Functional Role of Muscarinic Receptor Subtypes in Calcium Sensitization and Their Contribution to Rho-kinase and Protein Kinase C Pathways in Contraction of Human Detrusor Smooth Muscle

Abstract

We investigated the role of muscarinic receptor subtypes in calcium sensitization and their contribution to rho-kinase (ROK) and protein kinase C (PKC) pathways in carbachol (CCh)-induced contraction of human detrusor smooth muscle (DSM). α-toxin-permeabilized human DSM strips were prepared and mounted horizontally to record isometric force. The roles of M(2) and M(3) muscarinic receptors in Ca(2+) sensitization were studied using selective antagonists of M(2) (AF-DX116) and M(3) (4-DAMP) receptor subtypes. The effects of a selective inhibitor of ROK, Y-27632, and a selective inhibitor of PKC, bisindolylmaleimide I (GF-109203X), were also studied on contraction induced by 10 μM CCh with 100 μM guanosine triphosphate at a fixed 1 μM [Ca(2+)](i) after preincubation with 1 μM AF-DX116 or 1 μM 4-DAMP. Carbachol-induced Ca(2+) sensitization was predominantly inhibited by 4-DAMP compared with AF-DX116. Four-DAMP equivalently inhibited the relaxation effect of 5 μM GF-109203X as well as that of 5 μM Y-27632 on CCh-induced Ca(2+) sensitization. AF-DX116 reduced the relaxation effect of Y-27632 to a greater degree than GF-109203X. The results of the present study have demonstrated the predominant role of M(3) receptor subtype in Ca(2+) sensitization and the relative contribution to ROK and PKC pathways. Our study also shows that the ROK pathway is dominant compared with the PKC pathway after M(2) receptor activation, which in turn is inferior, but not negligible, in producing Ca(2+) sensitization.