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

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.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.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).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.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.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.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.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.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

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.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.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.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.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.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