Central administration of Orphanin FQ inhibits GnRH secretion by ORL1 receptor in the median eminence of freely moving ovariectomized rats

Objective

This study aimed to investigate the possible role of Orphanin FQ (OFQ) in the regulation of hypothalamic gonadotropin-releasing hormone (GnRH) secretion.

Methods

The method of push-pull perfusion and radioimmunoassay (RIA) were adopted to examine the secretory profile of GnRH in the median eminence (ME) in freely moving ovariectomized (OVX) rats after intracerebroventricular (icv) injection of OFQ and/or [Nphe¹]NC(1–13)NH² (NC13), a competitive antagonists of the opioid receptor-like 1 receptor (ORL1 receptor).

Results

GnRH release from ME significantly decreased from 40 min to 80 min after the administration of 20 and 200 nmol OFQ in OVX rats (P< 0.05). This inhibitory effect of 20 nmol OFQ could be abolished by pretreatment with equal dose of NC13. More interestingly, GnRH secretion from ME was increased markedly 60 min after icv injection of 100 and 200 nmol NC13 (P < 0.05).

Conclusion

Our results suggested central administration of OFQ could inhibit the release of GnRH in the ME of hypothalamus through ORL1 receptor, providing further in vivo evidence supporting the role of OFQ in the control of GnRH secretion.     

Concentration-dependent effects of muscimol to enhance pulsatile GnRH release from GT1-7 neurons in vitro

Immortalized GT1-7 neurons were used to characterize the effect of muscimol, a GABAA receptor agonist, to enhance pulsatile gonadotropin-releasing hormone (GnRH) release. GT1-7 neurons were grown on Cytodex-3 beads and placed in special superfusion microchambers. The cells were superfused at a rate of 6.2 ml x h-1 with Media 199 (pH 7.35) using a commercially available perfusion system. After a pre-muscimol period of 120 min, the cells were exposed for 5 min to 0.35, 1, 5 or 10 microM muscimol or 5 microM muscimol+20 microM of the GABAA receptor antagonist, bicuculline. Following removal of the muscimol (and bicuculline, in the case of the latter experiment), the superfusion was continued for another 115 min. Sample fractions were collected at 5 min intervals throughout the perfusion. Basal GnRH release from the GT1-7 neurons was pulsatile with an average interpulse interval of 45.4+/-0.5 min and an average pulse amplitude of 191.5+/-22.6 pg x min x ml-1. Our results also demonstrated that the GABAA receptor agonist, muscimol, enhances pulsatile GnRH release from GT1-7 neurons in culture. The response to muscimol was saturable and concentration-dependent with an EC50 of 0.47 microM. The effects of 5 microM muscimol to increase GnRH pulsatility were blocked by co-exposure to the GABAA receptor antagonist, bicuculline. The average GnRH interpulse intervals were 41.7+/-1.8 min, 32.5+/-2.9 min, 30.6+/-0.7 min and 25.5+/-0.4 min in the period following exposure to 0.35, 1, 5 and 10 microM of muscimol, respectively (post-muscimol period). GnRH pulse amplitude (mean-area for each pulse) was increased during exposure to muscimol but not during the pre- or post-muscimol periods. The GABAA receptor antagonist, bicuculline, itself had no effect on pulsatile GnRH release. These results are consistent with previously published reports suggesting that activation of the GABAA receptor stimulates hypothalamic GnRH release in embryonic and neonatal animals.     

Orphanin FQ inhibits synaptic transmission and long-term potentiation in rat hippocampus

It is known that opioid peptides acting on opioid receptors can modulate hippocampal synaptic functions. Although a novel member of the opioid receptor family, ORL₁ receptors, that displays high-sequence homology with classical opioid receptors is abundant in the hippocampus, little is known regarding its role in synaptic function. The present study was designed to investigate whether activation of the ORL₁ receptor by its natural ligand, orphanin FQ, could modulate synaptic transmission and synaptic plasticity in the hippocampus. The actions of orphanin FQ in the CA1 and dentate gyrus were examined by field potential recordings in response to stimulation of Schaffer collaterals and perforant path, respectively. Our results showed that orphanin FQ, but not the inactive analog des-Phe¹-orphanin FQ, reduced both the slope of the excitatory postsynaptic potentials and population spike amplitude. The inhibitory effect of orphanin FQ is dose dependent and probably involves a presynaptic mechanism, as suggested by the significantly increased paired-pulse facilitation evoked in the presence of orphanin FQ. In addition, orphanin FQ was found to inhibit the induction of long-term potentiation at the Schaffer collateral-CA1 synapse. These results demonstrate that orphanin FQ can function as an inhibitory modulator regulating synaptic transmission and synaptic plasticity in the hippocampus, suggesting that activation of ORL₁ receptors may play an important role in synaptic plasticity involved in learning and memory. Hippocampus 7:88–94, 1997. © 1997 Wiley-Liss, Inc.     

Concentration-dependent effects of muscimol to enhance pulsatile GnRH release from GT1–7 neurons in vitro

Immortalized GT1–7 neurons were used to characterize the effect of muscimol, a GABA_A receptor agonist, to enhance pulsatile gonadotropin-releasing hormone (GnRH) release. GT1–7 neurons were grown on Cytodex-3 beads and placed in special superfusion microchambers. The cells were superfused at a rate of 6.2 ml·h⁻¹ with Media 199 (pH 7.35) using a commercially available perfusion system. After a pre-muscimol period of 120 min, the cells were exposed for 5 min to 0.35, 1, 5 or 10 μM muscimol or 5 μM muscimol+20 μM of the GABA_A receptor antagonist, bicuculline. Following removal of the muscimol (and bicuculline, in the case of the latter experiment), the superfusion was continued for another 115 min. Sample fractions were collected at 5 min intervals throughout the perfusion. Basal GnRH release from the GT1–7 neurons was pulsatile with an average interpulse interval of 45.4±0.5 min and an average pulse amplitude of 191.5±22.6 pg·min·ml⁻¹. Our results also demonstrated that the GABA_A receptor agonist, muscimol, enhances pulsatile GnRH release from GT1–7 neurons in culture. The response to muscimol was saturable and concentration-dependent with an EC₅₀ of 0.47 μM. The effects of 5 μM muscimol to increase GnRH pulsatility were blocked by co-exposure to the GABA_A receptor antagonist, bicuculline. The average GnRH interpulse intervals were 41.7±1.8 min, 32.5±2.9 min, 30.6±0.7 min and 25.5±0.4 min in the period following exposure to 0.35, 1, 5 and 10 μM of muscimol, respectively (post-muscimol period). GnRH pulse amplitude (mean-area for each pulse) was increased during exposure to muscimol but not during the pre- or post-muscimol periods. The GABA_A receptor antagonist, bicuculline, itself had no effect on pulsatile GnRH release. These results are consistent with previously published reports suggesting that activation of the GABA_A receptor stimulates hypothalamic GnRH release in embryonic and neonatal animals.     

Partial characterization of K-induced increase in [Ca]cyt and GnRH release in GT1-7 neurons

Secretion of pituitary gonadotropins is regulated centrally by the hypothalamic decapeptide gonadotropin releasing hormone (GnRH). Using the immortalized hypothalamic GT1-7 neuron, we characterized pharmacologically the dynamics of cytosolic Ca²⁺ and GnRH release in response to K⁺-induced depolarization of GT1-7 neurons. Our results showed that K⁺ concentrations from 7.5 to 60 mM increased [Ca²⁺]_cyt in a concentration-dependent manner. Resting [Ca²⁺]_cyt in GT1-7 cells was determined to be 69.7 ± 4.0 nM (mean ± S.E.M.; N = 69). K⁺-induced increases in [Ca²⁺]_cyt ranged from 58.2 nM at 7.5 mM [K⁺] to 347 nM at 60 mM [K⁺]. K⁺-induced GnRH release ranged from about 10 pg/ml at 7.5 mM [K⁺] to about 60 pg/ml at 45 mM [K⁺]. K⁺-induced increases in [Ca²⁺]_cyt and GnRH release were enhanced by 1 μM BayK 8644, an L-type Ca²⁺ channel agonist. The BayK enhancement was completely inhibited by 1 μM nimodipine, an L-type Ca²⁺ channel antagonist. Nimodipine (1 μM) alone partially inhibited K⁺-induced increases in [Ca²⁺]_cyt and GnRH release. Conotoxin (1 μM) alone had no effect on K⁺-induced GnRH release or [Ca²⁺]_cyt, but the combination of conotoxin (1 μM) and nimodipine (1 μM) inhibited K⁺-induced increase in [Ca²⁺]_cyt significantly more (p < 0.02) than nimodipine alone, suggesting that N-type Ca²⁺ channels exist in GT1-7 neurons and may be part of the response to K⁺. The response of [Ca²⁺]_cyt to K⁺ was linear with increasing [K⁺] whereas the response of GnRH release to increasing [K⁺] appeared to be saturable. K⁺-induced increase in [Ca²⁺]_cyt and GnRH release required extracellular [Ca²⁺]. These experiments suggest that voltage dependent N- and L-type Ca²⁺ channels are present in immortalized GT1-7 neurons and that GnRH release is, at least in part, dependent on these channels for release of GnRH.     

Orphanin FQ/nociceptin modulates glutamate- and kainic acid-induced currents in acutely isolated rat spinal dorsal horn neurons

The heptadecapeptide orphanin FQ (OFQ), also known as nociceptin (NOC), is a newly discovered endogenous ligand for the opioid-like G-protein coupled receptor, ORL1. In the present study, the effects of OFQ/NOC on glutamate (Glu), kainic acid (KA) and quisqualic acid (QA) induced currents were examined in isolated rat spinal dorsal horn neurons of young rats using whole-cell patch-clamp techniques. Glu, KA and QA elicited rapid inward currents in 90%, 69%, 83% of tested neurons. OFQ/NOC(0.03300 nM) failed to induce any changes of membrane currents, but modulated Glu-, KA- and QA-elicited currents. OFQ/NOC inhibited and potentiated Glu-induced currents in 40.6% and 27.3% of examined cells (n=106) respectively. In about one third examined neurons, OFQ/NOC had no detectable effects on Glu responses. OFQ/NOC also inhibited and enhanced KA- and QA-induced currents (inhibition: KA, 67.1%, N=76; QA, 50%, N=36. Potentiation: KA, 23.7%, N=76; QA, 16.7%, N=36). In about 10% of tested cells, Gluinduced currents were potentiated after the application of OFQ/NOC, and lasted for 2030 min. The inhibitory effects of OFQ/NOC on KA and QA responses were naloxone-insensitive. The C-terminal fragment OFQ(8–17) presented same effects on EAA-induced responses. Taken together, OFQ/NOC primarily inhibited Glu-, KA- and QA-induced currents in isolated rat spinal dorsal horn neurons via non-opioid mechanism, which might contribute to nociceptive transmission in the spinal level.     

Interleukin-2 stimulates the secretion of arginine vasopressin but not corticotropin-releasing hormone from rat hypothalamic cells in vitro

Cytokines are involved in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis, thus providing a means of communication between the immune and neuroendocrine systems. In this study IL-2 caused a dose-dependent stimulation of AVP secretion from both the intact rat hypothalamus in vitro and hypothalamic cell cultures. IL-2 did not increase the secretion of CRH in either preparation, nor did it prime the cells to respond to a subsequent dose of IL-2. Both preparations, however, were able to respond to known CRH secretagogues such as 5-HT and 56 mM K⁺. This may provide yet another line of communication between the immune and neuroendocrine systems.     

Cholesterol secoaldehyde, an ozonation product of cholesterol, induces amyloid aggregation and apoptosis in murine GT1-7 hypothalamic neurons

Aldehydic products from ozonation of cholesterol and peroxidation of phospholipids have been shown to accelerate aggregation of amyloid-beta (Abeta) in vitro. Here, we show that 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al (ChSeco), an ozonation product of cholesterol, induces Abeta aggregation, generation of reactive oxygen species (ROS), and cytotoxicity in murine GT1-7 hypothalamic neurons. The formation of Abeta aggregates in situ was dose-dependent at ChSeco concentrations ranging from 1 to 20 microM. The increase in insoluble Abeta aggregates at increasing concentrations of ChSeco was accompanied by a decrease in soluble Abeta as evidenced by Western blot analysis. The formation of ROS in neuronal cells was found to be dose- and time-dependent with the magnitude being higher at 20 microM compared to 10 microM ChSeco or untreated controls. The increase in ROS was associated with depletion of GSH. The cytotoxicity induced by ChSeco involved changes in phosphatidylserine translocation, DNA fragmentation, and caspase 3/7 activity that are characteristic of apoptosis. Pretreatment of neuronal cells with Trolox, a water-soluble analog of alpha-tocopherol offered partial, but significant protection against ChSeco-induced cell death, whereas, N-acetyl-L-cysteine (NAC) completely prevented the cytotoxic effects of ChSeco. NAC and Trolox were without any effects on ChSeco-induced Abeta aggregation. Fibrillogenesis inhibitors, which inhibited Abeta aggregation, did not inhibit cell death induced by ChSeco, implying that ROS generation, and not Abeta aggregation, plays a major role in the observed cytotoxicity. However, since Alzheimer's and other neurodegenerative diseases are slow and progressive, the formation of Abeta aggregates in vivo by ChSeco may have long-term pathological consequences.     

Interleukin-1 inhibits NMDA-stimulated GnRH secretion: associated effects on the release of hypothalamic inhibitory amino acid neurotransmitters

Immune system activation is often accompanied by alterations in the reproductive axis. Interleukin-1 (IL-1), a polypeptide cytokine, has been postulated as a chemical messenger between the immune and the neuroendocrine systems. Using superfused hypothalamic fragments explanted from intact male rats, we evaluated the effects of IL-1 (0. 5 and 5 nM) on basal and N-methyl-D-aspartate (NMDA)-stimulated release of gonadotropin-releasing hormone (GnRH), and the associated modifications in the output of inhibitory amino acid neurotransmitters involved in the control of GnRH secretion. IL-1 did not modify basal GnRH release, but markedly restrained the stimulatory effect of NMDA on GnRH secretion. gamma-Aminobutyric acid, glycine and taurine concentrations significantly increased in the superfusion medium only after pretreatment with the higher dose of IL-1 (p < 0.05). Our results indicate that this cytokine inhibits NMDA- stimulated GnRH release, affecting the activity and/or the release of hypothalamic excitatory and inhibitory amino acid neurotransmitters participating in the regulation of GnRH secretion.     

Immunoreactivity to vasopressin- but not oxytocin-associated neurophysin antiserum in phasic neurons of rat hypothalamic paraventricular nucleus

Bursts of action potentials were recorded intracellularly from 11 phasically firing magnocellular neurons in the paraventricular nucleus in slices of rat hypothalamus. The bursts of overshooting, often broadening action potentials (63-87 mV peak-to-peak) were superimposed on depolarizing plateau potentials. Phasic activity was recorded before and/or after the neurons were injected with the fluorescent dye Lucifer Yellow CH. Injected neurons were first examined in whole slices, and subsequently, in sectioned material, characterized immunocytochemically using antisera to vasopressin- and oxytocin-associated neurophysins (VP-NP and OT-NP respectively). The 11 injections produced 8 single dye filled neurons and 3 pairs of dye-coupled neurons, 14 dye-filled cells in all. Six of the single cells and all the dye coupled pairs were immunoreactive with VP-NP antiserum and not reactive with OT-NP antiserum. Most of these neurons were in areas of the nucleus in which VP-NP reactive cells predominated, but two were surrounded by OT-NP reactive cells. Two single, dye-filled, phasically active, magnocellular neurons failed to show immunoreactivity to either antiserum.     

The effects of the phytoestrogen,coumestrol, on gonadotropin-releasing hormone (GnRH) mRNA expression in GT1-7 GnRH neurones

Phytoestrogens can produce inhibitory effects on gonadotropin secretion in both animals and humans, although little is known about the mechanisms and the role of direct action on oestrogen receptors (ER) in this process. We examined the effect of coumestrol, alone and combined with ER antagonists, on gonadotropin-releasing hormone (GnRH) mRNA expression in GT1-7 cells. Coumestrol was found to have an inhibitory effect compared to controls, which was blocked by R,R-THC, a selective ER beta antagonist. These results suggest that ER beta is involved in the suppression of GnRH mRNA expression by coumestrol.     

Medial hypothalamic stimulation suppresses nociceptive spinal dorsal horn neurons but not the tail-flick reflex in the rat

This study investigated the potential analgesic effects of medial hypothalamic stimulation (HS) on a measure of nocifensive behavior (tail-flick test (TF] in awake rats, and potential inhibitory effects of identical HS on spinal dorsal horn neuronal responses to noxious skin heating in the same animals anesthetized with sodium pentobarbital. Sixty-five male Sprague-Dawley rats implanted with a bipolar stimulation electrode in histologically verified medial hypothalamic sites were tested behaviorally for TF suppression during HS (100 ms trains at 100 Hz, 3/s, 100-1100 microA) in 2-4 consecutive weekly test sessions. Thirty-three of these rats were then used in electrophysiological experiments to record responses of 36 dorsal horn units to noxious skin heating (48-54 degrees C, 10 s/2 min) of the hindfoot pad in the absence of and during HS. Behaviorally, 31/65 rats had no TF suppression at the highest HS intensity tested (1100 microA), 24/65 rats exhibited aversive behavior or motor activity which disallowed reliable TF testing, and only 10/65 rats showed TF suppression in at least one test session. In electrophysiological experiments, the heat-evoked responses of 25/36 dorsal horn units were inhibited to at least 50% of control during HS. The responses of 11 units remained at 65-100% of the control responses during HS of up to 1100 microA. In rats demonstrating TF suppression, 4/7 units were inhibited. In rats with no TF suppression, 10/15 units were inhibited, and in rats showing aversive behavior, 11/14 units were inhibited by HS. These data indicate that although HS suppresses spinal nociceptive neurons, it does not cause reliable TF suppression in unanesthetized rats and bring into question the often-held assumption that stimulation-evoked descending inhibition of spinal nociceptive neurons implies behavioral analgesia.     

Oestrogen induces rhythmic expression of the Kisspeptin-1 receptor GPR54 in hypothalamic gonadotrophin-releasing hormone-secreting GT1-7 cells

Oestrogen-stimulated preovulatory gonadotrophin surges are temporally regulated in a way that remains not fully understood. Mammalian ovulation requires surges of gonadotrophin-releasing hormone (GnRH), released from specialised neurones in the hypothalamus. Surge regulation is mediated by ovarian oestrogen (17 β-oestradiol; E(2) ) feedback-acting as a negative signal until the early afternoon of the pro-oestrous phase, at which point it stimulates robust increases in GnRH release. Multiple lines of evidence suggest a role for the circadian clock in surge generation, although the presence of endogenous oscillators in several neuronal populations throughout the mediobasal hypothalamus complicates an elucidation of the underlying mechanisms of circadian regulation. In the present study, we propose that endogenous oscillators within GnRH neurones are modulated by oestrogen to elicit GnRH surge secretion. One mechanism by which this may occur is through the up-regulation of receptors of known stimulators of GnRH, such as kisspeptin's cognate receptor, GPR54. Through analysis of mRNA and protein abundance patterns, we found that high levels of E(2) elicit circadian expression profiles of GPR54 in vitro, and that disruption of endogenous GnRH oscillators of the clock dampens this effect. Additionally, although kisspeptin administration to GT1-7 cells does not result in surge-level secretion, we observed increased GnRH secretion from GT1-7 cells treated with positive feedback levels of E(2) . These results in this in vitro neuronal model system suggest a possible mechanism whereby receptor expression levels, and thus GnRH sensitivity to kisspeptin, may change dramatically over the pro-oestrous day. In this way, elevated ovarian E(2) may increase kisspeptidergic tone at the same time as increasing GnRH neuronal sensitivity to this neuropeptide for maximal surge release.     

Adrenoceptors in the preoptic-anterior hypothalamic area stimulate secretion of prolactin but not growth hormone in the male rat

Plasma growth hormone (GH) and prolactin concentrations were measured by radioimmunoassay in unanesthetized male rats after stereotaxic microinjection of adrenergic agents and 6-hydroxydopamine into the preoptic-anterior hypothalamic area (PO/AHA). Norepinephrine, epinephrine, isoprenaline and clonidine failed to stimulate GH, moreover, 16 nanomoles norepinephrine produced a decrease. However, these agents stimulated prolactin secretion and the mixed alpha antagonist phentolamine, administered systemically, inhibited the stimulatory action of epinephrine on prolactin secretion. GH and prolactin secretory patterns were not affected by 6-hydroxydopamine disruption of catecholamine terminals in the PO/AHA. GH responses to adrenergic agonists and the failure of 6-hydroxydopamine to affect GH secretory patterns indicate that PO/AHA norepinephrine afferents do not facilitate GH secretion. Taken in conjunction with previous studies, the results suggest that there must be an extra-hypothalamic site at which norepinephrine is stimulatory for GH. Prolactin responses suggest that alpha adrenoceptors in the PO/AHA may participate in prolactin secretion.     

Estrogen inhibits tuberoinfundibular dopaminergic neurons but does not cause irreversible damage

Dopaminergic neurons of the hypothalamic tuberoinfundibular dopaminergic (TIDA) system exert a tonic inhibitory control on prolactin (PRL) secretion whereas estrogen, known to inhibit TIDA neuron function, has been postulated to be toxic to TIDA neurons when it is chronically high. In order to determine whether estrogen in high doses can cause permanent damage to TIDA function, we submitted young female rats to continue high doses of estrogen administered, either centrally (intrahypothalamic estrogen implants) or peripherally (subcutaneous estrogen implants or weekly intramuscular (i.m.) injections for 7 weeks), subsequently withdrawing the steroid and observing the evolution of lactotrophes, serum PRL and TIDA neurons. Serum PRL was measured by radioimmunoassay whereas tyrosine hydroxylase positive (TH+) neurons and PRL cells were morphometrically assessed in sections of fixed hypothalami and pituitaries, respectively. After 30 days, hypothalamic estrogen implants induced a significant increase in serum PRL, whereas TH+ neurons were not detectable in the arcuate-periventricular hypothalamic (ARC) region of estrogen-implanted rats. Removal of implants on day 30 restored TH expression in the ARC and brought serum PRL back to basal levels 30 days after estrogen withdrawal. Subcutaneous or i.m. administration of estrogen for 7 weeks induced a marked hyperprolactinemia. However, 30 weeks after estrogen withdrawal, TH neuron numbers in the ARC were back to normal and serum PRL returned to basal levels. After peripheral but not central estrogen withdrawal, pituitary weight and lactotrophic cell numbers remained slightly increased. Our data suggest that estrogen even at high doses, does not cause permanent damage to TIDA neurons.     

Estrogen inhibits tuberoinfundibular dopaminergic neurons but does not cause irreversible damage

Dopaminergic neurons of the hypothalamic tuberoinfundibular dopaminergic (TIDA) system exert a tonic inhibitory control on prolactin (PRL) secretion whereas estrogen, known to inhibit TIDA neuron function, has been postulated to be toxic to TIDA neurons when it is chronically high. In order to determine whether estrogen in high doses can cause permanent damage to TIDA function, we submitted young female rats to continue high doses of estrogen administered, either centrally (intrahypothalamic estrogen implants) or peripherally (subcutaneous estrogen implants or weekly intramuscular (i.m.) injections for 7 weeks), subsequently withdrawing the steroid and observing the evolution of lactotrophes, serum PRL and TIDA neurons. Serum PRL was measured by radioimmunoassay whereas tyrosine hydroxylase positive (TH+) neurons and PRL cells were morphometrically assessed in sections of fixed hypothalami and pituitaries, respectively. After 30 days, hypothalamic estrogen implants induced a significant increase in serum PRL, whereas TH+ neurons were not detectable in the arcuate-periventricular hypothalamic (ARC) region of estrogen-implanted rats. Removal of implants on day 30 restored TH expression in the ARC and brought serum PRL back to basal levels 30 days after estrogen withdrawal. Subcutaneous or i.m. administration of estrogen for 7 weeks induced a marked hyperprolactinemia. However, 30 weeks after estrogen withdrawal, TH neuron numbers in the ARC were back to normal and serum PRL returned to basal levels. After peripheral but not central estrogen withdrawal, pituitary weight and lactotrophic cell numbers remained slightly increased. Our data suggest that estrogen even at high doses, does not cause permanent damage to TIDA neurons.     

Nociceptin/orphanin FQ inhibits ischaemia-induced glutamate efflux from rat cerebrocortical slices

Nociceptin/orphanin FQ (NC), the endogenous ligand for the G-protein coupled nociceptin receptor (NCR), has a modulatory role in various physiological processes including neurotransmitter release. We have examined the effects of NC, the analogues NC(1-13)NH2 and [F/G]NC(1-13)NH2 and the competitive antagonist [Nphe1]NC(1-13)NH2 (Nphe1) on glutamate efflux during an acute simulated ischaemic challenge in rat cerebrocortical slices. The increase in glutamate efflux seen with ischaemia was inhibited by NC (EC50 250 nM). At micromolar concentrations, the analogues were found to have a similar effect on glutamate efflux compared to NC. In all cases, inhibition of glutamate efflux was abolished by Nphe1 (30 microM). These results suggest a neuroprotective action for NC.     

Lead-induced cell signaling cascades in GT1-7 cells

The effects of lead on the signal transduction pathways that may be involved in the release of gonadotropin-releasing hormone (GnRH) from neurons in the hypothalamus have not been well defined. Using the GT1-7 cell line, an in vitro model for GnRH-secreting neurons, we examined signal transduction pathways directly affected by lead. We found that lead-induced phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1 and ERK2), as well as p90RSK and cAMP response element-binding protein (CREB), but did not induce IkappaB degradation. MEK1/2 inhibitor (PD98059) suppressed lead-induced ERK and p90RSK activation. Neither PKC inhibitors (Go6983, Go6976) nor CaMKII inhibitor (KN-62) had a pronounced effect on lead-induced ERK1 and ERK2 phosphorylation. However, MEK1/2 inhibitor, CaMKII inhibitor, and PKC inhibitor significantly suppressed lead-induced CREB phosphorylation. These results indicate that lead-activated PKC, CaMKII and MEK/ERK/p90RSK pathways simultaneously, all of which contributed to CREB phosphorylation. Our results also indicate that lead-induced p90RSK and CREB activation does not alter expression of early response genes like c-fos. We conclude that lead activates PKC, CaMKII or MEK-ERK-p90RSK pathways in GT1-7 cells, leading to CREB phosphorylation and modulation of gene expression.     

Exogenous cholecystokinin activates cFos expression in medullary but not hypothalamic neurons in neonatal rats

The central distribution of neurons activated to express cFos immunoreactivity in response to peripheral administration of cholecystokinin octapeptide (CCK) was examined in 2-day-old rats. Similar to previous results in adult rats, neurons in specific subregions of the area postrema and nucleus of the solitary tract (NST) expressed cFos after CCK treatment. However, in marked contrast to results in adult rats, CCK treatment in neonates did not stimulate cFos expression in hypothalamic neurons or in other forebrain areas, and did not increase plasma oxytocin levels. These results suggest that vagal sensory activation of intrinsic brainstem circuits may be sufficient for the known inhibitory effects of exogenous CCK on gastric motility and feeding in neonatal rats. The prominent forebrain activation produced by CCK administration in adult rats likely reflects later maturation of direct and relayed ascending neural projections from the NST.