Adenosine A1 Receptors Link to Smooth Muscle Contraction Via CYP4a, protein kinase C-, and ERK1/2

Adenosine A(1) receptor (A(1)AR) activation contracts smooth muscle, although signaling mechanisms are not thoroughly understood. Activation of A(1)AR leads to metabolism of arachidonic acid, including the production of 20-hydroxyeicosatetraenoic acid (20-HETE) by cytochrome P4504a (CYP4a). The 20-HETE can activate protein kinase C- (PKC-), which crosstalks with extracellular signal-regulated kinase (ERK1/2) pathway. Both these pathways can regulate smooth muscle contraction, we tested the hypothesis that A(1)AR contracts smooth muscle through a pathway involving CYP4a, PKC-, and ERK1/2. Experiments included isometric tension recordings of aortic contraction and Western blots of signaling molecules in wild type (WT) and A(1)AR knockout (A(1)KO) mice. Contraction to the A(1)-selective agonist 2-chloro-N cyclopentyladenosine (CCPA) was absent in A(1)KO mice aortae, indicating the contractile role of A(1)AR. Inhibition of CYP4a (HET0016) abolished 2-chloro-N cyclopentyladenosine-induced contraction in WT aortae, indicating a critical role for 20-HETE. Both WT and A(1)KO mice aortae contracted in response to exogenous 20-HETE. Inhibition of PKC- (Go6976) or ERK1/2 (PD98059) attenuated 20-HETE-induced contraction equally, suggesting that ERK1/2 is downstream of PKC-. Contractions to exogenous 20-HETE were significantly less in A(1)KO mice; reduced protein levels of PKC-, p-ERK1/2, and total ERK1/2 supported this observation. Our data indicate that A(1)AR mediates smooth muscle contraction via CYP4a and a PKC--ERK1/2 pathway.