Beta 1-adrenergic regulation of the GT1 gonadotropin-releasing hormone (GnRH) neuronal cell lines: stimulation of GnRH release via receptors positively coupled to adenylate cyclase.

The release of GnRH evoked by norepinephrine (NE) was studied in GT1 GnRH neuronal cell lines in superfusion and static cultures. GnRH release from static cultured GT1-7 cells was stimulated by NE in a dose-dependent fashion. This effect was mimicked by the nonsubtype-selective beta-adrenergic agonist isoproterenol and blocked by the beta-adrenergic antagonist propranolol and the beta 1-adrenergic subtype-specific antagonist CGP 20712A. However, the stimulation of GnRH release by NE was not affected by the beta 2-, alpha-, alpha 1-, or alpha 2-adrenergic antagonists ICI 118.551, phentolamine, prazosin, or yohimbine, respectively. Superfusion of GT1-1 cells with NE for 60-100 min resulted in rapid and sustained increases in GnRH secretion. The NE-stimulated GnRH release showed a higher amplitude and longer duration than the spontaneous GnRH pulses characteristic of GT1-1 cells. In parallel to the stimulation of GnRH release, NE also rapidly increased (first observed at 60 sec) the intracellular concentration of cAMP in isobutylmethylxanthine-pretreated GT1-1 and GT1-7 cells in a dose-dependent fashion. The stimulation of intracellular cAMP concentration was also mimicked by isoproterenol and blocked by propranolol and CGP 20712A. In addition, GT1 cells express beta 1- but not beta 2-adrenergic receptor mRNA, as probed by Northern blot analysis. These results demonstrate a direct stimulatory effect of NE on GnRH neurons. The pharmacological evidence and the mRNA analysis are consistent with NE acting through a beta 1-adrenergic receptor positively coupled to adenylate cyclase.     

Role of cAMP signaling in the mediation of dopamine-induced stimulation of GnRH secretion via D1 dopamine receptors in GT1-7 cells

Pharmacologically increasing cyclic adenosine monophosphate (cAMP) levels in GT1 gonadotropin-releasing hormone (GnRH) cell lines increased the secretion of GnRH. Dopamine (DA) increased the GnRH secretion in GT1 cells via a DA receptor positively coupled to adenylate cyclase. We then asked whether inhibition of the DA-induced increase in cAMP would block the stimulatory effect of DA on GnRH release. Expression of the cAMP-specific phosphodiesterase (PDE4D1) was used in a genetic approach to inhibit the DA-induced increase in cAMP levels. Cells were infected with an adenovirus vector (Ad) expressing PDE4D1 (PDE-Ad) or, for controls, with an empty Ad (Null-Ad). Infection with the PDE-Ad completely blocked the forskolin-induced stimulation of GnRH secretion and [Ca2+]i and decreased the majority of the release of cAMP into the culture medium. In contrast, although PDE-Ad infection blocked virtually all of the DA-induced increase in extracellular cAMP, the release of GnRH and the increase in [Ca2+]i were only delayed for approximately 15 min. GT1 cells express the D1 DA receptor which is positively coupled to adenylate cyclase but not the D5 DA receptor. These data suggest that the initial phase of the DA-induced secretion of GnRH is dependent on an increase in cAMP levels. However, it appears that an additional non-cAMP-regulated signaling pathway is involved in the stimulation of GnRH release via the D1 DA receptor.     

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

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

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.     

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)     

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

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

Hypothalamic D2 receptors mediate the preferential release of somatostatin-28 in response to dopaminergic stimulation

We have studied the effect of dopamine (DA) together with agonist and antagonist drugs of varying specificity on the release of immunoreactive forms of somatostatin (SS) from the perfused, adult rat hypothalamus in vitro. Levels of SS increased from 14.7 +/- 3.7 pg (mean +/- SE) under basal conditions to 137 +/- 23.0 pg after exposure to 10(-6) M DA. This dopaminergic effect was mimicked by the specific D2 agonists bromocriptine (10(-7) M) and LY 171555 (10(-6) M) but not by the D1 agonist SKF 38393A (10(-6) M). The stimulatory action of DA (10(-6) M) was blocked by the active (d) but not the inactive (l) isomer of butaclamol (10(-7) M). Similar blockade was achieved with the specific D2 antagonists metoclopramide (10(-8) M) and domperidone (10(-8) M), whereas the D1 antagonist SCH 23390 partially blocked the stimulation of DA but only when used at X100 greater concentration (10(-6) M). SCH 23390 (10(-8) M) did not affect the dopaminergic stimulation of SS release. HPLC characterization of the immunoreactive forms of SS yielded two peaks which corresponded to SS-28 and SS-14. The ratio of these forms varied significantly under different conditions. In the basal state the ratio of SS-28 to SS-14 was 1:4.4; in response to stimulation with DA, the ratio was 1:1.7 and in response to depolarization with 60 mM K+ the ratio was 1:3.1. In conclusion, the stimulatory action of DA on SS release is mediated via hypothalamic D2 receptors. Furthermore dopaminergic stimulation increases the molar ratio of SS-28 to SS-14 in the total immunoreactive SS which is released.     

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

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

Suppression of melatonin biosynthesis in the chicken pineal gland by retinally perceived light - involvement of D1-dopamine receptors.

In this study the role of retinal dopamine (DA) receptors in the light-induced suppression of melatonin biosynthesis in the chicken pineal gland was examined. Exposure of dark-adapted chickens to low intensity light (4 lux) at night significantly decreased the activity of serotonin N-acetyltransferase (AA-NAT; the penultimate and key regulatory enzyme in melatonin production) and melatonin content in the pineal gland. This suppressive action of light was blocked by intraocular (i.oc.) administration of SCH 23390 (a selective antagonist of D1-DA receptors), but was not affected by sulpiride (a selective antagonist of D2-DA receptors). Injection of DA (i.oc.) to dark-adapted chickens significantly decreased pineal AA-NAT activity and melatonin content in a dose- and time-dependent manner. The action of DA was mimicked by selective agonists of D1-DA receptors, SKF 38393 and SKF 81297, and non-hydrolyzable analogs of cyclic AMP (cAMP), dibutyryl-cAMP and 8-bromo-cAMP. However, i.oc. administration of quinpirole, a selective agonist of D2-DA receptors, did not modify pineal AA-NAT activity. In contrast, quinpirole potently decreased nocturnal AA-NAT activity in the retina. Systemic administration of SCH 23390 to chickens blocked the i.oc. DA-evoked decline in nighttime pineal AA-NAT activity, whereas sulpiride was ineffective. These findings indicate that light activation of retinal dopaminergic neurotransmission, with concomitant stimulation of D1-DA receptors positively coupled to the cAMP generating system, plays an important role in a cascade of events regulating pineal activity.     

Suppression of melatonin biosynthesis in the chicken pineal gland by retinally perceived light - involvement of D1-dopamine receptors.

In this study the role of retinal dopamine (DA) receptors in the light-induced suppression of melatonin biosynthesis in the chicken pineal gland was examined. Exposure of dark-adapted chickens to low intensity light (4 lux) at night significantly decreased the activity of serotonin N-acetyltransferase (AA-NAT; the penultimate and key regulatory enzyme in melatonin production) and melatonin content in the pineal gland. This suppressive action of light was blocked by intraocular (i.oc.) administration of SCH 23390 (a selective antagonist of D1-DA receptors), but was not affected by sulpiride (a selective antagonist of D2-DA receptors). Injection of DA (i.oc.) to dark-adapted chickens significantly decreased pineal AA-NAT activity and melatonin content in a dose- and time-dependent manner. The action of DA was mimicked by selective agonists of D1-DA receptors, SKF 38393 and SKF 81297, and non-hydrolyzable analogs of cyclic AMP (cAMP), dibutyryl-cAMP and 8-bromo-cAMP. However, i.oc. administration of quinpirole, a selective agonist of D2-DA receptors, did not modify pineal AA-NAT activity. In contrast, quinpirole potently decreased nocturnal AA-NAT activity in the retina. Systemic administration of SCH 23390 to chickens blocked the i.oc. DA-evoked decline in nighttime pineal AA-NAT activity, whereas sulpiride was ineffective. These findings indicate that light activation of retinal dopaminergic neurotransmission, with concomitant stimulation of D1-DA receptors positively coupled to the cAMP generating system, plays an important role in a cascade of events regulating pineal activity.     

Dopamine inhibits na,h-exchanger via D1-like receptor-mediated stimulation of protein kinase a in renal proximal tubules

Dopamine causes natriuresis and diuresis via activation of D1-like receptors located in the renal proximal tubules. It is reported that this response to dopamine results from the inhibition of Na,H-exchanger and Na,K-ATPase. Earlier studies have suggested a role of protein kinase A (PKA) in the inhibition of Na,H-exchanger, however, the effect of dopamine or the dopamine receptor subtype responsible for the stimulation of PKA has not been reported. Present study was designed to examine the effect of dopamine and D1-like receptor agonist, SKF 38393, on the stimulation of PKA activity in rat renal proximal tubules. Dopamine and SKF 38393 (1 nM - 1 microM) caused stimulation of PKA activity, an effect which was antagonized by a D1-like receptor antagonist, SCH 23390 (10 microM). Stimulation of PKA activity was also seen with forskolin and di-butyryl cAMP. We also observed that dopamine and SKF 38393 inhibited Na,H-exchanger activity in the proximal tubules. This response was blocked by SCH 23390 and Rp-cAMPS triethylamine, a selective inhibitor of PKA. Similarly, forskolin and di-butyryl cAMP inhibited Na,H-exchanger activity. The data provide direct evidence showing that dopamine, through the activation of D1-like receptors stimulates PKA activity which in turn inhibits Na,H-exchanger in the proximal tubules.     

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.     

Tetrodotoxin-insensitive relaxation of coronary arterial smooth muscle to electrical stimulation: possible involvement of a dopaminergic mechanism

Experiments were designed to determine the mechanism by which electrical stimulation causes tetrodotoxin-insensitive relaxation in isolated arteries. Rings of left anterior descending coronary arteries of dogs, pigs and calves were suspended in organ chambers between platinum electrodes. Experiments were performed after treatment with phenoxybenzamine and in the presence of propranolol. Calcium-free solution and calcium antagonists reduced the relaxation. Chemical denervation with 6-hydroxydopamine reduced the relaxation induced by electrical stimulation; in the presence of pargyline, the inhibitor of monoamine oxidase, it was virtually abolished. The nonselective dopaminergic antagonist droperidol and the selective DA1-dopaminergic antagonist SKF R83566 caused a concentration-dependent inhibition of the relaxation; the DA2-dopaminergic antagonist domperidone was ineffective. High concentrations of dopamine induced relaxation of the coronary smooth muscle; the relaxation was inhibited by SKF R83566 but not by droperidol. These results suggest that electrical stimulation causes relaxation by liberating an endogenous vasodilator substance, which acts on DA1-dopaminergic receptors of the coronary smooth muscle.     

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

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

3',5'-cyclic adenosine monophosphate augments intracellular Ca2+ concentration and gonadotropin-releasing hormone (GnRH) release in immortalized GnRH neurons in an Na+ -dependent manner

In GT1-7 cells, cAMP increases the intracellular Ca2+ concentration ([Ca2+](i)) through activation of the voltage-gated Ca2+ channels, thereby facilitating GnRH release. To activate these channels, the membrane potential must be depolarized. In the present study we hypothesize that cAMP depolarizes the cells by increasing the membrane Na+ permeability, as in the case of somatotrophs and pancreatic beta-cells. To examine this, we analyzed [Ca2+](i) and [Na+](i) in GT1-7 cells by an intracellular ion-imaging technique along with cAMP assay by RIA. Forskolin, a direct activator of adenylyl cyclase, increased [Ca2+](i) and [Na+](i) via cAMP formation. The forskolin-induced increase in [Ca2+](i) depended on the presence of Ca2+ and Na+ in the extracellular solution. This response was blocked by the voltage-gated Ca2+ channel blocker, nifedipine; the nonselective cation channel blocker, gadolinium (Gd3+); and the cyclic nucleotide-gated channel blocker, l-cis-diltiazem. In contrast, the forskolin-induced increase in [Na+](i) depended only on extracellular Na+, not on Ca2+. Gd3+ and l-cis-diltiazem also blocked the increase in [Na+](i). Furthermore, the forskolin-induced increase in GnRH release was blunted in both low Ca2+ and low Na+ media. The results indicate that cAMP increases the membrane Na+ permeability, probably through nonselective cation channels on GT1-7 cells, thereby promoting GnRH release.     

Role of the cAMP signaling pathway in the regulation of gonadotropin-releasing hormone secretion in GT1 cells

We studied the signaling pathways coupling gonadotropin-releasing hormone (GnRH) secretion to elevations in cAMP levels in the GT1 GnRH-secreting neuronal cell line. We hypothesized that increased cAMP could be acting directly by means of cyclic nucleotide-gated (CNG) cation channels or indirectly by means of activation of cAMP-dependent protein kinase (PKA). We showed that GT1 cells express the three CNG subunits present in olfactory neurons (CNG2, -4.3, and -5) and exhibit functional cAMP-gated cation channels. Activation of PKA does not appear to be necessary for the stimulation of GnRH release by increased levels of cAMP. In fact, pharmacological inhibition of PKA activity caused an increase in the basal secretion of GnRH. Consistent with this observation activation PKA inhibited adenylyl cyclase activity, presumably by inhibiting adenylyl cyclase V expressed in the cells. Therefore, the stimulation of GnRH release by elevations in cAMP appears to be the result of depolarization of the neurons initiated by increased cation conductance by cAMP-gated cation channels. Activation of PKA may constitute a negative-feedback mechanisms for lowering cAMP levels. We hypothesize that these mechanisms could result in oscillations in cAMP levels, providing a biochemical basis for timing the pulsatile release of GnRH.     

Dopamine D2-receptor messenger RNA is differentially regulated by dopaminergic agents in rat anterior and neurointermediate pituitary

Hypothalamic dopamine (DA), acting at DA D2-receptors (D2-R) on pituitary target cells, mediates peptide release and biosynthesis of rat pituitary anterior lobe (AL) prolactin, and neurointermediate lobe (NIL) pro-opiomelanocortin (POMC). We were interested in determining if dopamine agonists and antagonists were capable of modifying D2-R gene expression in these pituitary cells. Utilizing the recently published sequence of the rat D2-R, we isolated a rat D2-R cDNA clone by polymerase chain reaction, and have synthesized RNA probes to quantitate levels of D2-R mRNA by solution hybridization/nuclease protection assay. We report here that 5-day administration of the DA antagonist haloperidol led to significant increases in both D2-R mRNA and POMC mRNA in the NIL; the DA agonist bromocriptine caused a significant decrease in NIL POMC mRNA with no parallel change in D2-R mRNA. In contrast, no significant changes in D2-R mRNA in AL were observed following treatment with either the DA agonist or antagonist. These data provide evidence for tissue-specific regulation of D2-R mRNA in response to dopaminergic manipulation.     

DOPAMINE INHIBITS Na,H-EXCHANGER VIA D1-LIKE RECEPTOR-MEDIATED STIMULATION OF PROTEIN KINASE A IN RENAL PROXIMAL TUBULES

Dopamine causes natriuresis and diuresis via activation of D₁-like receptors located in the renal proximal tubules. It is reported that this response to dopamine results from the inhibition of Na,H-exchanger and Na,K-ATPase. Earlier studies have suggested a role of protein kinase A (PKA) in the inhibition of Na,H-exchanger, however, the effect of dopamine or the dopamine receptor subtype responsible for the stimulation of PKA has not been reported. Present study was designed to examine the effect of dopamine and D₁-like receptor agonist, SKF 38393, on the stimulation of PKA activity in rat renal proximal tubules. Dopamine and SKF 38393 (1 nM – 1 μM) caused stimulation of PKA activity, an effect which was antagonized by a D₁-like receptor antagonist, SCH 23390 (10 μM). Stimulation of PKA activity was also seen with forskolin and di-butyryl cAMP. We also observed that dopamine and SKF 38393 inhibited Na,H-exchanger activity in the proximal tubules. This response was blocked by SCH 23390 and Rp-cAMPS triethylamine, a selective inhibitor of PKA. Similarly, forskolin and di-butyryl cAMP inhibited Na,H-exchanger activity. The data provide direct evidence showing that dopamine, through the activation of D₁-like receptors stimulates PKA activity which in turn inhibits Na,H-exchanger in the proximal tubules.     

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)