P2X1 Receptors Localized in Lipid Rafts Mediate ATP Motor Responses in the Human Vas Deferens Longitudinal Muscles1

Abstract

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 were prepared from tissue biopsies to record isometric contractions. To ascertain its membrane distribution, tissue extracts were analyzed by immunoblotting 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 demonstrated 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 minute, 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. Immunoblot analysis of the human vas deferens extracts revealed a P2X1R oligomer of approximately 200 kDa under nonreducing conditions, whereas dithiothreitol-treated extracts showed a single band of approximately 70 kDa. The P2X1R was identified in ultracentrifugation fractions containing 15%-29% 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 human vas deferens contractile responses of the longitudinal smooth muscle layer, an effect mediated through P2X1Rs.

ATP as a cotransmitter in the autonomic nervous system

“…Lipid rafts are specialised cholesterol-rich lipid microdomains present in the plasma membrane, which are involved in receptor signalling and trafficking. P2X1 receptors are present in lipid rafts in several smooth muscle tissues, including rat tail artery, vas deferens and urinary bladder (Vial and Evans, 2005) and human vas deferens (Donoso et al, 2014). Chemical disruption of the lipid rafts selectively inhibited P2X1 receptor signalling, showing that the rafts play a very important role in maintaining the functional activity of P2X1 receptors.…”

The purinergic neurotransmitter revisited: A single substance or multiple players?

“…It is believed that ATP that is coreleased with NE from sympathetic nerve terminals acts through P2X receptors on smooth muscle to mediate membrane depolarization (e.g., excitatory junction potentials, EJP) and contraction (Stjarne & Astrand, 1984; Sneddon & Burnstock, 1984b; Allcorn et al, 1986; Mulryan et al, 2000). Although some studies report no purinergic component in the mechanical responses of the human vas deferens to nerve stimulation (Holmquist et al, 1990; Medina et al, 1998; Medina et al, 2010), other studies have suggested a role for the P2X1 receptor in human vas deferens motor activity (Banks et al, 2006), and in the longitudinal smooth muscle layer in particular (Amobi et al, 2012; Donoso et al, 2014). …”

ATP as a cotransmitter in sympathetic and parasympathetic nerves - another Burnstock legacy

“…They are ionotropic receptors that are permeable to Na + , K + and Ca 2+ and so mediate cellular depolarisation and excitation. Evidence suggests that the functional activity of P2X1R in smooth muscle cells of rat tail artery, vas deferens and urinary bladder (Vial and Evans, 2005) and human vas deferens (Donoso et al, 2014) requires them to be localised to lipid rafts, specialised cholesterol-rich lipid microdomains in the plasma membrane, as chemical disruption of the lipid rafts selectively inhibited P2X1R signalling.…”