1 P2Y receptors inhibiting adenylyl cyclase have been found in blood platelets, glioma cells, and endothelial cells. In platelets and glioma cells, these receptors were identi®ed as P2Y 12 . Here, we have used PC12 cells to search for adenylyl cyclase inhibiting P2Y receptors in a neuronal cellular environment. 2 ADP and ATP (0.1 ± 100 mM) left basal cyclic AMP accumulation unaltered, but reduced cyclic AMP synthesis stimulated by activation of endogenous A 2A or recombinant b 2 receptors. Forskolindependent cyclic AMP production was reduced by 41 mM and enhanced by 10 ± 100 mM ADP; this latter e ect was turned into an inhibition when A 2A receptors were blocked. 3 The nucleotide inhibition of cyclic AMP synthesis was not altered when P2X receptors were blocked, but abolished by pertussis toxin. 4 The rank order of agonist potencies for the reduction of cyclic AMP was (IC 50 values): 2-methylthio-ADP (0.12 nM)=2-methylthio-ATP (0.13 nM)4ADPbS (71 nM)4ATP (164 nM)=ADP (244 nM). The inhibition by ADP was not antagonized by suramin, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid, or adenosine-3'-phosphate-5'-phosphate, but attenuated by reactive blue 2, ATPaS, and 2-methylthio-AMP. 5 RT ± PCR demonstrated the expression of P2Y 2 , P2Y 4 , P2Y 6 , and P2Y 12 , but not P2Y 1 , receptors in PC12 cells. In Northern blots, only P2Y 2 and P2Y 12 were detectable. Di erentiation with NGF did not alter these hybridization signals and left the nucleotide inhibition of adenylyl cyclase unchanged.

Bradykinin has long been known to excite sympathetic neurons via B(2) receptors, and this action is believed to be mediated by an inhibition of M-currents via phospholipase C and inositol trisphosphate-dependent increases in intracellular Ca(2+). In primary cultures of rat superior cervical ganglion neurons, bradykinin caused an accumulation of inositol trisphosphate, an inhibition of M-currents, and a stimulation of action potential-mediated transmitter release. Blockade of inositol trisphosphate-dependent signaling cascades failed to affect the bradykinin-induced release of noradrenaline, but prevented the peptide-induced inhibition of M-currents. In contrast, inhibition or downregulation of protein kinase C reduced the stimulation of transmitter release, but not the inhibition of M-currents, by bradykinin. In cultures of superior cervical ganglia, classical (alpha, betaI, betaII), novel (delta, epsilon), and atypical (zeta) protein kinase C isozymes were detected by immunoblotting. Bradykinin induced a translocation of Ca(2+)-independent protein kinase C isoforms (delta and epsilon) from the cytosol to the membrane of the neurons, but left the cellular distribution of other isoforms unchanged. This activation of Ca(2+)-independent protein kinase C enzymes was prevented by a phospholipase C inhibitor. The bradykinin-dependent stimulation of noradrenaline release was reduced by inhibitors of classical and novel protein kinase C isozymes, but not by an inhibitor selective for Ca(2+)-dependent isoforms. These results demonstrate that bradykinin B(2) receptors are linked to phospholipase C to simultaneously activate two signaling pathways: one mediates an inositol trisphosphate- and Ca(2+)-dependent inhibition of M-currents, the other one leads to an excitation of sympathetic neurons independently of changes in M-currents through an activation of Ca(2+)-insensitive protein kinase C.

Bradykinin is known to stimulate neurons in rat sympathetic ganglia and to enhance transmitter release from their axons by interfering with the autoinhibitory feedback, actions that involve 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.

Background and Purpose-A ThrϾPro polymorphism at codon 715 in the coding region of the P-selectin gene has recently been described. Individuals carrying the Pro715 allele were reported to have a reduced risk of myocardial infarction. A possible association of this polymorphism with the risk of ischemic stroke is currently under discussion. Methods-We investigated the prevalence of the 715 ThrϾPro polymorphism in 450 patients aged younger than 60 years with ischemic stroke or transient ischemic attack and in 450 controls without vascular disease matched for age and gender. We also investigated possible interactions of the polymorphism with other vascular risk factors, stroke severity and stroke etiology. Results-The distribution of the two allelic variants of the 715ThrϾPro polymorphism did not differ significantly between patients and control subjects (78% versus 81% for Thr/Thr, 21% versus 18% for Thr/Pro and 1% versus 1% for Pro/Pro in patients and controls, respectively; adjusted odds ratio for carriers of the C allele: 1.0 [0.8 to 1.2; Pϭ0.695]). We found no significant interaction of this polymorphism with vascular risk factors, stroke severity, or stroke etiology. Conclusions-Our study supports results from previous investigation showing that the 715ThrϾPro polymorphism of the P-selectin gene was not associated with a risk or clinical characteristics of ischemic stroke.