UTP evokes noradrenaline release from rat sympathetic neurons by activation of protein kinase C.

The pathway involved in UTP-evoked noradrenaline release was investigated in cultures of rat superior cervical ganglia. Northern blots revealed an age-related increase in levels of mRNA for P2Y6 receptors in cultures obtained at postnatal days 1 and 5, respectively, but no change in transcripts for P2Y1 and P2Y2. Likewise, UTP-evoked overflow of previously incorporated [(3)H]noradrenaline was six-fold higher in neurons obtained at postanatal day 5. Various protein kinase C inhibitors diminished UTP-, but not electrically, induced tritium overflow by > 70%, as did down-regulation of protein kinase C by 24 h exposure to phorbol ester. beta-Phorbol-12,13-dibutyrate and dioctanoylglycerol caused concentration-dependent increases in [(3)H] outflow of up to 6% of total radioactivity, and the secretagogue actions of these agents were reduced in the presence of protein kinase C inhibitors and in neurons pretreated with phorbol ester. Overflow evoked by dioctanoylglycerol was attenuated in the absence of extracellular Ca(2+) and in the presence of tetrodotoxin or Cd(2+). In addition to triggering tritium overflow, UTP reduced currents through muscarinic K(+) channels which, however, were not affected by phorbol esters. This action of UTP was not altered by protein kinase C inhibitors. These results indicate that P2Y6 receptors mediate UTP-evoked noradrenaline release from rat sympathetic neurons via activation of protein kinase C, but not inhibition of K(M) channels.

P2Y receptor-mediated inhibition of voltage-activated Ca(2+) currents in PC12 cells.

To search for inhibitory nucleotide receptors in the sympathoadrenal cell lineage of the rat, voltage-activated Ca(2+) currents were recorded in PC12 cells after differentiation with nerve growth factor. ADP and ATP, but not uridine nucleotides, reduced Ca(2+) current amplitudes and slowed activation kinetics. This effect was mediated by GTP binding proteins, as it was abolished by intracellular GDP beta S and after treatment with pertussis toxin. Furthermore, depolarizations preceding the activation of Ca(2+) currents abolished the ADP-induced slowing of activation kinetics and attenuated its inhibitory action on current amplitudes. The modulatory effect of ADP was neither altered in the presence of adenosine receptor antagonists, nor mimicked by agonists at these receptors. In addition, the action of ADP was antagonized by reactive blue 2, but not by suramin or PPADS. Nucleotides tested for their inhibitory action on Ca(2+) currents displayed the following rank order of potency: 2-methylthio-ADP > or = 2-methylthio-ATP >> ADP beta S > ADP = ATP. When P2X receptors were blocked, the P2X agonists ATP and 2-methylthio-ATP still reduced Ca(2+) currents. The P2Y1 receptor antagonists adenosine-2'-phosphate-5'-phosphate and adenosine-3'-phosphate-5'-phosphate did not alter the inhibitory action of ADP, whereas the Sp-isomer of adenosine-5'-O-(1-thiotriphosphate) and 2'- and 3'-O-(4-benzoylbenzoyl)-ATP showed significant antagonistic activity. These results demonstrate that PC12 cells express an as yet unidentified P2Y receptor with pharmacological characteristics similar to those of P2Y1. As receptor-dependent modulation of Ca(2+) channels is a key event in presynaptic inhibition, this receptor may correspond to previously described presynaptic nucleotide receptors mediating autoinhibition of sympathetic transmitter release.

Two different signaling mechanisms involved in the excitation of rat sympathetic neurons by uridine nucleotides.

UTP stimulates transmitter release and inhibits M-type K(+) channels in rat superior cervical ganglion neurons via G protein-coupled P2Y receptors. To investigate the underlying signaling mechanisms, we treated the neurons with either pertussis or cholera toxin; neither treatment altered the inhibition of M-type K(+) channels by 10 microM UTP. However, pertussis toxin reduced UTP-evoked [(3)H]noradrenaline release by 66%. UTP, UDP, ATP, and ADP caused accumulation of inositol trisphosphate in a pertussis toxin-insensitive manner. Pharmacological inhibition of inositol trisphosphate-induced Ca(2+) release (by inhibition of phospholipase C, of inositol trisphosphate receptors, and of the endoplasmic Ca(2+)-ATPase) prevented the UTP-dependent inhibition of M currents but failed to alter UTP-evoked [(3)H]noradrenaline release. Chelation of intracellular Ca(2+) by 1,2-bis(2-aminophenoxy)ethane-N, N,N',N'-tetraacetic acid also reduced the inhibition of M currents by UTP. In addition, all these manipulations attenuated the inhibition of M currents by bradykinin, but hardly affected the inhibitory action of oxotremorine M. These results demonstrate that UTP inhibits M-type K(+) channels via an inositol trisphosphate-dependent signaling cascade that is also used by bradykinin but not by muscarinic acetylcholine receptors. In contrast, the secretagogue action of UTP is largely independent of this signaling cascade but involves pertussis toxin-sensitive G proteins. Thus, UTP-sensitive P2Y receptors excite sympathetic neurons via at least two different signal transduction mechanisms.