P2Y receptors are a family of G protein-coupled receptors that are activated by the endogenous nucleotides UDP and UTP. In arteries, endothelial P2Y receptors mediate vasodilation, whilst P2Y receptors on arterial smooth muscle cells mediate vasoconstriction (Chootip et al., 2002). We have shown that Ca2+ influx via Cav1.2 ion channels (Mitchell et al., 2007), Ca2+-sensitisation via rho kinase and protein kinase C (PKC) (Tengah & Kennedy, 2007) all contribute to nucleotide-evoked contractions of rat intrapulmonary arteries (IPA). The aim of this study was to determine the relative roles of these signalling components and Ca2+-dependent Cl- channels (ICl,Ca). 5mm rings of IPA were dissected from male Sprague-Dawley rats (200-250g), which were killed by cervical dislocation. The endothelium removed by gentle rubbing of the intima and the rings mounted under isometric conditions in 1ml baths at 37°C and a resting tension of 0.5g. Tension was recorded by Grass FT03 transducers connected to a Powerlab/4e system. Data were analysed using Student’s paired t test or 1-way ANOVA as appropriate. UDP and UTP (both 300µM) evoked slowly developing contractions that reached a peak within 2-3 min. Preincubation for 15 min with the ICl,Ca blocker, niflumic acid (1µM) had no effect on contractions to KCl (40mM, n=5), showing that this concentration does not block Cav1.2 ion channels or interact directly with the myofilaments to depress smooth muscle contractility. The peak amplitude of responses to UDP (P<0.05, n=5) and UTP (P<0.01, n=5) was, however, reduced significantly by approximately 40-55%. Nifedipine (1µM) also significantly inhibits the peak response to UDP and UTP by about 40-55% (Mitchell et al., 2007) and coadministration of niflumic acid (1µM) plus nifedipine (1µM) for 15 min inhibited contractions to UDP and UTP to the same extent as either agent alone (n=4 each). Pre-incubation for 15 min with the rho kinase inhibitor Y27632 (10µM) reduced significantly the peak responses to UDP (P<0.01, n=6) and UTP (P<0.01, n=5) by around 20-30%, whilst the PKC inhibitor, GF109203X (10µM) depressed the response to UDP by about 50% (P<0.01, n=6) and that of UTP (P<0.01, n=5) by about 20%. Coapplication of both inhibitors produced significantly greater inhibition than either alone (UDP: P<0.001, n=6; UTP: P<0.01, n=7). Additional co-application of nifedipine (1µM) with Y27632 (10µM) and GF109203X (10µM) caused no further inhibition of the contractions to UDP (n=5), but further depressed the response to UTP (P<0.05, n=5). These results indicate that ICl,Ca induces Ca2+ influx via Cav1.2 ion channels, which mediates about half of the peak contraction amplitude of rat IPA evoked by UDP and UTP. The remainder of the response is due to Ca2+-sensitisation via rho kinase and to PKC acting at an as yet unidentified site.