Running Title: P2X1Rs in human vas deferens localize in rafts Summary sentence: ATP elicits contractions of human vas deferens longitudinal muscles mediated by P2X1 receptors distributed in raft domains. Key words: P2X1 receptors, P2X receptor desensitization, human vas deferens, sympathetic co- transmission, ATP-induced contractility, P2X1 receptors in lipid rafts.

To assess the role of the P2X1 receptors (P2X1R) in the longitudinal and circular layers of the human vas deferens, ex-vivo isolated strips or rings from tissue biopsies were prepared to record isometric contractions. To ascertain its membrane distribution, tissue extracts were analyzed by immunoblots following sucrose gradient ultracentrifugation. ATP, alpha,beta-methylene ATP or electrical field stimulation elicited robust contractions of the longitudinal layer, but not of the circular layer which evidenced inconsistent responses. Alpha,beta-methylene ATP generated stronger and more robust contractions than ATP. In parallel, prostatic segments of the rat vas deferens were examined. The motor responses in both species were not sustained but decayed within the first min, showing desensitization to additional applications. Cross desensitization was established between alpha,beta-methylene ATP or ATP-evoked contractions and electrical field stimulation-induced contractions. Full recovery of the desensitized motor responses required more than 30 min and showed a similar pattern in human and rat tissues. Immuno blot analysis of the human vas deferens extracts revealed a P2X1R oligomer of approximately 200 kDa under non reducing conditions, whereas dithiothreitol-treated extracts showed a single band of approximately 70 kDa. The P2X1R was identified in ultracentrifugation fractions containing 1529 % sucrose; the receptor localized in the same fractions as flotillin-1, indicating that it regionalized into smooth muscle lipid rafts. In conclusion, ATP plays a key role in the human vas deferens contractile responses of the longitudinal smooth muscle layer, an effect mediated through P2X1Rs.