Nobuya Harayama

Evidence that multiple P2X purinoceptors are functionally expressed in rat supraoptic neurones

1 The expression, distribution and function of P2X purinoceptors in the supraoptic nucleus (SON) were investigated by reverse transcription‐polymerase chain reaction (RT‐PCR), in situ hybridization, and Ca2+‐imaging and whole‐cell patch‐clamp techniques, respectively. 2 RT‐PCR analysis of all seven known P2X receptor mRNAs in circular punches of the SON revealed that mRNAs for P2X2, P2X3, P2X4, P2X6 and P2X7 receptors were expressed in the SON, and mRNAs for P2X3, P2X4 and P2X7 were predominant. 3 In situ hybridization histochemistry for P2X3 and P2X4 receptor mRNAs showed that both mRNAs were expressed throughout the SON and in the paraventricular nucleus (PVN). 4 ATP caused an increase in [Ca2+]i in a dose‐dependent manner with an ED50 of 1.7 × 10−5m. The effects of ATP were mimicked by ATPγS and 2‐methylthio ATP (2MeSATP), but not by AMP, adenosine, UTP or UDP. αβ‐Methylene ATP (αβMeATP) and ADP caused a small increase in [Ca2+]i in a subset of SON neurones. 5 The P2X7 agonist 2′‐ & 3′‐O‐(4‐benzoylbenzoyl)‐ATP (BzATP) at 10−4m increased [Ca2+]i, but the potency of BzATP was lower than that of ATP. In contrast, BzATP caused a more prominent [Ca2+]i increase than ATP in non‐neuronal cells in the SON. 6 The effects of ATP were abolished by extracellular Ca2+ removal or by the P2 antagonist pyridoxal phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS), and inhibited by extracellular Na+ replacement or another P2 antagonist, suramin, but were unaffected by the P2X7 antagonist oxidized ATP, and the inhibitor of Ca2+‐ATPase in intracellular Ca2+ stores cyclopiazonic acid. 7 Two patterns of desensitization were observed in the [Ca2+]i response to repeated applications of ATP: some neurones showed little or moderate desensitization, while others showed strong desensitization. 8 Whole‐cell patch‐clamp analysis showed that ATP induced cationic currents with marked inward rectification. The ATP‐induced currents exhibited two patterns of desensitization similar to those observed in the [Ca2+]i response. 9 The results suggest that multiple P2X receptors, including P2X3, are functionally expressed in SON neurones, and that activation of these receptors induces cationic currents and Ca2+ entry. Such ionic and Ca2+‐signalling mechanisms triggered by ATP may play an important role in the regulation of SON neurosecretory cells.

Inhibition of N- and P/Q-type calcium channels by postsynaptic GABAB receptor activation in rat supraoptic neurones

1 Voltage‐dependent Ca2+ currents of dissociated rat supraoptic nucleus (SON) neurones were measured using the whole‐cell configuration of the patch‐clamp technique to examine direct postsynaptic effects of GABAB receptor activation on SON magnocellular neurones. 2 The selective GABAB agonist baclofen reversibly inhibited voltage‐dependent Ca2+ currents elicited by voltage steps from a holding potential of −80 mV to depolarized potentials in a dose‐dependent manner. The ED50 of baclofen for inhibiting Ca2+ currents was 1.4 × 10−6 M. Baclofen did not inhibit low threshold Ca2+ currents elicited by voltage steps from −120 to −40 mV. 3 Inhibition of high threshold Ca2+ currents by baclofen was rapidly and completely reversed by the selective GABAB antagonists, CGP 35348 and CGP 55845A, when the antagonists were added at the molar ratio vs. baclofen of 10 : 1 and 0.01 : 1, respectively. It was also reversed by a prepulse to +150 mV lasting for 100 ms. 4 The inhibition of Ca2+ currents was abolished when the cells were pretreated with pertussis toxin for longer than 20 h or with N‐ethylmaleimide for 2 min. It was also abolished when GDPβS was included in the patch pipette. When GTPγS was included in the patch pipette, baclofen produced irreversible inhibition of Ca2+ currents and this inhibition was again reversed by the prepulse procedure. 5 The inhibition of N‐, P/Q‐, L‐ and R‐type Ca2+ channels by baclofen (10−5 M) was 24.1, 10.5, 3.1 and 3.6 %, respectively, of the total Ca2+ currents. Only the inhibition of N‐ and P/Q‐types was significant. 6 These results suggest that GABAB receptors exist in the postsynaptic sites of the SON magnocellular neurones and mediate selective inhibitory actions on voltage‐dependent Ca2+ channels of N‐ and P/Q‐types via pertussis toxin‐sensitive G proteins, and that such inhibitory mechanisms may play a role in the regulation of SON neurones by the GABA neurones.

PACAP increases the cytosolic Ca2+ concentration and stimulates somatodendritic vasopressin release in rat supraoptic neurons.

Pituitary adenylate cyclase activating polypeptide (PACAP)‐like immunoreactivity and its receptor mRNA have been reported in the supraoptic and the paraventricular nucleus (SON and PVN, respectively) and PACAP has been implicated in the regulation of magnocellular neurosecretory cell function. To examine the site and the mechanism of the action of PACAP in the neurosecretory cells, we measured AVP release from SON slice preparations and the cytosolic Ca2+ concentration ([Ca2+]i) from single dissociated SON neurons. PACAP at concentrations from 10−12 to 10−7 M increased [Ca2+]i in dissociated SON neurons in a dose‐dependent manner. The patterns of the PACAP‐induced [Ca2+]i increase were either sustained increase or cytosolic Ca2+ oscillations. PACAP (10−7 M) increased [Ca2+]i in 27 of 27 neurons and glutamate (10−4 M) increased [Ca2+]i in 19 of 19 SON neurons examined, whereas angiotensin II (10−7 M) increased [Ca2+]i in only 15 of 60 SON neurons examined. PACAP at lower concentrations (10−10 to 10−8 M) increased [Ca2+]i in 70–80% of neurons examined. Although the onset and recovery of the PACAP‐induced [Ca2+]i increase were slower than those observed with glutamate, the spatial distribution of the [Ca2+]i increases in response to the two ligands were similar: [Ca2+]i increase at the proximal dendrites was larger and faster and that at the center of the soma was smaller and slower. The PACAP‐induced [Ca2+]i responseswere abolished by extracellular Ca2+ removal, the l‐type Ca2+‐channel blocker, nicardipine, or by replacement of extracellular Na+ with N‐methyl d‐glucamine, and were partially inhibited by the Na+‐channel blocker, tetrodotoxin. The N‐type Ca2+‐channel blocker, ω‐conotoxin GVIA did not significantly inhibit the PACAP‐induced [Ca2+]i responses. Furthermore, PACAP (10−7 M) as well as glutamate (10−4 M) increased AVP release from SON slice preparations, and extracellular Ca2+ removal or nicardipine inhibited the AVP release in response to PACAP.

The Mechanism of Inhibitory Actions of Propofol on Rat Supraoptic Neurons

BACKGROUND In the perioperative period, plasma osmotic pressure, systemic blood pressure, and blood volume often change dramatically. Arginine vasopressin is a key factor in the regulation of these parameters. This study was performed to evaluate the direct and the mechanism of the actions of propofol on arginine vasopressin release from magnocellular neurosecretory neurons in the rat supraoptic nucleus. METHODS Somatodendritic arginine vasopressin release from supraoptic nucleus slice preparations was measured by radioimmunoassay. Ionic currents were measured using the whole-cell mode of the patch-clamp technique in supraoptic nucleus slice preparations or in single dissociated supraoptic nucleus neurons of the rat. RESULTS Propofol at concentrations greater than 10(-5) M inhibited the arginine vasopressin release stimulated by potassium chloride (50 mM). This inhibition by propofol was not reversed by picrotoxin, a gamma-aminobutyric acid(A)(GABA(A)) receptor antagonist, whereas arginine vasopressin release induced by glutamate (10(-3) M) was also inhibited by propofol at a clinically relevant concentration (10(-6) M). The latter effect was reversed by picrotoxin. Propofol evoked Cl- currents at concentrations ranging 10(-6) to 10(-4) M. Propofol (10(-6) M) enhanced the GABA (10(-6) M)-induced current synergistically. Moreover, propofol (10(-6) M) prolonged the time constant of spontaneous GABA-mediated inhibitory postsynaptic currents. Furthermore, propofol (10(-5) M and 10(-4) M) reversibly inhibited voltage-gated Ca2+ currents, whereas it did not affect currents induced by glutamate (10(-3) M). CONCLUSIONS Propofol inhibits somatodendritic arginine vasopressin release from the supraoptic nucleus, and the enhancement of GABAergic inhibitory synaptic inputs and the inhibition of voltage-gated Ca2+ entry are involved in the inhibition of arginine vasopressin release.

Patch-clamp analysis of the mechanism of PACAP-induced excitation in rat supraoptic neurones.

In neurosecretory cells of the supraoptic nucleus (SON) of rats, pituitary adenylate cyclase activating polypeptide (PACAP)causes an increase in [Ca2+]i, and stimulates somatodendritic vasopressin (VP) release. In this report, to elucidate the ionic mechanism of the action of PACAP, membrane potentials and ionic currents were measured from SON neurones in slice preparations or from dissociated SON neurones. In the current clamp mode, PACAP depolarized membrane potentials of both phasic and non‐phasic neurones and increased the firing rate. Moreover, simultaneous measurements of membrane potentials and [Ca2+]i revealed that the membrane depolarization correlated well with increases in [Ca2+]i. In the voltage‐clamp mode, PACAP induced inward currents at a holding potential of −70 or −80 mV in a dose‐dependent manner and the time course of the currents was similar to that of the PACAP‐induced membrane depolarization. The averaged reversal potential of the PACAP‐induced currents obtained from dissociated SON neurones was −33 mV, which was close to the reversal potential of non‐selective cation currents in SON neurones. The currents were rapidly and reversibly inhibited by a cation‐channel blocker, gadolinium. Analysis of synaptic inputs into SON neurones in slice preparations revealed that PACAP had little or no effects on the frequency of spontaneous excitatory and inhibitory postsynaptic currents. These results suggest that pituitary adenylate cyclase activating polypeptide (PACAP) activates PACAPreceptors in the postsynaptic membrane of the supraoptic nucleus (SON) neurones, and that the activation of PACAP receptors leads to opening of non‐selective cation channels, depolarization of the membrane potential, and increase in the firing rate in SON neurones. Such mechanisms may account for the PACAP‐induced increase in [Ca2+]i and vasopressin (VP) release observed in SON neurones.

Involvement of Postsynaptic EP4 and Presynaptic EP3 Receptors in Actions of Prostaglandin E2 in Rat Supraoptic Neurones

We have reported that supraoptic nucleus (SON) neurones are excited by prostaglandin E2 (PGE2) presumably via dual postsynaptic PG receptors, FP receptors and unidentified EP receptors, and that presynaptic EP receptors may also be involved in the excitation. In the present study, to clarify the receptor mechanism of the PGE2‐mediated actions on SON neurones, we studied the pre‐ and postsynaptic effects of four newly developed EP agonists that are selective for each of the four EP receptors, EP1−4, on rat SON neurones using extracellular recording and whole‐cell patch‐clamp techniques. The EP4 agonist ONO‐AE1‐329 mimicked the excitatory effects of PGE2, whereas the EP1 agonist ONO‐DI‐004, the EP2 agonist ONO‐AE1‐257 and the EP3 agonist ONO‐AE‐248 had little or no effect. The effects of ONO‐AE1‐329 were unaffected by the EP1/FP/TP antagonist, ONO‐NT‐012, which potently suppressed the excitation caused by the FP agonist fluprostenol and PGE2. ONO‐AE1‐329 caused marked excitation when responses to fluprostenol were desensitized by repeated applications of fluprostenol. Patch‐clamp analysis in SON neurones showed that ONO‐AE1‐329 induced inward currents at a holding potential of −70 mV and the reversal potential of the currents was −35.1 ± 2.3 mV. On the other hand, the frequency of spontaneous inhibitory postsynaptic currents recorded from SON slice preparations was suppressed by ONO‐AE‐248, but unaffected by the other three EP agonists. These results suggest that SON neurones possess postsynaptic EP4 receptors and that γ‐aminobutyric acid neurones innervating SON neurones possess presynaptic EP3 receptors in their terminals. Activation of the two EP receptors may be involved in the excitatory regulation of SON neurones by PGE2.

Proadrenomedullin N-terminal 20 peptide (PAMP) reduces inward currents and Ca2+ rises induced by nicotine in bovine adrenal medullary cells

It has been recently reported that proadrenomedullin N-terminal 20 peptide (PAMP), which is secreted with adrenomedullin and catecholamines from the adrenal medulla, inhibits catecholamine release stimulated with nicotine. In the present study, to elucidate anticholinergic mechanisms of PAMP we employed the whole-cell patch-clamp and the intracellular Ca2+ imaging techniques in cultured bovine adrenal medullary cells. PAMP inhibited nicotinic currents and [Ca2+]i rises induced by nicotine in a dose-dependent manner (10(-9)-10(6) M). These inhibitions were selective, since PAMP alone did not induce any ionic currents, moreover it did not affect voltage-dependent Ba2+ currents or high K+ (50 mM)-induced [Ca2+]i rises. The onset of the inhibitory effect of PAMP (10(-6) M) was very rapid and reached a steady-state level within 10 sec. The effect of PAMP (10(-6) M) lasted for about 10-15 min. Desensitization process of the nicotinic current fitted to a single exponential function with a time constant of 6.4 +/- 0.3 sec. When PAMP (10(-6) M) simultaneously added with nicotine (10(-5) M), the desensitization process was facilitated and fitted to two exponentials with time constants of 0.46 +/- 0.08 and 2.5 +/- 0.8 sec. From the present results, the inhibition by PAMP of nicotinic currents which was well associated with that of nicotine induced [Ca2+]i rises leads to the attenuation of catecholamine release probably, at least in part, due to the facilitation of the desensitization process of the nicotinic currents.

Mechanisms of Cytosolic Ca2+ Suppression By Prostaglandin E2 Receptors in Rat Melanotrophs

We have previously reported that voltage‐dependent Ca2+ (VDC) channels of rat melanotrophs are inhibited by prostaglandin E2 (PGE2). In this study, mechanisms involved in the inhibitory actions of PGE2 receptors of rat melanotrophs were analysed using reverse transcriptase‐polymerase chain reaction (RT‐PCR), Ca2+‐imaging and whole‐cell, patch‐clamp techniques with recently developed EP agonists, each of which is selective for the known four subclasses of EP receptors (EP1–4). PGE2 reversibly suppressed the cytosolic Ca2+ concentration ([Ca2+]i). The maximum reduction in [Ca2+]i by PGE2 was comparable to that by dopamine or to that by extracellular Ca2+ removal. RT‐PCR analysis of all four EP receptors revealed that EP3 and EP4 receptor mRNAs were expressed in the intermediate lobe. The effects of PGE2 to suppress [Ca2+]i were mimicked by the selective EP3 agonist, ONO‐AE‐248, whereas three other EP agonists, ONO‐DI‐004 (EP1), ONO‐AE1‐259 (EP2) and ONO‐AE1‐329 (EP4), had little or no effect on [Ca2+]i. All four G‐protein activated inward rectifying K+ (GIRK) channel mRNAs were identified in intermediate lobe tissues by RT‐PCR. Dopamine concentration‐dependently activated GIRK currents, whereas PGE2 did not activate GIRK currents, even at the concentration causing maximal inhibition of VDC channels. These results suggest that PGE2 acts on EP3 receptors to suppress Ca2+ entry of rat melanotrophs by selectively inhibiting VDC channels of these cells. We have compared the possible cellular and molecular mechanisms of inhibition by dopamine and PGE2.

Analysis of G-protein-activated inward rectifying K+ (GIRK) channel currents upon GABAB receptor activation in rat supraoptic neurons

While magnocellular neurons in the supraoptic nucleus (SON) possess rich Gi/o-mediated mechanisms, molecular and cellular properties of G-protein-activated inwardly rectifying K(+) (GIRK) channels have been controversial. Here, properties of GIRK channels are examined by RT-PCR and whole-cell patch-clamp techniques in rat SON neurons. Patch clamp experiments showed that the selective GABAB agonist, baclofen, enhanced currents in a high K(+) condition. The baclofen-enhanced currents exhibited evident inward rectification and were blocked by the selective GABAB antagonist, CGP55845A, the IRK channel blocker, Ba(2+), and the selective GIRK channel blocker, tertiapin, indicating that baclofen activates GIRK channels via GABAB receptors. The GIRK currents were abolished by N-ethylmaleimide pretreatment, and prolonged by GTPγS inclusion in the patch pipette, suggesting that Gi/o proteins are involved. RT-PCR analysis revealed mRNAs for all four GIRK 1-4 channels and for both GABABR1 and GABABR2 receptors in rat SON. However, the concentration-dependency of the baclofen-induced activation of GIRK currents had an EC50 of 110 µM, which is about 100 times higher than that of baclofen-induced inhibition of voltage-dependent Ca(2+) channels. Moreover, baclofen caused no significant changes in the membrane potential and the firing rate. These results suggest that although GIRK channels can be activated by GABAB receptors via the Gi/o pathway, this occurs at high agonist concentrations, and thus may not be a physiological mechanism regulating the function of SON neurons. This property that the membrane potential receives little influence from GIRK currents seems to be uncommon for CNS neurons possessing rich Gi/o-coupled receptors, and could be a special feature of rat SON neurons.

2307 Simultaneous measurements with Ca2+ imaging and patch-clamp techniques in rat supraoptic magnocellular neurons

Tatsushi Onaka, Xiu Liu, Kinji Yagi Noxious stimuli activate the Xl and A2 noradrenergic neurones in the brainstem. The noradrenergic neurones project to hypothalamic magnocellular neurosecretory cells. Oytocin release from the neurohypophysis is facilitated by noxious stimuli. We have previously shown that an i.c.v. alphai-adrenoceptor antagonist or destruction of the ascending noradrenergic projection to the hypothalamus blocks oxytocin release after noxious stimuli. These data suggest that the noradrenergic afferents to oxytocin neurones mediate the hormone release after noxious stimuli. If this is the case, noradrenaline release in the supraoptic nucleus is expected to increase after noxious stimuli. We collected microdialysates of the nucleus before and after electric footshocks in urethane-anaesthetised rats and measured noradrenaline contents by high performance liquid chromatography with electrochemical detection. The results show that noxious stimuli increased noradrenaline release in the supraoptic nucleus.