Involvement of cyclic AMP-dependent and -independent mechanisms in the relaxation of rat detrusor muscle via β-adrenoceptors

Abstract

We investigated the cAMP-dependent and -independent mechanisms of relaxation via β-adrenoceptor in rat detrusor muscle with and without pre-contraction. A microdialysis technique was used to measure detrusor tension and cAMP level on the same detrusor tissue. In non-contracted tissue, isoproterenol, clenbuterol (β₂-adrenoceptor agonist) and FR165101, ((8S)-8-{[(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino}-6,7,8,9-tetrahydro-5H-benzocyclohepten-2-yl)oxy]acetic acid hydrochloride (β₃-adrenoceptor agonist) relaxed detrusor muscle and cAMP levels also increased in a concentration dependent manner. SQ22536 (adenylyl cyclase inhibitor) markedly suppressed relaxation, suggesting that β-adrenoceptor-mediated relaxation may be attributed mainly to cAMP-dependent mechanism. In high K⁺ pre-contracted tissue, although relaxation advanced in a concentration dependent manner, cAMP production reached a plateau at concentrations of more than 10^− 7 M. SQ22536 had only a small inhibitory effect. However, large-conductance, Ca²⁺-activated K⁺ (BK_Ca) channel inhibitors, charybdotoxin and iberiotoxin markedly suppressed relaxation. These results suggest that in addition to cAMP-dependent pathway, BK_Ca channels are involved in the β-adrenoceptor agonists-induced relaxation in pre-contracted detrusor muscle.

Introduction

The detrusor muscle is relaxed by the activation of β-adrenoceptors distributed on bladder smooth muscle in various species including humans (Yamazaki et al., 1998, Yamaguchi, 2002, Nomiya and Yamaguchi, 2003). This relaxation of detrusor muscle via β-adrenoceptors is thought to contribute to the urine storage during bladder filling.

The mechanism by which β-adrenoceptor agonists induce relaxation of smooth muscle is not fully understood, but an intracellular pathway for smooth muscle relaxation is believed to be activated by adenosine 3′ : 5′-cyclic monophosphate (cAMP). Activation of β-adrenoceptors couples via Gs proteins to adenylyl cyclase, leading to an increase in intracellular cAMP levels and a subsequent activation of cAMP-dependent protein kinase A (PKA) (Gilman, 1987, Murray, 1990). Then, PKA phosphorylates myosin light chain kinase, which suppresses a calcium-calmodulin-dependent interaction of myosin with actin. The increase in cAMP production also results in attenuation of cytoplasmic calcium ion concentration ([Ca²⁺]_I) by removal of Ca²⁺ from cytoplasm. Besides the above cAMP-dependent mechanisms, it has been suggested that in vascular (Scornik et al., 1993), gastrointestinal (Horinouchi and Koike, 2002) and airway smooth muscle (Kume et al., 1993), β-adrenergic transduction pathways exist that are independent of cAMP formation, involving direct interaction of Gs proteins with potassium (K⁺) channels. However, with regard to bladder smooth muscle, the cAMP-independent mechanism remains unclear.

Most in vitro relaxation studies are performed using pre-contracted muscle strips because the basal tone of smooth muscle is very low. Therefore, the relaxing effect of β-adrenoceptor agonists has been studied in the presence of various pharmacological constrictors (carbachol, prostaglandin, high K⁺, etc.) that produce pre-contraction. However, the cellular signaling processes in response to β-adrenoceptor agonists may differ according to whether the detrusor muscle is contracted or not.

Thus, in the present study, we investigated the cAMP-dependent and -independent mechanisms of β-adrenoceptor agonist-induced relaxation in the two different states of detrusor muscle, i.e., with or without pre-contraction.