicited outward currents. The algebraic sum of the individual currents elicited by morphine and clonidine significantly exceeded the actual current elicited by their co-administration. This nonadditivity, as well as the observation that cAMP analogues reverse the morphine- and clonidine-induced hyperpolarizations, suggests that these compounds hyperpolarize locus ceruleus neurons through a shared ionic mechanism the activation of which might be signaled by a decrease in intracellular cAMP.     

Alpha2-adrenoreceptor activation inhibits LTP and LTD in the basolateral amygdala: involvement of Gi/o-protein-mediated modulation of Ca2+-channels and inwardly rectifying K+-channels in LTD

Activation of adrenoreceptors modulates synaptic transmission in the basolateral amygdala. Here, we investigated the effects of alpha2-adrenoreceptor activation on long-term depression and long-term potentiation in an in vitro slice preparation of the mouse basolateral amygdala. Field potentials and excitatory postsynaptic currents were evoked in the basolateral amygdala by stimulating the lateral amygdala. Norepinephrine (20 micro m) reduced synaptic transmission and completely blocked the induction of long-term potentiation and long-term depression. The alpha2-adrenoreceptor antagonist yohimbine (2 micro m) reversed this effect. The alpha2-adrenoreceptor agonist clonidine (10 micro m) mimicked the effects of norepinephrine. The Gi/o-protein inhibitor pertussis toxin (5 micro g/mL) reversed the effect of clonidine. Long-term depression was blocked in the presence of omega-conotoxin GVIA, but not omega-agatoxin IVA. Clonidine inhibited voltage-activated Ca2+ currents mediated via N- or P/Q-type Ca2+-channels. The inhibitory action of clonidine on long-term depression was reversed when inwardly rectifying K+-channels were blocked by Ba2+ (300 micro m). The present data suggest that alpha2-adrenoreceptor activation impairs the induction of long-term depression in the basolateral amygdala by a Gi/o-protein-mediated inhibition of presynaptic N-type Ca2+-channels and activation of inwardly-rectifying K+-channels.     

Intracellular calcium involvement in pituitary adenylate cyclase-activating polypeptide stimulation of growth hormone and gonadotrophin secretion in goldfish pituitary cells

The involvement of intracellular Ca(2+) stores and their regulatory mechanisms in mediating pituitary adenylate cyclase-activating polypeptide (PACAP) stimulation of growth hormone (GH) and maturational gonadotrophin (GTH-II) secretion from goldfish pituitary cells was investigated using a cell column perifusion system. Pretreatment with caffeine abolished the GH and GTH-II responses to PACAP. Dantrolene attenuated PACAP-elicited GTH-II release but did not affect the GH response, whereas ryanodine and 8-bromo-cADP ribose did not alter PACAP-induced GH and GTH-II release. Two endoplasmic/sarcoplasmic reticulum Ca(2+) ATPase (SERCA) inhibitors, thapsigargin and cyclopiazonic acid, augmented PACAP-induced GTH-II release; similarly, thapsigargin elevated GH responses to PACAP. Treatment with carbonyl cyanide m-chlorophenylhydrazone, a mitochondrial uncoupler, reduced PACAP-stimulated GH release; however, inhibition of the mitochondrial Ca(2+) uniport by Ru360 did not affect GH and GTH-II responses. The phosphatidyl inositol (PI)-specific phospholipase C (PLC) inhibitor ET-18-OCH(3) inhibited, whereas the phosphatidyl-choline (PC)-specific PLC inhibitor D609 enhanced, PACAP-stimulated GH and GTH-II responses. On the other hand, the IP(3) receptor blocker xestospongin D had no effect on PACAP-induced GTH-II response and potentiated the GH response. These results suggest that, despite some differences between GH and GTH-II cells, PACAP actions in both cell types generally rely on a caffeine-sensitive, but a largely ryanodine receptor-independent, mechanism. PC-PLC and some SERCA negatively modulate PACAP actions but mitochondrial Ca(2+) stores per se are not important. A novel PI-PLC mechanism, which does not involve the traditional IP(3)/Ca(2+) pathway, is also suggested.     

Three Native Somatostatin Isoforms Differentially Affect Membrane Voltage‐Sensitive Ion Currents in Goldfish Somatotrophs

Message encoding for three isoforms of somatostatin (SS) peptides, SS‐14, goldfish brain (gb)SS‐28 and [Pro²]SS‐14, are expressed in goldfish hypothalamus and pituitary tissues. All three native goldfish SSs are active in reducing basal and stimulated growth hormone (GH) responses in cultured goldfish pituitary cells, although with different potencies and efficacies. In the present study, we examined the effects of these three endogenous SSs on electrophysiological properties of goldfish somatotrophs and their physiological relevance. Voltage‐sensitive K⁺, Ca²⁺ and Na⁺ channels in identified goldfish somatotrophs in primary culture were isolated using whole‐cell, amphotericin B‐perforated patch‐clamp techniques. None of the three SSs affected Na⁺ currents but all three SSs increased maximal K⁺ current magnitude, with SS‐14 being the most effective. [Pro²]SS14 did not affect Ba²⁺ currents through voltage‐sensitive Ca²⁺ channels but SS14 decreased the magnitude of early and late Ba²⁺ currents, whereas gbSS‐28 reduced that of the late Ba²⁺ current. Under current‐clamp conditions, SS14 and gbSS28 attenuated evoked action potential magnitudes by 34% and 18%, respectively, although [Pro²]SS14 had no effects. However, all three SSs decreased basal intracellular Ca²⁺ levels ([Ca²⁺]_i) and suppressed basal GH release. These data suggest that, although the ability of SS‐14 and gbSS‐28 to decrease basal [Ca²⁺]_i and GH release can be explained, at least in part, by their attenuating effects on cell excitability and current flow through voltage‐sensitive Ca²⁺channels, [Pro²]SS14‐induced reduction in GH responses and [Ca²⁺]_i cannot be explained by changes in Ca²⁺ channel properties.     

Differential involvement of protein kinase C and protein kinase A in ghrelin-induced growth hormone and gonadotrophin release from goldfish (Carassius auratus) pituitary cells

Ghrelin (GRLN) and its receptor have been identified and characterised in goldfish brain and the pituitary, and recent evidence shows that goldfish (g)GRLN₁₉ induces both growth hormone (GH) and maturational gonadotrophin (LH) release through an extracellular Ca²⁺‐dependent mechanism in goldfish. To further understand the role of GRLN in hormone release, the present study examined the involvement of protein kinase C (PKC) and protein kinase A (PKA) in gGRLN₁₉‐induced GH and LH release and corresponding Ca²⁺ signals in primary cultures of goldfish pituitary cells. Treatments with PKC inhibitors, Bis‐II and Gö 6976, significantly reduced gGRLN₁₉‐induced GH and LH release and their corresponding intracellular Ca²⁺ signals in identified somatotrophs and gonadotrophs, respectively. gGRLN₁₉ was unable to further stimulate hormone release or Ca²⁺ signals when cells were pretreated with the PKC agonist, DiC8. PKA inhibitors, H‐89 and KT 5720, inhibited gGRLN₁₉‐induced LH release and Ca²⁺ signals in gonadotrophs but not GH release or Ca²⁺ signals in somatotrophs. Interestingly, pretreatment of pituitary cells with the adenylate cyclase activator forskolin potentiated gGRLN₁₉‐induced GH, but not LH, release, although it had no effect on intracellular Ca²⁺ signals in either cell type. Taken together, the results suggest that PKC is an important intracellular component in gGRLN₁₉‐induced GH and LH release, whereas PKA is involved in gGRLN₁₉‐elicited LH release. Furthermore, the PKA pathway potentiates gGRLN₁₉‐induced GH release via a Ca²⁺‐independent mechanism. Overall, the present study provides insight into the neuroendocrine regulation of GH and LH release by elucidating the mechanistic aspects of GRLN, a hormone involved in many critical physiological processes, including pituitary functions.     

Effects of noradrenergic agonists and antagonists on growth hormone secretion under gamma-hydroxybutyrate narco-analgesia in the rat

(1) The administration of gamma-hydroxybutyrate (GHB) has no effect on the hypothalamic concentration of norepinephrine (NE), dopamine (DA) and serotonin (5-HT) in the rat. (2) The injection of GHB induces a consistent growth hormone (immunoreactive GH) secretory episode, followed by basal levels for several hr. (3) During this period, the infusion of clonidine, an α-adrenergic agonist, elicits a dramatic GH increment, whereas basal levels are not modified by isoproterenol, a β-adrenergic agonist. (4) In animals pretreated with α- and β-adrenergic agonists, clonidine enhances the GH peak induced by GBH, which is clearly inhibited by isoproterenol. The effects of α- and β-adrenergic antagonists were also investigated in this study. (5) The data reported here could support the hypothesis that α-adrenergic receptors mediate the secretion of the GH-releasing hormone and that β-adrenergic receptors are involved in the release of somatostatin.     

The paradox of alpha 2 adrenergic regulation of prolactin (PRL) secretion. I. The PRL-releasing action of the alpha 2 receptor agonists

Systemic (IV) administration of the alpha 2 receptor agonist clonidine is known to stimulate secretion of PRL and growth hormone (GH) suggesting a stimulatory role of the central alpha 2 receptors in the regulation of the two hormones. The present work confirms this notion for GH but indicates that the alpha 2 agonists stimulate PRL release by a peripheral action not involving central alpha 2 receptors. This conclusion is based on the following findings: 1) The minimum effective IV dose of clonidine or UK 14304 was four times larger for activation of PRL than GH secretion and had already manifest extracentral effects (elevation of arterial BP). 2) Subcutaneous injection of UK 14304 (220 micrograms/kg) elevated plasma GH but not PRL levels indicating that an effective activation of the central alpha 2 receptors does not stimulate PRL release. 3) Peripherally acting alpha 2 agonists (p-aminoclonidine, oxymetazoline) had no effect on GH secretion but stimulated PRL release in a manner identical with the effect of clonidine or UK 14304. 4) The peripherally acting alpha 2 antagonist DG-5128 blocked only the PRL secretory response to UK 14304 whereas the peripherally and centrally active yohimbine blocked the PRL and GH responses.     

Seasonal Variation of Neuropeptide Y Actions on Growth Hormone and Gonadotropin-ll Secretion in the Goldfish: Effects of Sex Steroids

The effects of neuropeptide Y (NPY) on growth hormone (GH) and gonadotropin-II (GtH-II) release in different reproductive stages were studied using perfused pituitary fragments of female goldfish. The GH and GtH-II release responses to 5-min pulses of NPY were relatively small in sexually regressed fish (July), intermediate in recrudescent fish (December), and maximal in sexually mature (= prespawning) fish (May). To test if sex steroids can modulate NPY action, the effects of in vivo implantation of 17β-estradiol (E₂) and testosterone (T) (both at 100 μg/g dosage) on NPY-induced GH and GtH-II secretion were examined. In sexually regressed goldfish, implantation of T significantly enhanced NPY-induced GH and GtH-ll release from perfused pituitary fragments; implantation of E₂ potentiated the NPY-induced GtH-II, but not GH release. However, steroid implantation did not affect responses to NPY when this experiment was repeated using pituitaries from sexually mature fish. To test the hypothesis that steroids may act directly at the level of the pituitary to potentiate NPY action, pituitary fragments taken from sexually regressed goldfish were incubated with 100 nM T for 24 h, and the GH and GtH-ll responses to 5-min challenges of NPY assessed in the presence of T. Both GH and GtH-ll responses to NPY were not affected by treatment with T in vitro, suggesting that T does not act directly at the level of the pituitary. Since we have found that gonadotropin-releasing hormone (GnRH) in part mediates the effects of NPY on GH and GtH-ll release, the possibility that steroids may potentiate the actions of NPY on GnRH release were also examined. In sexually regressed fish, NPY did not alter GnRH release either from pituitary fragments or preoptic anterior hypothalamic slices. When fish were pretreated with E₂ and T by in vivo implantation, NPY significantly stimulated the release of GnRH. Taken together, these results demonstrate that: 1) there is a seasonal variation of NPY action on GH and GtH-ll release in the female goldfish; 2) sex steroids, especially T, potentiate the effects of NPY on GH and GtH-ll release in sexually regressed fish, when the endogenous steroid levels are low; and 3) the seasonality of NPY actions and the potentiation by steroids may be mediated, at least in part, by enhanced stimulation of GnRH release.     

Diverse Effects of Norepinephrine on Vasopressin Release may Reflect Modulation by Hypotonicity

Previous studies in this laboratory and others have demonstrated both stimulation and inhibition of vasopressin release by norepinephrine. In other regions of the central nervous system, diverse effects of norepinephrine reflect activation of different types of adrenergic receptors and the interaction of norepinephrine with other regulatory signals. In this study, the role of these two mechanisms in the diverse action of norepinephrine on vasopressin release were examined using organ-cultured explants of the hypothalamo-neurohypophyseal system. Receptor-specific adrenergic agonists were found to have different effects on vasopressin release, and their effects were dependent upon whether they were delivered in isotonic saline or distilled water. When delivered in isotonic saline at a concentration of 10^?5 M, norepinephrine and phenylephrine, an α₁-adrenergic agonist, increased vasopressin release (P< 0.005 and P< 0.025, respectively); isoproterenol (10^?5 M), a β-adrenergic agonist, did not alter vasopressin release; and clonidine, an α₂-adrenergic agonist, was ineffective at 10^?5 M, but stimulated vasopressin release at 10^?4 M (P<0.024). However, when these agonists were delivered in 10 μl of distilled water, their effects on vasopressin release were different. This amount of distilled water lowered the osmolality of the culture medium by 3 to 5 mosmol/kg H2O, but did not significantly alter vasopressin release. Vasopressin release was reduced below basal levels when norepinephrine (10^?5 M) or clonidine (10^?6 M) were delivered in distilled water (P<0.025), but it was not significantly different from basal when phenylephrine or isoproterenol (10^?5 M) were delivered in distilled water. These data support the hypothesis that osmotic signals modulate the effect of norepinephrine on vasopressin release, and suggest that, at least in part, this reflects modulation of the type of adrenergic receptor dominating the regulation of vasopressin release.     

GABAA receptor mediated elevation of Ca2+ and modulation of gonadotrophin-releasing hormone action in alphaT3-1 gonadotropes

gamma-amino butyric acid (GABA) is the major inhibitory neurotransmitter in the CNS, mediating fast inhibitory synaptic transmission, by activating GABAA receptors. However, these GABA-gated Cl- channels can also be excitatory, causing depolarization, and increasing Ca2+ entry via voltage-operated Ca2+ channels (VOCCs). Evidence exists for excitatory ionotropic GABA receptors in anterior pituitary cells, including gonadotropes, but these have not been directly characterized and their pharmacology remains controversial. Here we have measured the cytosolic Ca2+ concentration ([Ca2+]i) in alphaT3-1 gonadotropes, to test for expression of excitatory GABA receptors. The GABAA agonists, GABA and muscimol, both caused rapid, robust and dose-dependent increases in [Ca2+]i (EC50 values 2.7 and 1 microM), whereas the GABAB agonist, baclofen, did not. The GABAA antagonist, bicuculline, inhibited muscimol's effect, whereas the GABAB antagonist, phaclofen, did not. The neuroactive steroid 5alpha-pregnan-3alpha-ol-11,20-dione (an allosteric activator of GABAA receptors) increased [Ca2+]i, and this effect, like that of muscimol, was inhibited by picrotoxin. The muscimol effect on [Ca2+]i was blocked by the VOCC antagonist, nifedipine, or by Ca2+-free medium. When cells were pretreated with muscimol this increased the spike phase of the [Ca2+]i response to subsequent stimulation with gonadotropin-releasing hormone (GnRH). Similar amplification was seen in muscimol-pretreated cells stimulated with GnRH in Ca2+-free medium, but not when cells were pretreated with muscimol in Ca2+-free medium. The amplification was not, however, GnRH receptor-specific, because the spike response to ionomycin was also increased by muscimol pretreatment. These data provide the first direct evidence for expression of excitatory GABAA receptors, and the first demonstration of acute steroid effects, on GnRH-responsive pituitary cells. They also reveal a novel mechanism by which GABAA activation modulates GnRH action, raising the possibility that this may also influence gonadotrophin secretion from non-immortalized gonadotropes.     

Dopamine and full‐field illumination activate D1 and D2‐D5‐type receptors in adult rat retinal ganglion cells

Dopamine can regulate signal generation and transmission by activating multiple receptors and signaling cascades, especially in striatum, hippocampus, and cerebral cortex. Dopamine modulates an even larger variety of cellular properties in retina, yet has been reported to do so by only D1 receptor‐driven cyclic adenosine monophosphate (cAMP) increases or D2 receptor‐driven cAMP decreases. Here, we test the possibility that dopamine operates differently on retinal ganglion cells, because the ganglion cell layer binds D1 and D2 receptor ligands, and displays changes in signaling components other than cAMP under illumination that should release dopamine. In adult rat retinal ganglion cells, based on patch‐clamp recordings, Ca²⁺ imaging, and immunohistochemistry, we find that 1) spike firing is inhibited by dopamine and SKF 83959 (an agonist that does not activate homomeric D1 receptors or alter cAMP levels in other systems); 2) D1 and D2 receptor antagonists (SCH 23390, eticlopride, raclopride) counteract these effects; 3) these antagonists also block light‐induced rises in cAMP, light‐induced activation of Ca²⁺/calmodulin‐dependent protein kinase II, and dopamine‐induced Ca²⁺ influx; and 4) the Ca²⁺ rise is markedly reduced by removing extracellular Ca²⁺ and by an IP3 receptor antagonist (2‐APB). These results provide the first evidence that dopamine activates a receptor in adult mammalian retinal neurons that is distinct from classical D1 and D2 receptors, and that dopamine can activate mechanisms in addition to cAMP and cAMP‐dependent protein kinase to modulate retinal ganglion cell excitability. J. Comp. Neurol. 520:4032–4049, 2012. © 2012 Wiley Periodicals, Inc.     

Twenty-four hour growth hormone secretion in patients with panic disorder

BACKGROUND: Patients with panic disorder have blunted growth hormone (GH) responses to clonidine, suggesting subsensitivity of post-synaptic alpha(2)-adrenoreceptors, presumably in response to excessive central noradrenergic outflow. However, basal levels of GH release over a full circadian cycle have not been examined in panic. Reduced basal GH release would suggest an overall hypo-active GH system rather than a specific alpha-adrenergic abnormality. METHODS: To determine whether panic patients show reduced basal GH secretion, 20 patients and 12 healthy controls were studied. Blood samples were drawn every 15 min for 24 h and plasma was assayed for GH. Patients were restudied during successful treatment with alprazolam. Groups were compared on overnight and daytime GH secretion and circadian patterns of release. RESULTS: Patients showed normal levels on all measures of GH release. Treatment may have reduced nocturnal GH release slightly, but treated patients still did not differ from controls. The normal predominance of sleep over waking GH secretion was seen in both groups. CONCLUSIONS: Panic patients, in contrast to depressed patients, have normal somatotrophic axis activity when measured in a resting state over a full circadian cycle. GH dysregulation may only be evident in these patients in activation paradigms and has been most consistently demonstrated by challenges with the alpha(2)-noradrenergic agonist, clonidine.     

Pharmacological identification of the alpha-adrenergic receptor type which inhibits the beta-adrenergic activated adenylate cyclase system in cultured astrocytes

The adrenergic agonist norepinephrine can exert its influence on cell function by activating both alpha- and beta-adrenergic receptors. In astrocytes, the alpha-adrenergic receptor activity of norepinephrine is known to inhibit the cyclic AMP response elicited by its action at beta-adrenergic receptors. Pharmacological studies were conducted to identify the subtype of alpha-adrenergic receptor which mediates this inhibitory action. The alpha 2-adrenergic antagonist yohimbine potentiated the cyclic AMP response elicited by norepinephrine, whereas the alpha 1-adrenergic antagonist prazosin did not affect the response. The alpha 2-adrenergic agonist clonidine inhibited the cyclic AMP response elicited by the beta-adrenergic agonist isoproterenol and this inhibition could be blocked by yohimbine but not by prazosin. In contrast, the alpha 1-adrenergic agonist phenylephrine did not inhibit the cyclic AMP response to isoproterenol. These studies indicate that the inhibitory action of norepinephrine is mediated by its action at alpha 2-adrenergic receptors.