Study of purinergic modulation of contractile activity in guinea-pig vas deferens and the interaction with superoxide anion and adrenergic activity

This study has been conducted & submitted in partial fulfillment of the Requirements for the master's degree in Pharmaceutical sciences (Pharmacology), Department of Pharmacology, College of Pharmacy, King Saud University 1428H- 2007GThe present study was designed to investigate the mechanisms of the purinergic transmitters (AP4A and ATP) and their contractile activity in the vas deferens of guinea-pig. In addition, the relationships between these compounds and noradrenaline (NA), superoxide anion, Myosin Light Chain Phosphatase (MLCP) and Protein Kinase C (PKC) were studied. The concentration-response curves (CRCs) to AP4A and ATP showed that AP4A is more potent than ATP in causing contraction. Furthermore, using different purinoceptor blockers such as: a) 8- cyclopentyl-1,3-dipropylxanthine (DPCPX) as A1-purinoceptor blocker, b) Pyridoxal phosphate 6-azophenyl-2’,4’-disulphonic acid (PPADS) as P2X–purinoceptor blocker and c) H,.-meATP as P2X-purinoceptor desensitizer produced a significant inhibition of AP4A and ATP contractile activities. The facilitation of AP4A- and ATP-induced contraction by NA was determined by measuring the contractile activities of AP4A and ATP in presence of NA. Our results indicate that NA significantly increased the contractile activities of AP4A and ATP in a dose-dependent manner. This facilitation could be due to an interaction between PKC and MLCP. Using MLCP inhibitor, canthiridin, leads to a significant stimulation on the contractile activity of AP4A and ATP. On the other hand, applying cheleythrine, a PKC Inhibitor, produced a significant inhibition of AP4A- and ATP-induced contraction activities in presence NA. These results suggest that the interaction between PKC and MLCP has a critical role in the facilitation of AP4A- and ATP-induced contraction by NA. Furthermore, we investigate the effect of superoxide anion on contractile activities of AP4A and ATP using endogenous and exogenous models of superoxide anion stress. The endogenous superoxide anion stress was generated by the Cu/Zn superoxide dismutase (SOD) inhibitor, Diethylthiocarbamate (DETCA), whereas the exogenous superoxide anion stress was induced by the superoxide anion generator pyrogallol. Both models inhibited the AP4A- and ATP-induced contraction, which suggests that superoxide anion has an inhibitory effect on the purinergicmediated contraction. In conclusion, the study presented in this thesis produced some potential understanding regarding the role of purine (mono- and dinucleotides) in the contractile activity of vas deferens and the interdependence of some mediators as follows: (a)P2X1 –purinoceptor has the major role in the contraction of vas deferens to AP4A and ATP, while A1–purinoceptor plays a partial role in the contraction of guinea-pig vas deferens to AP4A and ATP. (b)Inhibition of MLCP by Canthiridin resulted in significant enhancement of purine mediated contractions. (c) Prior application of NA produces an enhancement of both AP4A and ATP at postsynaptic sites of guinea-pig vas deferens. (d)Stimulation of PKC pathway by NA binding to G-protein coupled adrenergic receptors may involved in the postsynaptic synergism between NA and the studied purines (AP4A and ATP). (e)Superoxide anion stress has an inhibitory effect on the purinergic contraction in the guinea-pig vas deferens. This effect could be due to inhibition of extracellular Ca2+ influx or other sites of cellular Ca2+ handling