Regulation of Ca2+-sensitive adenylyl cyclase in gonadotropin-releasing hormone neurons.

In immortalized GnRH neurons, cAMP production is elevated by increased extracellular Ca2+ and the Ca2+ channel agonist, BK-8644, and is diminished by low extracellular Ca2+ and treatment with nifedipine, consistent with the expression of adenylyl cyclase type I (AC I). Potassium-induced depolarization of GT1-7 neurons causes a dose-dependent monotonic increase in [Ca2+]i and elicits a bell-shaped cAMP response. The inhibitory phase of the cAMP response is prevented by pertussis toxin (PTX), consistent with the activation of G(i)-related proteins during depolarization. Agonist activation of the endogenous GnRH receptor in GT1-7 neurons also elicits a bell-shaped change in cAMP production. The inhibitory action of high GnRH concentrations is prevented by PTX, indicating coupling of the GnRH receptors to G(i)-related proteins. The stimulation of cAMP production by activation of endogenous LH receptors is enhanced by low (nanomolar) concentrations of GnRH but is abolished by micromolar concentrations of GnRH, again in a PTX-sensitive manner. These findings indicate that GnRH neuronal cAMP production is maintained by Ca2+ entry through voltage-sensitive calcium channels, leading to activation of Ca2+-stimulated AC I. Furthermore, the Ca2+ influx-dependent activation of AC I acts in conjunction with AC-regulatory G proteins to determine basal and agonist-stimulated levels of cAMP production.

Amplitude-dependent spike-broadening and enhanced Ca(2+) signaling in GnRH-secreting neurons.

In GnRH-secreting (GT1) neurons, activation of Ca(2+)-mobilizing receptors induces a sustained membrane depolarization that shifts the profile of the action potential (AP) waveform from sharp, high-amplitude to broad, low-amplitude spikes. Here we characterize this shift in the firing pattern and its impact on Ca(2+) influx experimentally by using prerecorded sharp and broad APs as the voltage-clamp command pulse. As a quantitative test of the experimental data, a mathematical model based on the membrane and ionic current properties of GT1 neurons was also used. Both experimental and modeling results indicated that inactivation of the tetrodotoxin-sensitive Na(+) channels by sustained depolarization accounted for a reduction in the amplitude of the spike upstroke. The ensuing decrease in tetraethylammonium-sensitive K(+) current activation slowed membrane repolarization, leading to AP broadening. This change in firing pattern increased the total L-type Ca(2+) current and facilitated AP-driven Ca(2+) entry. The leftward shift in the current-voltage relation of the L-type Ca(2+) channels expressed in GT1 cells allowed the depolarization-induced AP broadening to facilitate Ca(2+) entry despite a decrease in spike amplitude. Thus the gating properties of the L-type Ca(2+) channels expressed in GT1 neurons are suitable for promoting AP-driven Ca(2+) influx in receptor- and non-receptor-depolarized cells.

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.

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.

Coordinate regulation of gonadotropin-releasing hormone neuronal firing patterns by cytosolic calcium and store depletion.

Elevation of cytosolic free Ca2+ concentration ([Ca2+]i) in excitable cells often acts as a negative feedback signal on firing of action potentials and the associated voltage-gated Ca2+ influx. Increased [Ca2+]i stimulates Ca2+-sensitive K+ channels (IK-Ca), and this, in turn, hyperpolarizes the cell and inhibits Ca2+ influx. However, in some cells expressing IK-Ca the elevation in [Ca2+]i by depletion of intracellular stores facilitates voltage-gated Ca2+ influx. This phenomenon was studied in hypothalamic GT1 neuronal cells during store depletion caused by activation of gonadotropin-releasing hormone (GnRH) receptors and inhibition of endoplasmic reticulum (Ca2+)ATPase with thapsigargin. GnRH induced a rapid spike increase in [Ca2+]i accompanied by transient hyperpolarization, followed by a sustained [Ca2+]i plateau during which the depolarized cells fired with higher frequency. The transient hyperpolarization was caused by the initial spike in [Ca2+]i and was mediated by apamin-sensitive IK-Ca channels, which also were operative during the subsequent depolarization phase. Agonist-induced depolarization and increased firing were independent of [Ca2+]i and were not mediated by inhibition of K+ current, but by facilitation of a voltage-insensitive, Ca2+-conducting inward current. Store depletion by thapsigargin also activated this inward depolarizing current and increased the firing frequency. Thus, the pattern of firing in GT1 neurons is regulated coordinately by apamin-sensitive SK current and store depletion-activated Ca2+ current. This dual control of pacemaker activity facilitates voltage-gated Ca2+ influx at elevated [Ca2+]i levels, but also protects cells from Ca2+ overload. This process may also provide a general mechanism for the integration of voltage-gated Ca2+ influx into receptor-controlled Ca2+ mobilization.

Mediation of cyclic AMP signaling by the first intracellular loop of the gonadotropin-releasing hormone receptor.

The gonadotropin-releasing hormone (GnRH) receptor, which is a unique G protein-coupled receptor without a C-terminal cytoplasmic domain, activates both inositol phosphate (InsP) and cAMP signaling responses. The function of the highly basic first intracellular (1i) loop of the GnRH receptor in signal transduction was evaluated by mutating selected residues located in its N and C termini. Replacements of Leu58, Lys59, Gln61, and Lys62 at the N terminus, and Leu73, Ser74, and Leu80 at the C terminus, caused no change in binding affinity. The agonist-induced InsP and cAMP responses of the Q61E and K59Q,K62Q receptors were also unaffected, but the L58A receptor showed a normal InsP response and an 80% decrease in cAMP production. At the C terminus, the InsP response of the L73R receptor was normal, but cAMP production was reduced by 80%. The EC50 for GnRH-induced InsP responses of the S74E and L80A receptors was increased by about one order of magnitude, and the cAMP responses were essentially abolished. These findings indicate that cAMP signaling from the GnRH receptor is dependent on specific residues in the 1i loop that are not essential for activation of the phosphoinositide signaling pathway.

Muscarinic regulation of intracellular signaling and neurosecretion in gonadotropin-releasing hormone neurons.

Agonist activation of cholinergic receptors expressed in perifused hypothalamic and immortalized GnRH-producing (GT1-7) cells induced prominent peaks in GnRH release, each followed by a rapid decrease, a transient plateau, and a decline to below basal levels. The complex profile of GnRH release suggested that acetylcholine (ACh) acts through different cholinergic receptor subtypes to exert stimulatory and inhibitory effects on GnRH release. Whereas activation of nicotinic receptors caused a transient increase in GnRH release, activation of muscarinic receptors inhibited basal GnRH release. Nanomolar concentrations of ACh caused dose-dependent inhibition of cAMP production that was prevented by pertussis toxin (PTX), consistent with the activation of a plasma-membrane Gi protein. Micromolar concentrations of ACh also caused an increase in phosphoinositide hydrolysis that was inhibited by the M1 receptor antagonist, pirenzepine. In ACh-treated cells, immunoblot analysis revealed that membrane-associated G(alpha q/11) immunoreactivity was decreased after 5 min but was restored at later times. In contrast, immunoreactive G(alpha i3) was decreased for up to 120 min after ACh treatment. The agonist-induced changes in G protein alpha-subunits liberated during activation of muscarinic receptors were correlated with regulation of their respective transduction pathways. These results indicate that ACh modulates GnRH release from hypothalamic neurons through both M1 and M2 muscarinic receptors. These receptor subtypes are coupled to Gq and Gi proteins that respectively influence the activities of PLC and adenylyl cyclase/ion channels, with consequent effects on neurosecretion.

Visual detection of transport-P in peptidergic neurones.

1. Hypothalamic peptidergic neurones possess an uptake process for amines (transport-P), for which prazosin is a substrate. It is characterized by a paradoxical increase in the accumulation of [3H]-prazosin when the concentration of unlabelled prazosin is increased above 10(-7) M. This increase is due to activation of a proton-dependent, vacuolar-type ATPase-linked pump that is blocked by tricyclic antidepressants. This study utilized a fluorescence method to detect amine uptake in individual cells. 2. Prazosin is fluorescent but most of its emission spectrum is in the ultraviolet range. We therefore used an analogue of prazosin in which the furan ring had been substituted with a fluorescent group, BODIPY FL. This compound's emission maximum is in the green part of the visible spectrum. 3. BODIPY FL prazosin accumulated in immortalised peptidergic neurones and the characteristic emission spectrum of the compound was evident in these cells. Accumulation of BODIPY FL prazosin was saturable and was inhibited by the tricyclic antidepressant desipramine and by unlabelled prazosin. As previously described for prazosin, uptake of BODIPY FL prazosin was blocked by cold temperature and by the organic base chloroquine. Thus, prazosin and BODIPY FL prazosin were accumulated by the same uptake process. 4. BODIPY FL prazosin accumulated in a granular distribution, which is compatible with storage in intracellular vesicles. 5. Hypothalamic cells from foetal rats in primary culture also accumulated BODIPY FL prazosin by a desipramine-sensitive process. Uptake was predominantly in neurones and glial cells did not accumulate the amine. 6. Fluorescent detection provides visual evidence for amine uptake in peptidergic neurones and should enable detailed study of the distribution of this process in the brain.

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.

Activation of LH receptors expressed in GnRH neurons stimulates cyclic AMP production and inhibits pulsatile neuropeptide release.

The GT1-7 cell line of immortalized GnRH neurons has been shown to express receptors for GnRH, LH, and prolactin, as well as a variety of other hormones and transmitters. Treatment of GT1-7 cells with hCG caused a dose-dependent increase in cAMP production, with a rapid increase during the first 15 min and a subsequent decrease that was prevented by pre-treatment with pertussis toxin. Furthermore, the stimulatory effect of cholera toxin on cAMP production was inhibited by hCG in a dose-dependent manner. These data indicate that the LH receptors expressed in GT1-7 cells are coupled to adenylyl cyclase both stimulatory (Gs) and inhibitory (Gi) proteins. In perifused cell cultures, treatment with forskolin and 8-bromo cAMP increased the amplitude of spontaneous GnRH release. However, treatment with nanomolar concentrations of hCG abolished pulsatile GnRH release from both GT1-7 cells and rat hypothalamic cells. The similarity of hCG action on pulsatile GnRH release to that of extracellular Ca2+ depletion and calcium channel antagonists, and its partial resistance to potassium-induced depolarization, suggest that it results from inhibition of plasma-membrane ion channel activity. It is probable that the inhibitory action of hCG on pulsatile GnRH release is responsible for initiation of the suppression of pituitary LH secretion during pregnancy.

L-type Ca2+ channels mediate joint modulation by gamma-amino-butyric acid and glutamate of [Ca2+]i and neuropeptide secretion in immortalized gonadodropin-releasing hormone neurons.

To examine the role of calcium signaling in the joint modulation of gonadotropin-releasing hormone (GnRH) secretion by gamma-aminobutyric acid (GABA) and glutamate, cytoplasmic calcium ([Ca2+]i) responses to the two transmitters were analyzed in monolayer networks of the GT1-7 line of immortalized GnRH neurons. [Ca2+]i was increased by GABA and the GABAA receptor agonist, muscimol, and these responses were inhibited by the GABAA receptor antagonist, bicuculline. In contrast, the GABAB receptor agonist, baclofen, and the GABAB receptor antagonist, phaclofen, had no effect on basal and GABA- and glutamate-induced Ca2+ levels in GT1-7 neurons. The GABA- and muscimol-induced responses consisted of a spike increase in [Ca2+]i followed by a decrease to a plateau; both the increase and the subsequent decrease in [Ca2+]i depended on agonist concentration. Glutamate, N-methyl-D-aspartate (NMDA), and kainate also increased [Ca2+]i, but were less effective than GABA. GABA-, glutamate-, NMDA-, and kainate-induced [Ca2+]i responses were almost abolished in Ca(2+)-free medium and were markedly attenuated by nifedipine. The [Ca2+]i response to GABA was unaffected by prior application of glutamate, and vice versa. This additive effect of glutamate on the GABA-induced [Ca2+]i response was mimicked by prior or simultaneous application of low (10 mM) KCl. The [Ca2+]i response to simultaneous application of GABA and glutamate was also equal to the sum of the individual responses, whereas the GnRH secretory response was larger. However, the secretory responses to GABA and glutamate applied individually or together, were markedly attenuated by nifedipine.(ABSTRACT TRUNCATED AT 250 WORDS)

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)

Sequential activation of phospholipase-C and -D in agonist-stimulated gonadotrophs.

The contributions of phospholipase-C and -D to diacylglycerol (DG) formation during agonist-induced cell signaling were investigated in rat pituitary cells and alpha T3-1 gonadotrophs. In both cell types, GnRH caused a biphasic increase in DG formation, with an initial spike within 60 sec, followed by a larger and sustained rise to reach a second peak after 15 min of stimulation. Both phases of DG production were temporally correlated with inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] formation, consistent with the dependence of DG formation on phospholipase-C-mediated phosphoinositide hydrolysis. However, the ability of GnRH to stimulate phosphatidylethanol (PEt) in the presence of ethanol suggested that phospholipase-D may also participate in DG formation. Two inhibitors of phospholipase-C-dependent phosphoinositide hydrolysis, U73122 and neomycin sulfate, reduced the PEt as well as the Ins(1,4,5)P3 response to GnRH, indicating that phospholipase-D is activated during phospholipase-C-dependent signaling in pituitary gonadotrophs. The production of both DG and PEt was increased by treatment with the active phorbol ester phorbol 12-myristate 13-acetate (PMA), but not with inactive 4 alpha-phorbol 13-didecanoate, indicating that stimulation of protein kinase-C leads to activation of phospholipase-D. In accord with this, GnRH- and PMA-induced elevations of DG and PEt production were attenuated or abolished in protein kinase-C-depleted cells. In contrast, short and long term stimulation with PMA had no effect on basal inositol phosphate production. Also, GnRH-induced inositol phosphate production was not affected by protein kinase-C depletion. Finally, U73122 and neomycin sulfate did not inhibit PMA-induced PEt formation. These data indicate that GnRH activates a dual phospholipase pathway in a sequential and synchronized manner; phospholipase-C initiates the biphasic increase in Ins(1,4,5)P3 and DG formation, and protein kinase-C mediates the integration of phospholipase-D into the signaling response during the sustained phase of agonist stimulation.

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.

Autocrine induction of c-fos expression in GT1 neuronal cells by gonadotropin-releasing hormone.

Activation of GnRH receptors in GnRH neuronal (GT1-7) cells causes marked and transient increases in c-fos expression, with peak response at 30 min. GnRH and des-Gly10-[D-Ala6]GnRH N-ethylamide induced concentration-dependent c-fos responses with EC50s of approximately 0.1 and approximately 1 nM, respectively. GnRH action was mimicked by phorbol 12-myristate-13-acetate (PMA), but stimulation of Ca2+ entry by K(+)-induced depolarization and Bay K 8644 was much less effective. In protein kinase C-depleted cells, the c-fos response to GnRH was reduced to that elicited by increased Ca2+ entry, and the effect of PMA was abolished. Thus, GnRH-induced c-fos expression in GT1 cells appears to be mediated predominantly by protein kinase C, and to a lesser extent by Ca2+. These findings demonstrate that c-fos expression can be induced in a peptidergic neuron by activation of receptors for its neurosecretory product. It is possible that the expression of c-fos in GnRH hypothalamic neurons during the proestrous surge of gonadotropins could likewise be stimulated by a positive feedback action of GnRH on its neuronal receptors.

High-affinity uptake of noradrenaline in postsynaptic neurones.

1. Neurotransmitters released from nerve endings are inactivated by re-uptake into the presynaptic nerve terminals and possibly into neighbouring glial cells. While analysing the functional properties of alpha 1-adrenoceptors in the hypothalamus, we observed a high-affinity uptake process for noradrenaline in postsynaptic peptidergic neurones. 2. In primary hypothalamic cell cultures and in a hypothalamic neuronal cell line, [3H]-prazosin bound with high affinity and was displaced by unlabelled prazosin in concentrations of 10(-10) to 10(-7) M. However, at concentrations of unlabelled prazosin above 10(-7) M, there was a paradoxical increase in apparent [3H]-prazosin binding. 3. Methoxamine, an alpha 1-adrenoceptor ligand that is not subject to significant neuronal uptake, displaced [3H]-prazosin but did not cause the paradoxical increase in the apparent binding of [3H]-prazosin. Cooling the cells to 4 degrees C reduced the total amount of prazosin associated with the cells; under these conditions, methoxamine almost completely inhibited [3H]-prazosin binding to the cells. 4. In the presence of desipramine (DMI), unlabelled prazosin displaced [3H]-prazosin as before, but no paradoxical increase in apparent binding was seen above 10(-7) M. 5. The paradoxical increase of [3H]-prazosin binding was not observed in membrane preparations of hypothalamic neurones. These findings indicated that the paradoxical increase in apparent [3H]-prazosin binding was due to a cellular uptake process that becomes evident at high concentrations of the ligand. 6. DMI (10(-5) M) had no effect on the specific binding of [3H]-prazosin. The presence of alpha1-adrenoceptors was confirmed by binding of [125]-HEAT, but [3H]-idazoxan (an alpha2- ligand) did not bind to the cells.7. The uptake of prazosin obeyed the Michaelis-Menten model, with similar Km and Vmax values in both types of cultures.8. Noradrenaline was taken up with high affinity by both types of cultures. (+/-)-[3H]-noradrenaline uptake was reduced by DMI and by excluding sodium from the medium, indicating that this process has some of the properties of uptake 1. (+/-)-[3H]-noradrenaline uptake in the cell line was unaffected by testosterone.9. The measured uptake of (-)-noradrenaline in the cell line was considerably increased by blockade of catechol-omicron-methyl-transferase and monoamine oxidase, suggesting that (-)-noradrenaline is metabolized to lipophilic products that escape across the plasma membrane.10. Studies in rats, in which the noradrenaline isomer 6-hydroxydopamine was used, suggested that the post synaptic uptake process is operative in hypothalamic CRH and vasopressin neurones in vivo.11. The Km for (-)-noradrenaline was within the range for the high affinity uptake, process in noradrenergic neurones. Uptake takes place in concentrations at which noradrenaline activates alpha1-adrenoceptors.Removal of noradrenaline from the vicinity of the receptors may prevent desensitization,thus maintaining the responsiveness of postsynaptic neurones to the actions of the neurotransmitter.

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.

Actions of acetyl-L-carnitine on the hypothalamo-pituitary-gonadal system in female rats.

Acetyl-L-carnitine (ALC) is known to affect several aspects of neuronal activity. To evaluate the neuroendocrine actions of this compound, several endocrinological parameters were followed in ALC-treated and control animals during recovery from dark-induced anestrus. In treated animals, serum luteinizing hormone (LH) and prolactin levels were higher than those of controls during the proestrous and estrous phases of the cycle, and serum estradiol levels were higher during estrus. No significant changes were observed in serum levels of follicle-stimulating hormone and progesterone. Uterine weight was increased in ALC-treated rats during proestrus and estrus, but not in diestrus. The basal release of gonadotropin-releasing hormone (GnRH) from perifused hypothalamic slices of ALC-treated animals was elevated at proestrus and diestrus, and GnRH release elicited by high K+ was higher during all three phases of the cycle. The basal release of LH from perifused pituitaries of treated animals was elevated in diestrus, and the LH response to GnRH was higher in estrus and diestrus I. Depolarization with K+ caused increased LH secretion during proestrus and estrus in treated animals. In contrast to these effects of ALC treatment in vivo, no direct effects of ALC were observed during short- or long-term treatment of cultured pituitary cells. These results indicate that ALC treatment influences hypothalamo-pituitary function in a cycle stage-dependent manner, and increases the secretory activity of gonadotrophs and lactotrophs. Since no effects of ALC on basal and agonist-induced secretory responses of gonadotrophs were observed in vitro, it is probable that its effects on gonadotropin release are related to enhancement of GnRH neuronal function in the hypothalamus.