GABA inhibition of cyclic AMP production in immortalized GnRH neurons is mediated by calcineurin-dependent dephosphorylation of adenylyl cyclase 9

The neurotransmitter gamma-aminobutyric acid (GABA) is an important modulator of gonadotropin-releasing hormone (GnRH), and consequently of reproduction. GABA, acting via ionotropic GABAA receptors, exerts a biphasic effect on GnRH secretion in immortalized GnRH cells. The initial increase in GnRH secretion is triggered by a sharp rise in [Ca2+]i, while the progressive decline of GnRH levels that follows is paralleled by reduced levels of intracellular cAMP. The experiments described here were designed to explore the potential signaling pathways involved in this novel GABAA ionotropic inhibition of cAMP synthesis in GT1-7 cells. Using RT-PCR and real-time PCR, we found that GT1-7 cells express 8 of 9 known membrane adenylyl cyclase (AC) isoforms, including a large proportion of AC3 and AC9, as well as AC5 and AC6, all of which are negatively regulated by increases in [Ca2+]i. In contrast, isoforms of AC that are positively regulated by [Ca2+]i were barely detectable (AC1) or undetectable (AC8). Pharmacological activation of L-type voltage-operated calcium channels with BayK 8644 produced a decrease in [cAMP]i similar to that induced by GABA, while blocking these calcium channels with verapamil reversed the effect of GABA on cAMP synthesis. Furthermore, blocking calcineurin with deltamethrin, FK-506 or cyclosporin A blocked the inhibitory effect of GABA on [cAMP]i, supporting the involvement of AC9 in this effect. In addition, blocking Ca2+/calmodulin-dependent protein kinase II (CamKII) with KN-62 partially reversed the action of GABA, suggesting that AC3 may also be involved in this effect. Finally, GABA increased phosphatase activity in a calcium-dependent manner, an effect blocked by calcineurin inhibitors. Collectively, our results show that the ionotropic action of GABA via the activation of GABAA receptors can decrease AC activity in immortalized GnRH neurons, and that the effect of GABA appears to be mediated by a transient increase in [Ca2+]i followed by activation of calcineurin and CamKII, leading to dephosphorylation of AC9 and phosphorylation of AC3, respectively, and subsequently reducing the synthesis of cAMP.     

Structure, function and transforming potential of the epidermal growth factor receptor

We have examined the pharmacology of the voltage-sensitive Ca2+ channels (VSCCs) that mediate gonadotropin secretion from primary cultures of rat pituitary cells, stimulated by either cell depolarization or by binding of gonadotropin-releasing hormone (GnRH). We also measured single-cell [Ca2+]i transients using fura-2 in gonadotropes identified by a reverse hemolytic plaque assay employing an antiserum to luteinizing hormone (LH). Cell depolarization evoked by either 50 mM K+ or 30 microM veratridine induced 2- to 6-fold increases in gonadotropin secretion over basal levels. GnRH caused 6- to 20-fold increases in follicle-stimulating hormone (FSH) and LH secretion, respectively, with maximal stimulation at 100 nM GnRH. K(+)- or GnRH-induced FSH release was largely prevented by co-incubation with 1 mM CdCl. Tetrodotoxin (TTX, 5 microM) prevented the veratridine-, but not the K(+)- or GnRH-induced, stimulation of FSH secretion. Nitrendipine (Ntd, 1 microM) produced 35-50% inhibition (NS) of both FSH and LH release stimulated by either 50 mM K+ or 100 nM GnRH. Ntd also inhibited the K(+)-induced [Ca2+]i rise (greater than 90%), as well as the secondary, plateau phase of the GnRH-induced elevation of [Ca2+]i (100% inhibition). Omega-conotoxin (omega-CgTx, 100 nM) partially suppressed FSH and LH release (NS) due to both K+ (33% each) and GnRH (44% and 18%, respectively). omega-CgTx showed variable effects on [Ca2+]i transients evoked by K+ or GnRH ranging from clear inhibition to no effect. We conclude that influx of extracellular Ca2+ is one of several fundamental events underlying the depolarization- or receptor-activated release of LH and FSH, and that this influx can be inhibited by dihydropyridine-sensitive ('L') Ca2+ channels. Two classes of L-channels may exist in gonadotropes, that differ in their sensitivity to omega-CgTx.     

Gonadotropin-releasing hormone-induced Ca2+ transients in single identified gonadotropes require both intracellular Ca2+ mobilization and Ca2+ influx.

We examined the effects of gonadotropin-releasing hormone (GnRH) on the intracellular free Ca2+ concentration ([Ca2+]i) in single rat anterior pituitary gonadotropes identified by a reverse hemolytic plaque assay. Concentrations of GnRH greater than 10 pM elicited increases in [Ca2+]i in identified cells but not in others. In contrast, depolarization induced by 50 mM K+ increased [Ca2+]i in all cells. Ca2+ transients induced by GnRH exhibited a complex time course. After an initial rapid rise, the [Ca2+]i fell to near basal levels only to be followed by a secondary extended rise and fall. Analysis of the Ca2+ transients on a rapid time base revealed that responses frequently consisted of several rapid oscillations in [Ca2+]i. Removal of extracellular Ca2+ or addition of the dihydropyridine Ca2+-channel blocker nitrendipine completely blocked the secondary rise in [Ca2+]i but had no effect whatsoever on the initial spike. Nitrendipine also blocked 50 mM K+-induced increases in [Ca2+]i in identified gonadotropes. The secondary rise induced by GnRH could be enhanced by a phorbol ester in a nitrendipine-sensitive fashion. Multiple spike responses to GnRH stimulation of the same cell could only be obtained if subsequent Ca2+ influx was permitted either by allowing a secondary rise to occur or by producing a Ca2+ transient by depolarizing the cells with 50 mM K+. It therefore appears that the response to GnRH consists of an initial phase of Ca2+ mobilization, probably mediated by inositol trisphosphate, and a subsequent phase of Ca2+ influx through nitrendipine-sensitive Ca2+ channels that may be activated by protein kinase C. The relative roles of these phases in the control of gonadotropin secretion are discussed.     

Stimulation of luteinizing hormone release by gamma-aminobutyric acid (GABA) agonists: mediation by GABAA-type receptors and activation of chloride and voltage-sensitive calcium channels

The mechanism by which gamma-aminobutyric acid (GABA) stimulates the release of LH was analyzed in cultured female rat pituitary cells. In 3-h incubations, GABA (1-100 microM) caused a dose-dependent increase in LH release, with the maximal response about 16% of that evoked by 10 nM GnRH. GABA action was independent of the GnRH receptor, since 1 microM GnRH antagonist [( N-acetyl-D-p-Cl-Phe1,2,D-Trp3,D-Lys6,D-Ala10] GnRH), which completely inhibits GnRH action, did not affect the response to GABA. In studies on the effects of GABA receptor agonists and antagonists, 4,5,6,7-tetrahydoisoxazolo-[5,4-c]pyridin-3(2H)-one (THIP) and muscimol (GABAA agonists) gave similar response patterns, with the same maximal stimulation as GABA but much higher potencies. In contrast, the GABAB receptor agonist baclofen did not stimulate LH release. The GABAA receptor antagonist SR95531 caused dose-dependent inhibition of the LH-releasing effects of GABA and muscimol (10 microM), with complete blockade at 10 microM SR95531. T-Butylbicyclophosphorothionate, an inhibitor of the GABAA receptor-associated chloride channel, also dose-dependently reduced the releasing effect of 100 microM GABA. These results indicate that GABA action is mediated by the chloride channel-associated GABAA receptor. However, the other GABAA receptor antagonists, including bicuculline, picrotoxin, and strychnine, did not attenuate the LH-releasing effect of 100 microM GABA in concentrations up to 100 microM, suggesting that GABA action is mediated by nonclassical GABAA receptors. Incubation in the presence of nifedipine (1 microM) or in calcium-free medium inhibited the LH-releasing action of GABA, indicating that calcium influx through voltage-sensitive calcium channels (VSCC) is required for GABA-induced LH release. Such entry of Ca2+ would result from activation of VSCC by depolarization due to the increased Cl- conductance caused by GABAA receptor activation. In cell perfusion studies, the actions of GABA and muscimol were attenuated or abolished after repetitive stimulation, consistent with desensitization of the GABA receptors. These findings have demonstrated that the stimulation of LH release by GABA is independent of GnRH action, occurs via binding to nonclassical GABAA receptors, which rapidly desensitize, and is mediated by the activation of VSCC.     

Endogenous GABA release inhibits the firing of adult gonadotropin-releasing hormone neurons

The effect of endogenous gamma-aminobutyric acid (GABA)(A) receptor-mediated signaling on the excitability of adult male and female GnRH neurons was examined using gramicidin perforated-patch electrophysiology in GnRH-LacZ and GnRH-GFP (green fluorescent protein) transgenic mouse models. In both lines of mice, approximately 80% of GnRH neurons (n = 42) responded to the selective GABA(A) receptor antagonist bicuculline (20 microm) with a rapid and reversible membrane depolarization and/or increase in firing rate. Approximately 16% of GnRH neurons gave no response, and two neurons were inhibited by bicuculline. The same depolarizing responses (78%) were obtained from adult gonadectomized GnRH-GFP mice. The depolarizing response to bicuculline persisted in the presence of tetrodotoxin, demonstrating that even action potential-independent GABA release was acting to reduce GnRH neuron membrane potential. These observations show that endogenous GABA signaling through the GABA(A) receptor exerts a powerful net inhibitory effect upon the excitability of mature GnRH neurons.     

GABA(B) receptors in anterior pituitary cells. Mechanism of action coupled to endocrine effects

The activation of pituitary GABA(B) receptors by the specific agonist baclofen inhibits pituitary hormone secretion in vitro. Here we studied the mechanism of action of GABA(B) receptors in rat adenohypophysis. Anterior pituitary cells were obtained by trypsinization and were either plated for hormonal studies and cAMP determination or incubated in FURA 2AM for calcium measurements. Baclofen (BACL: 1 x 10(-5) M) significantly inhibited basal and thyrotropic releasing hormone (TRH)-stimulated (1 x 10(-7) M) PRL secretion in anterior pituitary cells from proestrous rats. In the presence of pertussis toxin (PTX: 150 ng/ml, 20 h), which leads to the uncoupling of the G(i/o)-protein from the receptor, both effects of BACL were abolished while the effect of dopamine (DA: 1 x 10(-8) M), used as an inhibitory control, was reduced from 70 to 25%. PTX also reversed BACL-induced inhibition of gonadotropin-releasing hormone (GnRH)-elicited luteinizing hormone (LH) secretion in anterior pituitary cells from 15-day-old female rats. In addition, though working in a pituitary mixed cell population, in which only some cell types possess GABA(B) receptors, BACL (1 x 10(-5) M) attenuated the forskolin-induced (0.5 microM) increase in cAMP. This effect was prevented by co-incubation with the antagonist 2 hydroxysaclofen and by preincubation with PTX. BACL (5 x 10(-5) M) and DA (5 x 10(-7) M) inhibited basal intracellular calcium concentrations ([Ca(2+)](i)) in pituitary cells and the effect of the latter was significantly stronger. The effect of BACL on [Ca(2+)](i) was abolished after preincubation with PTX. In the presence of the potassium channel blocking agents barium (200 microM and 1 mM) and tetraethylammonium (10 mM), BACL was still able to inhibit [Ca(2+)](i). Blockade of voltage-sensitive calcium channels (VSCC) with either verapamil (5 x 10(-6) M) or nifedipine (1 x 10(-6) M) completely abolished the effect of BACL on [Ca(2+)](i). In the presence of 12.5 mM potassium concentration baclofen significantly inhibited [Ca(2+)](i). In conclusion, our results describe the negative coupling of adenohypophyseal GABA(B) receptors to VSCC through PTX-sensitive G-proteins. These characteristics suggest a resemblance of these receptors to the typical presynaptic GABA(B) sites described in the central nervous system.     

Enhancement by veratridine and 60 mM KCl of vasopressin-induced 3',5'-cyclic adenosine monophosphate production and cellular free calcium concentration in rat renal papillary collecting tubule cells in culture

The effect of extracellular calcium (Ca2+) on the cellular action of arginine vasopressin (AVP) was examined using depolarizing agents in rat renal papillary collecting tubule cells in culture. One-hour exposure of cells to veratridine enhanced AVP-induced cAMP production in a dose-dependent manner. This enhancement by veratridine of cellular cAMP production in response to AVP was totally blunted by cotreatment with 5 X 10(-4) M verapamil, 3 X 10(-3) M cobalt, or Ca2+-free medium containing 1 X 10(-3) M EGTA. These agents block cellular Ca2+ uptake by different mechanisms. Similarly, 60 mM KCl enhanced AVP-induced cAMP production, and this effect was blocked by pretreatment with verapamil, cobalt or Ca2+-free medium containing 1 X 10(-3) M EGTA. When cellular free Ca2+ concentrations [Ca2+]i were measured by the fluorescence dye fura-2, both 1 X 10(-4) M veratridine and 60 mM KCl significantly increased [Ca2+]i from 67.6 to 141.8 nM and from 74.6 to 166.2 nM, respectively. Such rises in [Ca2+]i depended on extracellular Ca2+ since the increase in [Ca2+]i was completely blocked in Ca2+-free medium or in the presence of 3 X 10(-3) M cobalt. In addition, veratridine and 60 mM KCl significantly augmented the AVP-induced increase in [Ca2+]i. The possible mechanisms by which depolarizing agents induce cellular Ca2+ mobilization include the opening of voltage-sensitive Ca2+ channels in the plasma membrane. The present results indicate that veratridine and 60 mM KCl enhance AVP-induced cAMP production and cellular free Ca2+ concentration through cellular Ca2+ uptake in renal papillary collecting tubule.     

Potassium depolarization elevates cytosolic free calcium concentration in rat anterior pituitary cells through 1,4-dihydropyridine-sensitive, omega-conotoxin-insensitive calcium channels

Changes in membrane potential may influence Ca2+-dependent functions through changes in cytosolic free calcium concentration [( Ca2+]i). This study characterized pharmacologically those voltage-dependent Ca2+ channels in normal rat anterior pituitary cells that are involved in the elevation of [Ca2+]i upon high potassium-induced membrane depolarization. The [Ca2+]i was monitored directly by means of the intracellularly trapped fluorescent indicator fura-2. The addition of K+ (6-100 mM) increased [Ca2+]i in a concentration-dependent manner. The fluorescent signal reached a peak within seconds and then decayed to form a new elevated plateau. K+ at the highest concentration used (100 mM) raised [Ca2+]i by about 450 nM. The K+-induced increase in [Ca2+]i was absent in a Ca2+-free medium. BAY K 8644, a 1,4-dihydropyridine Ca2+ channel agonist, also caused an increase in [Ca2+]i. The maximum response in [Ca2+]i upon stimulation with BAY K 8644 (100 nM) was about 40 nM. The half-maximally effective concentration of BAY K 8644 (100 nM) was about 20 nM. The response in [Ca2+]i upon BAY K 8644-stimulation was abolished in a Ca2+-free medium. Predepolarization with various K+ concentrations enhanced the effect of BAY K 8644 (1 microM) on [Ca2+]i. Pretreatment with BAY K 8644 (1 microM) enhanced the response in [Ca2+]i induced by K+ (25 mM). The addition of Mg2+ (30 mM) and nifedipine (1 microM) lowered the resting [Ca2+]i by about 40 and 20 nM, respectively. Mg2+, nifedipine, nimodipine, G? 5438, verapamil, and diltiazem inhibited the K+ (25 mM)-induced increase in [Ca2+]i; the order of potency (and half-maximally inhibitory concentrations) were nimodipine = G? 5438 = nifedipine (approximately 100 nM) greater than verapamil (900 nM) greater than diltiazem (greater than 10 microM) greater than Mg2+ (6 mM). Omega-Conotoxin (100 nM) did not inhibit the K+ (25 mM)-induced increase in [Ca2+]i. These data demonstrate that, over a wide range, membrane depolarization induced by high potassium concentration is indeed associated with increases in [Ca2+]i in normal rat anterior pituitary cells. This elevation of [Ca2+]i is mainly due to an influx of Ca2+ through 1,4-dihydropyridine-sensitive, omega-conotoxin-insensitive calcium channels (L-type).     

Multiple intracellular pathways for regulation of chloride secretion in cultured pig tracheal submucosal gland cells.

Tracheal submucosal glands are of great relative importance in the secretion of chloride and water to the airway lumen. This study aimed to examine whether the cystic fibrosis transmembrane conductance regulator (CFTR) is involved in cyclic adenosine monophosphate (cAMP) or Ca2+-activated Cl- secretion. Regulation of Cl- secretion in cell cultures derived from pig tracheal submucosal gland acini was investigated by X-ray microanalysis. With or without preincubation with CFTR antisense oligodeoxynucleotide (5 microM). A significant decrease in cellular Cl and K concentration was induced by 5 mM 8-bromo-adenosine 3': 5'-cyclic monophosphate (8-bromo-cAMP), 3 microM calcium ionophore ionomycin, 200 microM 5'-uridine triphosphate (UTP) and 200 microM 5'-adenosine triphosphate (ATP), respectively. The decrease in cellular Cl content was significantly inhibited by the Cl- channel blocker 5-nitro-2-(3-phenylpropyl-amino)-benzoic acid (NPPB; 50 microm). Preincubation of the cells with CFTR antisense oligodeoxynucleotide significantly inhibited the 8-bromo-cAMP-induced decrease in Cl, whereas CFTR sense oligodeoxynucleotide had no effect. The effects of ionomycin, ATP or UTP were not blocked by either CFTR antisense oligodeoxynucleotide or CFTR sense oligodeoxynueleotide. To measure the cytosolic free calcium concentration ([Ca2+]i) the cells grown on glass coverslips were loaded with fura-2 tetraoxymethylester (fura-2 AM; 5 microM). The [Ca2+]i was measured as the fluorescence ratio of emission (340/380 nm). Ionomycin (3 microM) caused a rapid increase in [Ca2+]i followed by a sustained plateau, but 8-bromo-cAMP had a more complex effect on [Ca2+]i. Exposure to ATP or UTP caused a rapid increase in [Ca2+]i followed by a decrease. In conclusion, cystic adenosine monophosphate and ionomycin induced Cl- secretion through different intracellular pathways. Adenosine triphosphate and uridine triphosphate also induced Cl- secretion probably with Ca as an intracellular messenger. The cystic fibrosis transmembrane conductance regulator is not involved in Cl- secretion activated by extracellular adenosine triphosphate and uridine triphosphate.     

The relationship between pulsatile secretion and calcium dynamics in single, living gonadotropin-releasing hormone neurons

It is well established that pulsatile release of GnRH regulates the reproductive axis, but little is known about the mechanisms underlying this pulsatility. Recent findings that GT1 cells, a line derived from the mouse embryonic hypothalamus, release GnRH in a pulsatile manner indicates that this rhythmic activity is an intrinsic property of GnRH neurons. In several attempts to uncover the intracellular basis for this pulsatile phenomenon, it was revealed that intracellular calcium concentrations change in a rhythmic fashion in GnRH neurons and that cellular depolarization, which triggers a secretory event, is associated with profound calcium changes in the cells. These findings raised the intriguing possibility that periodic alterations in intracellular calcium concentrations may underlie the phenomenon of pulsatile secretion in GnRH neurons. To address this, we first adapted the use of FM1-43 fluorescence to monitor changes of secretion in individual GT1-7 cells and then combined this approach with simultaneous measurement ofintracellular free calcium ([Ca2+]i, fura 2 method). In initial validation experiments, we found that stimulation of exocytosis with K+ (75 mM) or N-methyl-D-aspartate (NMDA, 100 microM) predictably evoked dynamic increases of both FM1-43 and fura 2 fluorescence. Later measurement of calcium dynamics and exocytotic activity in unstimulated cells revealed that [Ca2+]i underwent transitions from quiescence to high oscillatory behavior, and that these shifts were frequently associated with exocytotic events. Moreover, these calcium oscillatory transitions and associated changes in secretory activity occurred synchronously among most adjacent cells and at a frequency similar to that reported for pulsatile release of GnRH by entire cultures of GnRH neurons. Taken together, these results indicate that the intrinsic secretory pulsatility of GnRH neurons appears to be a consequence of coordinated, periodic changes in the pattern of calcium oscillations within individual cells.     

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.     

Mechanisms of vasodilation induced by NKH477, a water-soluble forskolin derivative, in smooth muscle of the porcine coronary artery.

To study the mechanism of vasodilation induced by 6-(3-dimethylaminopropionyl) forskolin (NKH477), a water-soluble forskolin derivative, its effects on the acetylcholine (ACh)-induced contraction of muscle strips of porcine coronary artery were examined. [Ca2+]i, isometric force, and cellular concentrations of cAMP and inositol 1,4,5-trisphosphate were measured. NKH477 (0.1-1.0 microM), isoproterenol (0.01-0.1 microM), or forskolin (0.1-1.0 microM) increased cAMP and attenuated the contraction induced by 128 mM K+ or 10 microM ACh in a concentration-dependent manner. These agents, at concentrations up to 0.3 microM, did not change the amount of cGMP. NKH477 (0.1 microM) attenuated the contraction induced by 128 mM K+ without corresponding changes in the evoked [Ca2+]i responses. ACh (10 microM) produced a large phasic increase followed by a small tonic increase in [Ca2+]i and produced a sustained contraction. The ACh-induced phasic increase in [Ca2+]i, but not the tonic increase, disappeared after application of 0.1 microM ionomycin. NKH477 (0.1 microM) attenuated both the increase in [Ca2+]i and the force induced by 10 microM ACh in muscle strips that were not treated with ionomycin and inhibited the ACh-induced contraction without corresponding changes in [Ca2+]i in ionomycin-treated muscle strips. These results suggest that NKH477 inhibits ACh-induced Ca2+ mobilization through its action on ionomycin-sensitive storage sites. In ionomycin-treated and 128 mM K(+)-treated muscle strips, 0.1 microM NKH477 shifted the [Ca2+]i-force relation to the right in the presence or absence of 10 microM ACh. In beta-escin-skinned smooth muscle strips, 0.1 microM NKH477 shifted the pCa-force relation to the right but had no effects on Ca(2+)-independent contraction. We conclude that in smooth muscle of porcine coronary artery, NKH477 inhibits ACh-induced contraction by both attenuating ACh-induced Ca2+ mobilization and reducing the sensitivity of the contractile machinery to Ca2+, possibly by activating cAMP-dependent mechanisms.     

Na(+)-Ca2+ exchange, myoplasmic Ca2+ concentration, and contraction of arterial smooth muscle.

Na(+)-Ca2+ exchange is proposed to be an important regulator of myoplasmic intracellular Ca2+ concentration ([Ca2+]i) and contraction in vascular smooth muscle. We investigated the role of Na(+)-Ca2+ exchange in regulating [Ca2+]i in swine carotid arterial tissues that were loaded with aequorin to allow simultaneous measurement of [Ca2+]i and force. Reversal of Na(+)-Ca2+ exchange, by reduction of extracellular Na+ concentration ([Na+]o) to 1.2 mM, induced a large increase in aequorin-estimated [Ca2+]i and a low [Ca2+]i sensitivity. The contraction induced by 1.2 mM [Na+]o was partially caused by depolarization and opening of L-type Ca2+ channels because 10 microM diltiazem partially attenuated the 1.2 mM [Na+]o-induced increases in [Ca2+]i. High dose ouabain (10 microM), a putative endogenous Na+,K(+)-ATPase inhibitor, increased both [Ca2+]i and force. However, the increases in [Ca2+]i and force were mostly blocked by 10 microM phentolamine, suggesting the predominant effect of ouabain was to increase norepinephrine release from nerve terminals. In the presence of 10 microM phentolamine, 10 microM ouabain slightly accentuated 1 microM histamine-induced increases in [Ca2+]i and force. The ouabain dose necessary to induce contraction in the absence of phentolamine was significantly less than the ouabain dose necessary to accentuate histamine-induced contractions in the presence of phentolamine. These results suggest that Na(+)-Ca2+ exchange exists in swine arterial smooth muscle. These data also suggest that ouabain (which should increase [Na+]i and inhibit Na(+)-Ca2+ exchange) primarily enhances contractile function in the swine carotid artery by releasing catecholamines from nerve terminals; direct action of Na+,K(+)-ATPase inhibitors on smooth muscle appears to occur only with very high doses.     

Role of membrane depolarization and T-type Ca2+ channels in angiotensin II and K+ stimulated aldosterone secretion

The hypothesis that Ca2+ influx necessary for angiotensin II (AngII) and K+ stimulation of aldosterone secretion is primarily mediated by membrane depolarization and activation of T-type Ca2+ channels was examined in isolated rat adrenal glomerulosa cells. Perforated-patch clamp recordings of membrane potential (Vm) demonstrated that AngII and K+ induce concentration-dependent depolarizations capable of activating T channels and, at high K+ and AngII concentrations, activating L channels and inactivating T channels. K+-induced depolarizations were stable and readily reversible. Vm was proportional to K+ concentration, exhibiting a linear slope of 53.7 mV per 10-fold increase in K+. AngII-induced depolarizations were complex, consisting of a slow maintained component superimposed with small amplitude depolarizing fluctuations. Slow oscillations in Vm were occasionally observed in response to 10(-9) M AngII or greater. The slow, maintained component of depolarization coincided with inhibition of K+ conductance. Neither rapid fluctuations nor slow oscillations in Vm were blocked by mibefradil or other treatments that inhibit voltage-gated Ca2+ channels. Perforated-patch clamp experiments also demonstrated that AngII (10(-8) M) inhibited L channels by 45.6% without affecting T channels. Thus AngII activates T channels by depolarization rather than T channel modulation in rat cells. The concentration dependencies of mibefradil inhibition of T channels and AngII- and K+-induced aldosterone secretion were compared. Under whole-cell patch clamp mibefradil induced a concentration-dependent inhibition of T channels, exhibiting a K(app) of 0.62 microM. Mibefradil inhibition was use-dependent but mibefradil neither acted as an open channel blocker nor significantly affected T channel inactivation or activation. Mibefradil inhibited K+- and AngII-induced secretion at concentrations similar to that for T channel inhibition; at high concentrations (10 microM) mibefradil inhibited AngII-induced secretion by 88% and completely inhibited K+-induced secretion. The IC50 for K+-induced secretion was dependent on K+ concentration, increasing from 0.2 microM for 6 mM K+ to 2.5 microM for 10 mM K+ or greater. Mibefradil exhibited an IC50 of 1.1 microM for inhibition of secretion at all AngII concentrations examined (0.1, 1.0, and 10 nM). Mibefradil also exhibited multiple nonspecific effects, which complicated the assessment of T channel function, including; inhibition of leak and voltage-dependent K+ conductances, inhibition of Ca2+-independent aldosterone secretion, and inhibition of secretion under conditions expected to completely inactivate T channels (10 nM AngII or 20 mM K+). In summary, these results indicate that voltage-gated T channels represent the primary Ca2+ influx pathway activated by physiological concentrations of AngII and K+ but other Ca2+ influx pathways must mediate aldosterone secretion induced by high K+ or AngII concentrations.     

Effect of GABA on GnRH neurons switches from depolarization to hyperpolarization at puberty in the female mouse

The amino acid gamma-aminobutyric acid (GABA) plays an important role in the regulation of the GnRH neurons. We examined whether GABA depolarizes or hyperpolarizes GnRH neurons over postnatal development using gramicidin, perforated-patch electrophysiology combined with GnRH-LacZ transgenic mice in whom GnRH neurons can be made to fluoresce. The basic membrane properties and GABA responsiveness of GnRH neurons were not altered by transgene expression or fluorescence. Ten of 12 immature GnRH neurons (10-17 d) were depolarized by GABA in a direct and dose-dependent manner that was blocked by a GABA(A) receptor antagonist. In peripubertal GnRH neurons (25-30 d), GABA exerted depolarizing (4/11) as well as hyperpolarizing (5/11) effects on GnRH neurons. In adult female mice, GABA was found to exert exclusively hyperpolarizing actions on GnRH neurons (9/10) that were direct and mediated by the GABA(A) receptor. GABA switched from depolarizing to hyperpolarizing actions around postnatal d 31, the time of vaginal opening. Unidentified preoptic area neurons exhibited predominantly hyperpolarizing responses to GABA at all three postnatal stages. These findings demonstrate that GnRH neurons display an unusually late postnatal switch in their response to GABA. They also provide the first direct evidence that GABA inhibits the electrical activity of postpubertal GnRH neurons.     

Alpha-adrenergic receptor stimulation during simulated ischemia and reperfusion in canine cardiac Purkinje fibers

We studied the effects of alpha-adrenergic receptor stimulation and calcium on automaticity of isolated canine Purkinje fibers during simulated ischemia and reperfusion. Ischemia included acidosis (pH 6.7), hypoxia (PO2 = 10-25 mm Hg), hyperkalemia (10 mM K+), and either normal or elevated [Ca2+]o (2.7 or 10.8 mM). Control automatic rate and maximum diastolic potential were 18 +/- 2 beats/min and -94 +/- 1 mV, respectively. Simulated ischemia led to depolarization (to -60 +/- 1 mV), cessation of normal automaticity, and in 21% of fibers, bursts of an abnormal automatic rhythm. Phenylephrine, 5 X 10(-8) M, increased the incidence of the automatic rhythm during ischemia to 44%; this effect was blocked by prazosin but not by propranolol. During reperfusion after simulated ischemia at 2.7 mM [Ca2+]o, automatic rhythm and maximum diastolic potential returned toward control values; after simulated ischemia at 10.8 mM [Ca2+]o, automatic rates were greater than those seen after normal Ca2+ ischemia and were associated with sustained membrane depolarization. Phenylephrine (5 X 10(-8) M) at 2.7 mM [Ca2+]o rapidly restored membrane potential during reperfusion, an effect that was blocked by prazosin. At 10.8 mM [Ca2+]o, phenylephrine also restored membrane potential during reperfusion and blunted the increase in reperfusion rate induced by high [Ca2+]o alone. These effects were blocked by propranolol but not by prazosin. Our results show that the effects of phenylephrine on automatic rhythms during simulated ischemia are blocked by alpha-adrenergic receptor antagonists and that rhythms occurring during reperfusion have alpha- and beta-adrenergic receptor components.     

Glucagon-like peptide 1 elevates cytosolic calcium in pancreatic beta-cells independently of protein kinase A.

Glucagon-like peptide 1 (7-36)amide (GLP-1) is an insulinotropic intestinal peptide hormone with a potential role as antidiabetogenic therapeutic agent. It mediates a potentiation of glucose-induced insulin secretion, by activation of adenylate cyclase and subsequent elevation of cytosolic free calcium, [Ca2+]cyt. We investigated the role of protein kinase A (PKA) in GLP-1 signal transduction, using isolated mouse islets as well as the differentiated beta-cell line INS-1. Two specific inhibitors of PKA, (Rp)-adenosine cyclic 3',5'-phosporothioate (Rp-cAMPS, up to 3 mM) and KT5720 (up to 10 microM), did not inhibit the GLP-1-induced [Ca2+]cyt elevation. Another PKA inhibitor, H-89, reduced the [Ca2+]cyt elevation only when applied at high concentrations (10-40 microM), higher than sufficient for PKA inhibition in many cell types. Furthermore, at these concentrations, H-89 also inhibited presumably PKA-independent processes such as glucose-induced [Ca2+]cyt elevations and intracellular calcium storage. This suggests a PKA-independent action of H-89. Similarly to H-89, the potent but unselective protein kinase inhibitor staurosporine inhibited the GLP-1-induced [Ca2+]cyt elevation only at high concentrations, at which it also inhibited glucose-induced [Ca2+]cyt elevations. The same observations as with GLP-1 were made when adenylate cyclase was stimulated with forskolin, for selective examination of signal transduction downstream of receptor and G protein. Our results suggest that the GLP-1-induced [Ca2+]cyt elevation is mediated independently of PKA and thus belongs to the yet-little-characterized ensemble of effects that are mediated by binding of cAMP to other target proteins.     

Characterization of alphaT3-1 cells stably transfected with luteininzing hormone beta-subunit complementary deoxyribonucleic acid

Luteinizing hormone (LH) consists of alpha- and beta-subunits, and synthesis and secretion of LH are regulated by gonadotropin-releasing hormone (GnRH). In order to examine the molecular mechanisms by which GnRH regulates LH secretion, we transfected alphaT3-1 cells with rat LHbeta-subunit cDNA under the control of a constitutive promoter and established a stable cell line of LH2 cells which secreted LH in response to GnRH. Pulsatile and continuous GnRH pretreatments increased gene expression of the alpha-subunit and synthesis of LH, and enhanced the LH secretion by brief treatments with GnRH and 56 mM KCl. The LH secretions were partially blocked by elimination of extracellular Ca2+. GnRH-induced LH secretion was completely inhibited by calphostin C (a protein kinase C inhibitor) and 1 microM wortmannin. In contrast to the GnRH induction, high K+-induced LH secretion was inhibited by KN93, a Ca2+/calmodulin-dependent protein kinase II inhibitor, as well as by 1 microM wortmannin. We also confirmed that activation of cAMP-pathway induced LH secretion, but activation of mitogen-activated protein (MAP) kinase pathway was not involved in LH secretion. These results suggest that GnRH directly regulates LH secretion as well as LHbeta-subunit synthesis, and that LH2 cells are a useful model for the study of LH secretion induced by several secretagogues.     

Complex correlation between excitatory amino acid-induced increase in the intracellular Ca2+ concentration and subsequent loss of neuronal function in individual neocortical neurons in culture.

Primary cultures of cerebral cortical neurons and single-cell imaging of intracellular free Ca2+ concentration ([Ca2+]i) with the ratiometric dye fura-2 were used to assess excitatory amino acid (EAA)-induced neurotoxicity; the loss of neuronal function as defined by the ability of the cells to respond to K(+)-induced depolarization by a transient increase in Ca2+ influx was measured. The responsiveness of individual neurons was measured quantitatively as the [Ca2+]i values of the second KCl (2.KCl) stimulation divided by those of the first KCl (1.KCl) stimulation, giving the value of the ratio (2.KCl/1.KCl). Exposure to EAAs led to an increase in [Ca2+]i, but no simple correlation between the increase in [Ca2+]i and neuronal responsiveness could be demonstrated. Rather, below a threshold level of [Ca2+]i (ca. 1 microM), the neuronal responsiveness was largely independent of the glutamate receptor-agonist-induced increase in [Ca2+]i. However, when [Ca2+]i increased above this threshold level, the neurons almost invariably lost the ability to respond to a K(+)-induced depolarization, particularly after exposure to glutamate. Therefore, the cortical neurons were found to be exceptionally vulnerable to the glutamate-induced loss of function when compared with the effect induced by the glutamate receptor subtype-specific agonists, N-methyl-D-aspartate, quisqualate, and 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl) propionate. The findings suggest that the loss of neuronal membrane polarization precedes plasma membrane disruption and is a sensitive marker of EAA-induced neurodegeneration observed at the single-cell level.