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 Ca(2+)-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 P2X(2), P2X(3), P2X(4), P2X(6) and P2X(7) receptors were expressed in the SON, and mRNAs for P2X(3), P2X(4) and P2X(7) were predominant. 3. In situ hybridization histochemistry for P2X(3) and P2X(4) receptor mRNAs showed that both mRNAs were expressed throughout the SON and in the paraventricular nucleus (PVN). 4. ATP caused an increase in [Ca(2+)](i) in a dose-dependent manner with an ED(50) of 1.7 x 10(-5) M. The effects of ATP were mimicked by ATP gamma S and 2-methylthio ATP (2MeSATP), but not by AMP, adenosine, UTP or UDP. alpha beta-Methylene ATP (alpha beta MeATP) and ADP caused a small increase in [Ca(2+)](i) in a subset of SON neurones. 5. The P2X(7) agonist 2'- &3'-O-(4-benzoylbenzoyl)-ATP (BzATP) at 10(-4) M increased [Ca(2+)](i), but the potency of BzATP was lower than that of ATP. In contrast, BzATP caused a more prominent [Ca(2+)](i) increase than ATP in non-neuronal cells in the SON. 6. The effects of ATP were abolished by extracellular Ca(2+) 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 P2X(7) antagonist oxidized ATP, and the inhibitor of Ca(2+)-ATPase in intracellular Ca(2+) stores cyclopiazonic acid. 7. Two patterns of desensitization were observed in the [Ca(2+)](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 [Ca(2+)](i) response. 9. The results suggest that multiple P2X receptors, including P2X(3), are functionally expressed in SON neurones, and that activation of these receptors induces cationic currents and Ca(2+) entry. Such ionic and Ca(2+)-signalling mechanisms triggered by ATP may play an important role in the regulation of SON neurosecretory cells.