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Vaginal lubrication, an indicator of sexual arousal and tissue health, increases significantly during genital sexual arousal. Adrenergic alpha-receptors (AR) are an important regulator of genital physiological responses involved in mediating vascular and nonvascular smooth muscle contractility; the role of β-AR in sexual arousal, however, has not yet been investigated.The goal of this study was to reveal the functional role of β-AR in modulating vaginal lubrication during sexual arousal and the mechanisms underlying the process.The effects of adrenaline on vaginal epithelial ion transport, intracellular cyclic adenosine monophosphate (cAMP) content ([cAMP]i), and vaginal lubrication were investigated using short-circuit current (ISC) of rat vaginas incubated in vitro, enzyme-linked immunosorbent assay (ELISA), and measurement of vaginal lubrication in vivo, respectively. The expressions of β-AR in vaginal epithelium were analyzed by reverse transcription-polymerase chain reaction, western blot, and immunofluorescence.Changes of ISC responses; mRNA, protein expressions and localization of β-AR; [cAMP]i; vaginal lubrication.Serosal application of adrenaline induced an increase of ISC across rat vaginal epithelium that blocked by propranolol, a β-AR antagonist, rather than phentolamine, an α-AR antagonist. β1/2-AR were both present in rat and human vaginal epithelial cells. Removing Cl− or application of CFTR(inh)-172, an inhibitor of cystic fibrosis transmembrane conductance regulator (CFTR), abolished adrenaline-induced ISC responses. The elevated levels of [cAMP]i induced by adrenaline were prevented by the pretreatment with propranolol. Vaginal lubrication measured in vivo showed that adrenaline or pelvic nerve stimulation caused a marked increase in vaginal lubrication, whereas pretreatment with propranolol or CFTR(inh)-172 reduced the effect.Activation of epithelial β-AR facilitates vaginal lubrication during sexual arousal by stimulating vaginal epithelial Cl− secretion in a cAMP-dependent pathway. Thus, vaginal epithelial β-AR might be another regulator of vaginal sexual arousal responses. Sun Q, Huang J, Yang D-L, Cao X-N, and Zhou W-L. Activation of β-adrenergic receptors during sexual arousal facilitates vaginal lubrication by regulating vaginal epithelial Cl− secretion. J Sex Med **;**:**–**.