Autocrine regulation of calcium influx and gonadotropin-releasing hormone secretion in hypothalamic neurons.

Gonadotropin-releasing hormone (GnRH) receptors are expressed in hypothalamic tissues from adult rats, cultured fetal hypothalamic cells, and immortalized GnRH-secreting neurons (GT1 cells). Their activation by GnRH agonists leads to an overall increase in the extracellular Ca2+-dependent pulsatile release of GnRH. Electrophysiological studies showed that GT1 cells exhibit spontaneous, extracellular Ca2+-dependent action potentials, and that their inward currents include Na+, T-type and L-type Ca2+ components. Several types of potassium channels, including apamin-sensitive Ca2+-controlled potassium (SK) channels, are also expressed in GT1 cells. Activation of GnRH receptors leads to biphasic changes in intracellular Ca2+ concentration ([Ca2+]i), with an early and extracellular Ca2+-independent peak and a sustained and extracellular Ca2+-dependent plateau phase. During the peak [Ca2+]i response, electrical activity is abolished due to transient hyperpolarization that is mediated by SK channels. This is followed by sustained depolarization and resumption of firing with increased spike frequency and duration. The agonist-induced depolarization and increased firing are independent of [Ca2+]i and are not mediated by inhibition of K+ currents, but by facilitation of a voltage-insensitive and store depletion-activated Ca2+-conducting inward current. The dual control of pacemaker activity by SK and store depletion-activated Ca2+ channels facilitates voltage-gated Ca2+ influx at elevated [Ca2+]i levels, but also protects cells from Ca2+ overload. This process accounts for the autoregulatory action of GnRH on its release from hypothalamic neurons.

Local regulation of gonadotroph function by pituitary gonadotropin-releasing hormone.

Cultured rat pituitary cells and immortalized pituitary gonadotrophs (alphaT3-1 cells) express specific messenger RNA transcripts for GnRH and exhibit positive immunostaining for the GnRH peptide. Each cell type released GnRH during both static culture and perifusion, albeit in lesser amounts than cultured hypothalamic cells and GT1-7 neurons. In perifused pituitary cells, exposure to a GnRH agonist stimulated the release of GnRH as well as LH. In contrast, treatment with a GnRH receptor antagonist or with GnRH antiserum decreased basal LH release. In pituitary cell cultures, a small proportion of gonadotrophs exhibited high amplitude and low frequency baseline Ca2+ oscillations in the absence of GnRH stimulation. Such spontaneous oscillations were comparable to those induced by picomolar concentrations of GnRH and could be abolished by treatment with a GnRH antagonist. These in vitro findings indicate that locally produced GnRH causes low level activation of pituitary GnRH receptors, induces spontaneous intracellular Ca2+ oscillations, and contributes to basal LH secretion in cultured pituitary cells. In vivo, such autocrine or paracrine actions of pituitary-derived GnRH could provide a mechanism for the maintenance of optimal responsiveness of the gonadotrophs to pulses of GnRH arising in the hypothalamus. The presence and actions of GnRH in the anterior pituitary gland, the major site of expression of GnRH receptors, suggest that local regulatory effects of the neuropeptide could supplement the primary hypothalamic mechanism for the control of episodic gonadotropin secretion.

Modeling of membrane excitability in gonadotropin-releasing hormone-secreting hypothalamic neurons regulated by Ca2+-mobilizing and adenylyl cyclase-coupled receptors.

Gonadotropin-releasing hormone (GnRH) secretion from native and immortalized hypothalamic neurons is regulated by endogenous Ca(2+)-mobilizing and adenylyl cyclase (AC)-coupled receptors. Activation of both receptor types leads to an increase in action potential firing frequency and a rise in the intracellular Ca(2+) concentration ([Ca(2+)](i)) and neuropeptide secretion. The stimulatory action of Ca(2+)-mobilizing agonists on voltage-gated Ca(2+) influx is determined by depletion of the intracellular Ca(2+) pool, whereas AC agonist-stimulated Ca(2+) influx occurs independently of stored Ca(2+) and is controlled by cAMP, possibly through cyclic nucleotide-gated channels. Here, experimental records from immortalized GnRH-secreting neurons are simulated with a mathematical model to determine the requirements for generating complex membrane potential (V(m)) and [Ca(2+)](i) responses to Ca(2+)-mobilizing and AC agonists. Included in the model are three pacemaker currents: a store-operated Ca(2+) current (I(SOC)), an SK-type Ca(2+)-activated K(+) current (I(SK)), and an inward current that is modulated by cAMP and [Ca(2+)](i) (I(d)). Spontaneous electrical activity and Ca(2+) signaling in the model are predominantly controlled by I(d), which is activated by cAMP and inhibited by high [Ca(2+)](i). Depletion of the intracellular Ca(2+) pool mimics the receptor-induced activation of I(SOC) and I(SK), leading to an increase in the firing frequency and Ca(2+) influx after a transient cessation of electrical activity. However, increasing the activity of I(d) simulates the experimental response to forskolin-induced activation of AC. Analysis of the behaviors of I(SOC), I(d), and I(SK) in the model reveals the complexity in the interplay of these currents that is necessary to fully account for the experimental results.

Control of action potential-driven calcium influx in GT1 neurons by the activation status of sodium and calcium channels.

An analysis of the relationship between electrical membrane activity and Ca2+ influx in differentiated GnRH-secreting (GT1) neurons revealed that most cells exhibited spontaneous, extracellular Ca(2+)-dependent action potentials (APs). Spiking was initiated by a slow pacemaker depolarization from a baseline potential between -75 and -50 mV, and AP frequency increased with membrane depolarization. More hyperpolarized cells fired sharp APs with limited capacity to promote Ca2+ influx, whereas more depolarized cells fired broad APs with enhanced capacity for Ca2+ influx. Characterization of the inward currents in GT1 cells revealed the presence of tetrodotoxin-sensitive Na+, Ni(2+)-sensitive T-type Ca2+, and dihydropyridine-sensitive L-type Ca2+ components. The availability of Na+ and T-type Ca2+ channels was dependent on the baseline potential, which determined the activation/inactivation status of these channels. Whereas all three channels were involved in the generation of sharp APs, L-type channels were solely responsible for the spike depolarization in cells exhibiting broad APs. Activation of GnRH receptors led to biphasic changes in cytosolic Ca2+ concentration ([Ca2+]i), with an early, extracellular Ca(2+)-independent peak and a sustained, extracellular Ca(2+)-dependent phase. During the peak [Ca2+]i response, electrical activity was abolished due to transient hyperpolarization. This was followed by sustained depolarization of cells and resumption of firing of increased frequency with a shift from sharp to broad APs. The GnRH-induced change in firing pattern accounted for about 50% of the elevated Ca2+ influx, the remainder being independent of spiking. Basal [Ca2+]i was also dependent on Ca2+ influx through AP-driven and voltage-insensitive pathways. Thus, in both resting and agonist-stimulated GT1 cells, membrane depolarization limits the participation of Na+ and T-type channels in firing, but facilitates AP-driven Ca2+ influx.

Autocrine regulation of gonadotropin-releasing hormone secretion in cultured hypothalamic neurons.

Episodic hormone secretion is a characteristic feature of the hypothalamo-pituitary-gonadal system, in which the profile of gonadotropin release from pituitary gonadotrophs reflects the pulsatile secretory activity of GnRH-producing neurons in the hypothalamus. Pulsatile release of GnRH is also evident in vitro during perifusion of immortalized GnRH neurons (GT1-7 cells) and cultured fetal hypothalamic cells, which continue to produce bioactive GnRH for up to 2 months. Such cultures, as well as hypothalamic tissue from adult rats, express GnRH receptors as evidenced by the presence of high-affinity GnRH binding sites and GnRH receptor transcripts. Furthermore, individual GnRH neurons coexpress GnRH and GnRH receptors as revealed by double immunostaining of hypothalamic cultures. In static cultures of hypothalamic neurons and GT1-7 cells, treatment with the GnRH receptor antagonist, [D-pGlu1, D-Phe2, D-Trp(3,6)]GnRH caused a prominent increase in GnRH release. In perifused hypothalamic cells and GT1-7 cells, treatment with the GnRH receptor agonist, des-Gly10-[D-Ala6]GnRH N-ethylamide, reduced the frequency and increased the amplitude of pulsatile GnRH release, as previously observed in GT1-7 cells. In contrast, exposure to the GnRH antagonist analogs abolished pulsatile secretion and caused a sustained and progressive increase in GnRH release. These findings have demonstrated that GnRH receptors are expressed in hypothalamic GnRH neurons, and that receptor activation is required for pulsatile GnRH release in vitro. The effects of GnRH agonist and antagonist analogs on neuropeptide release are consistent with the operation of an ultrashort-loop autocrine feedback mechanism that exerts both positive and negative actions that are necessary for the integrated control of GnRH secretion from the hypothalamus.

Dependence of intracellular signaling and neurosecretion on phospholipase D activation in immortalized gonadotropin-releasing hormone neurons.

The excitability of gonadotropin-releasing hormone (GnRH) neurons is essential for episodic neuropeptide release, but the mechanism by which electrical activity controls GnRH secretion is not well characterized. The role of phospholipase D (PLD) in mediating the activity-dependent secretory pathway was investigated in immortalized GT1 neurons, which both secrete GnRH and express GnRH receptors. Activation of these Ca2+-mobilizing receptors was associated with transient hyperpolarization of GT1 cells, followed by sustained firing of action potentials. This was accompanied by an increase in PLD activity, as indicated by elevated phosphatidylethanol (PEt) production. GnRH-induced PEt production was reduced by inhibition of phospholipase C-dependent phosphoinositide hydrolysis by U73122 and neomycin, suggesting that signaling from phospholipase C led to activation of PLD. The intermediate role of protein kinase C (PKC) in this process was indicated by the ability of phorbol 12-myristate 13-acetate to induce time- and dose-dependent increases in PEt and diacylglycerol, but not inositol trisphosphate, and by reduction of GnRH-induced PEt accumulation in PKC-depleted cells. Consistent with the role of action potential-driven Ca2+ entry in this process, agonist-induced PLD activity was also reduced by nifedipine and low extracellular Ca2+. Inhibition of the PLD pathway by ethanol and propranolol reduced diacylglycerol production and caused a concomitant fall in GnRH release. These data indicate that voltage-gated Ca2+ entry and PKC act in an independent but cooperative manner to regulate PLD activity, which contributes to the secretory response in GT1 cells. Thus, the electrical activity of the GnRH-secreting neuron participates in the functional coupling between GnRH receptors and PLD pathway.

GnRH-induced cytosolic calcium oscillations in pituitary gonadotrophs: phase resetting by membrane depolarization.

Cultured rat pituitary gonadotrophs under whole-cell voltage clamp conditions respond to the hypothalamic hormone GnRH with synchronized oscillatory changes in both cytosolic Ca2+ concentration ([Ca2+]i) and [Ca2+]i-activated, apamin-sensitive K+ current (IK(Ca)). We found, and report here for the first time, that in GnRH-stimulated cells a brief depolarizing pulse can elicit a transient [Ca2+]i rise similar to the endogenous cycle. Furthermore, Ca2+ entry during a single depolarizing pulse was found to shift the phase of subsequent endogenous [Ca2+]i oscillations, which thereafter continue to occur at their previous frequency before the pulse. Application of two consecutive depolarizing pulses showed that the size of the [Ca2+]i rise evoked by the second pulse depended on the time lapsed between two consecutive pulses, indicating that each endogenous or evoked [Ca2+]i rise cycle leaves the Ca2+ release mechanism of the gonadotroph in a refractory state. Recovery from this condition can be described by an exponential function of the time lapsed between the pulses (time constant of ca. 1 s). We propose that the underlying mechanism in both refractoriness after endogenous cycles and phase resetting by a brief pulse of Ca2+ entry involves the InsP3 receptor-channel molecule presumed to be located on the cytosolic aspect of the endoplasmic reticulum membrane.

Coordinate actions of calcium and protein kinase-C in the expression of primary response genes in pituitary gonadotrophs.

Activation of GnRH receptors in cultured pituitary cells and alpha T3-1 gonadotrophs caused prominent, but transient, increases in messenger RNAs for primary response genes (PRGs) including c-fos, c-jun, and junB. GnRH-induced stimulation peaked at 30 min and was dose related, with similar EC50 values (approximately 1 nM) for all three PRGs and higher maximum responses for junB than for c-jun and c-fos. The agonist-induced expression of PRGs was mimicked by activation of protein kinase-C with the phorbol ester phorbol 12-myristate 13-acetate (PMA), which acted additively with GnRH at low concentrations of both stimuli. Depletion of cellular protein kinase-C by prior treatment with PMA reduced GnRH- and PMA-induced expression of PRGs. The protein kinase-C inhibitor staurosporine also attenuated agonist- and phorbol ester-induced PRG expression. Activation of Ca2+ entry by the calcium channel agonist BayK 8644 or high K(+)-induced depolarization caused a concentration-dependent rise in intracellular Ca2+ ([Ca2+]i) and a concentration-dependent and transient expression of PRGs, albeit of smaller amplitudes than those elicited by GnRH and PMA. Ca(2+)-dependent PRG expression was abolished by the calmodulin inhibitor W-7. Parallel measurements of [Ca2+]i and steady-state levels of PRG messenger RNAs indicated that intracellular Ca2+ exerted both additive and suppressive actions over its physiological concentration range on GnRH- and PMA-induced PRG expression. At lower intracellular calcium concentrations, calcium acted additively with low concentrations of GnRH and PMA. However, high calcium concentrations suppressed high agonist- and phorbol ester-induced PRG expression. In contrast, omission of Ca2+ from the extracellular medium significantly enhanced induction of PRGs. These findings indicate that GnRH-induced PRG expression in gonadotrophs is mediated by protein kinase-C and calcium, and that protein kinase-C-dependent induction of PRGs is modulated both positively and negatively by physiological changes in [Ca2+]i. Such coordinate actions of the two signaling molecules provide a mechanism for the control of PRG expression by preferential integration of low strength, and attenuation of high strength, extracellular signals.

Glutamate modulates [Ca2+]i and gonadotropin-releasing hormone secretion in immortalized hypothalamic GT1-7 neurons.

Glutamate and its receptors are present in the hypothalamus and have been proposed to participate in neuroendocrine regulation, including the control of GnRH secretion. To address the mechanism of glutamate action, we measured [Ca2+]i, inositol phosphate, and secretory responses to glutamate receptor subtype agonists and antagonists in the immortalized GT1-7 cell line of GnRH-secreting hypothalamic neurons. Glutamate, N-methyl-D-aspartate (NMDA), kainate, and trans-(+/-)-1-amino-(1S,3R)-cyclopentanedicarboxylic acid increased GnRH secretion. In monolayer cultures of GT1-7 cells, L- but not D-glutamate induced a moderate, concentration-dependent rise in [Ca2+]i. The action of glutamate on [Ca2+]i was mimicked by NMDA, alpha-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazolepropanoic acid (AMPA), and kainate. Responses to NMDA were potentiated by the coagonist, glycine, and were inhibited by an antagonist of the glycine site on the NMDA receptor, 5,7-dichlorokynurenic acid (DCKA). NMDA-induced [Ca2+]i responses were also inhibited by Mg2+ and by the NMDA receptor antagonist, (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,1 0-imine hydrogen maleate (MK-801), but not by the AMPA/kainate antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). In contrast, responses to AMPA and kainate were inhibited by CNQX but not by Mg2+, DCKA, or MK-801. Responses to glutamate were more inhibited by MK-801 plus CNQX than by either antagonist alone. All [Ca2+]i responses were nearly abolished in Ca(2+)-free solution. None of the agonists stimulated inositol phosphate formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of gonadotropin-releasing hormone secretion by acetyl-L-carnitine in hypothalamic neurons and GT1 neuronal cells.

Pulsatile gonadotropin-releasing hormone (GnRH) secretion from perifused hypothalamic cells and GT1-1 neuronal cells was significantly increased after culture in medium containing 100 microM acetyl-L-carnitine (ALC). This action of ALC was largely due to an increase in the spike amplitude of GnRH release. In addition, the receptor-mediated release of GnRH by N-methyl-D-aspartic acid and endothelin was significantly increased in perifused cells cultured in ALC-enriched medium. Stimulatory effects of ALC on basal, high K(+)- and agonist-induced GnRH release were also observed during long-term culture of primary hypothalamic neurons. Similar effects of ALC were evident in cultured GT1-1 cells and were accompanied by a significant increase in cell number. These observations in normal and transformed GnRH neurons demonstrate that ALC promotes the growth and secretory activity of neuropeptide-producing cells of the hypothalamus.

Expression of gonadotropin-releasing hormone receptors and autocrine regulation of neuropeptide release in immortalized hypothalamic neurons.

The hypothalamic control of gonadotropin secretion is mediated by episodic basal secretion and midcycle ovulatory surges of gonadotropin-releasing hormone (GnRH), which interacts with specific plasma membrane receptors in pituitary gonadotrophs. Similar GnRH receptors and their mRNA transcripts were found to be expressed in immortalized hypothalamic neurons, which release GnRH in a pulsatile manner in vitro. Activation of these neuronal GnRH receptors elicited dose-related intracellular Ca2+ concentration responses that were dependent on calcium mobilization and entry and were inhibited by GnRH antagonists. Exposure of perifused neurons to a GnRH agonist analog caused a transient elevation of GnRH release and subsequent suppression of the basal pulsatile secretion. This was followed by dose-dependent induction of less frequent but larger GnRH pulses and ultimately by single massive episodes of GnRH release. The ability of GnRH to exert autocrine actions on its secretory neurons, and to promote episodic release and synchronized discharge of the neuropeptide, could reflect the operation of the endogenous pulse generator and the genesis of the preovulatory GnRH surge in vivo.

Calcium signaling and episodic secretion of gonadotropin-releasing hormone in hypothalamic neurons.

Gonadotropin-releasing hormone (GnRH) is released episodically into the pituitary portal vessels and from hypothalamic tissue of male and female rats in vitro. Perifused primary cultures of rat hypothalamic neurons, as well as the GT1-1 GnRH neuronal cell line, spontaneously exhibited episodic GnRH secretion of comparable frequency to that observed with perifused hypothalami. Such pulsatile GnRH release from GT1 cells indicates that GnRH neurons generate rhythmic secretory activity in the absence of input from other cell types. In primary hypothalamic cultures, the frequency of GnRH pulses increased with the duration of culture. The spontaneous pulsatility in GnRH release was abolished in Ca(2+)-deficient medium and was markedly attenuated in the presence of nifedipine, an antagonist of voltage-sensitive Ca2+ channels. The basal intracellular Ca2+ level of perifused GT1-1 cells cultured on coverslips was also dose-dependently reduced by nifedipine. Conversely, depolarization with high K+ increased intracellular Ca2+ and GnRH release in an extracellular Ca(2+)-dependent and nifedipine-sensitive manner. The dihydropyridine Ca2+ channel agonist Bay K 8644 increased basal and K(+)-induced elevations of intracellular Ca2+ concentration and GnRH secretion. These findings demonstrate that pulsatile neuropeptide secretion is an intrinsic property of GnRH neuronal networks and is dependent on voltage-sensitive Ca2+ influx for its maintenance.

Receptors and neurosecretory actions of endothelin in hypothalamic neurons.

Primary cultures of rat hypothalamic neurons were found to secrete the potent calcium-mobilizing and mitogenic peptide endothelin (ET) and to contain specific ET binding sites with higher affinity for ET-1 and ET-2 than ET-3. ET receptors of similar specificity were also identified in two gonadotropin-releasing hormone (GnRH) neuronal cell lines (GT1-1 and GT1-7). In both primary cultures and GnRH neurons, receptor binding of ETs led to marked and dose-dependent increases of inositol phosphates; inositol bis-, tris-, and tetrakisphosphates increased promptly, reached a peak within 2 min, and returned toward the steady-state levels during the next 10 min. ET-1 was more potent than ET-3 in mobilizing inositol phosphates, consistent with its greater affinity for the ET receptors in these cells. ET also stimulated GnRH secretion from perifused hypothalamic cultures and GnRH cell lines, with a sharp increase followed by a prompt decline to the basal level. These data show that ET is produced in the hypothalamus and acts through calcium-mobilizing ET receptors in normal and transformed secretory neurons to stimulate GnRH release. These actions of locally produced ETs upon GnRH-secreting neurons indicate that the vasoconstrictor peptides have the capacity to regulate neurosecretion and could participate in the hypothalamic control of anterior pituitary function and gonadotropin secretion.

Opiate receptor subtypes in the rat hypothalamus and neurointermediate lobe.

The potent opiate radioligands [3H]etorphine, [3H]ethylketocyclazocine (EKC), and [3H]naloxone, bound specifically and saturably to a single class of membrane-binding sites in rat neurointermediate lobe (NIL), with Kd values of 3.7, 24, and 51 nM, respectively. In the hypothalamus (Ht), [3H]etorphine bound to specific and saturable sites with a Kd of 2.9 nM. Binding-inhibition studies with [3H]etorphine and unlabeled etorphine-HCl as well as [3H]EKC and unlabeled EKC, revealed high and low affinity binding sites in rat Ht and NIL as well as in the neural lobe of the bovine pituitary gland. [3H]naloxone also bound specifically to two classes of sites in Ht membranes, but to only a single class of low affinity sites in NIL membranes. Specific binding represented 80-90% of total [3H]etorphine binding, about 75% of total [3H]EKC binding, and 45-55% of total [3H]naloxone binding at 22 C in NIL and Ht, respectively. Relative binding potencies derived from Ki values for binding-inhibition studies of [3H]etorphine with opioid peptides and opiates were: NIL, etorphine-HCl greater than dynorphin A greater than naloxone-HCl greater than dynorphin-(1-9) greater than beta-endorphin much greater than alpha-neoendorphin approximately (Leu5)enkephalin approximately DAGO (Tyr-D-Ala-Gly-NMe-Phe-Gly-ol); Ht, etorphine HCl greater than naloxone-HCl greater than beta-endorphin greater than dynorphin A much greater than DAGO greater than morphiceptin much greater than (Leu5)enkephalin. Specific [3H]etorphine binding was also demonstrable after preincubation of NIL membranes with DAGO and (Leu5)enkephalin and after preincubation of Ht membranes with morphiceptin and (Leu5)enkephalin; such binding could be displaced by nonradioactive dynorphin A. In addition, [3H]etorphine binding to bovine neural lobe was displaceable by naloxone-HCl, with an ED50 of 43 nM. Specific ligands for sigma-opiate receptors, such as (+)SKF 10,047 (N-allylnorcyclazocine), phencyclidine (PCP), and (-)cyclazocine, displaced specifically bound [3H]etorphine and [3H]EKC from NIL membranes only at high (micromolar) concentrations. However, specific [3H]PCP sites were of higher affinity in NIL and Ht membranes, with similar Kd values of 102 and 190 nM respectively, and different concentrations (0.15 and 1.32 pmol/mg protein, respectively). These data have revealed several differences in the opiate-binding properties of rat Ht and NIL membranes.(ABSTRACT TRUNCATED AT 400 WORDS)