Characterisation of signalling mechanisms that underlie P2Y receptor-mediated vasoconstriction of rat intrapulmonary artery.

Nawazish-i-Husain Syed, Asrin Tengah, Charles Kennedy. University of Strathclyde, Glasgow, UK.

P2Y receptors are a family of G protein-coupled receptors that are activated by the endogenous nucleotides UTP and UDP. In the vascular system, endothelial P2Y receptors mediate vasodilation, whilst P2Y receptors located on arterial smooth muscle cells mediate vasoconstriction (Chootip et al., 2002). Previously we showed that Ca2+ influx via Cav1.2 ion channels (Mitchell et al., 2007), Ca2+-sensitisation via rho kinase and protein kinase C (PKC) (Tengah et al., 2007) contribute to the nucleotide-evoked vasoconstriction 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).

5 mm rings of IPA were dissected from male Sprague-Dawley rats (200-250g). The endothelium was removed by gentle rubbing of the intima and the rings were 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 (AD Instruments). Contractions were elicited by addition of agonists to the bath. Data were analysed using Student’s paired t test or one way ANOVA with Tukey’s comparison, as appropriate. Values of P < 0.05 were considered to be statistically significant.

UTP and UDP (both 300 µM) evoked slowly developing contractions, which reached a peak within 2-3 minutes. Preincubation for 15 min with the ICl,Ca blocker, niflumic acid (1 µM) had no effect on contractions to KCl (40 mM, n = 5), indicating that at this concentration it does not act in a non-specific manner to depress smooth muscle contractility. It did, however, reduce significantly the peak responses to UDP (P<0.05, n = 5) and UTP (P<0.01, n = 5) by approximately 40-55% of their control values. The degree of inhibition did not differ significantly between the nucleotides. We reported previously that nifedipine (1 µM) also significantly inhibited the peak response to UDP and UTP by approximately 40-55% (Mitchell et al., 2007). Coadministration of niflumic acid (1 µM) and nifedipine (1 µM) 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 (10 µM) reduced significantly the peak responses to UDP (P<0.01, n = 6) and UTP (P<0.01, n = 5) by approximately 20-30%, whilst the PKC inhibitor, GF109203X (10 µM) depressed the response to UTP by around 20% (P<0.01, n = 5) and that of UDP (P<0.01, n = 6) by about 50%. 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 (1 µM) 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 UTP and UDP. The remainder of the response is due to Ca2+-sensitisation via rho kinase and to PKC acting at an as yet unidentified site.

Chootip, K., Ness, K., Wang, J., Gurney, A.M. & Kennedy, C. (2002). Br. J. Pharmacol., 137, 637-646.

Mitchell, C., Gurney, A.M. and Kennedy, C. (2007). Life Sciences 2007, PC369.

Tengah, A. and Kennedy, C. (2007). Life Sciences 2007, PC366.