Signaling By Protease-Activated Receptors in Gastrointestinal Smooth Muscle

In the present study, we have examined the expression of protease-activated receptors (PARS) and characterized their signaling pathways in rabbit gastric muscle cells. Immunoblot analysis revealed expression of PARl and PAR2 but not PAR3 or PAR4 in smooth muscle. The PARl agonist TFLLR activated Gq, G12, and Gi3, but not Gil, Gi2, G13, Gs or Gz, whereas the PAR2 agonist SLIGRL activated Gq, G13, Gil, and Gi2, but not Gi3, G12, Gs, or Gz. Both PARl and PAR2 agonists stimulated PI hydrolysis and Rho kinase activity and inhibited cAMP formation. PAR1-stimulated PI hydrolysis was abolished in cells expressing Gαq minigene, but was not affected in cells expressing Gαi minigene or in cells treated with pertussis toxin (PTx). PAR2-stimulated PI hydrolysis was partially inhibited in cells expressing Gαq or Gαi minigene and in cells treated with PTx. PAR1- and PAR2-stimulated Rho kinase activity was abolished in cells expressing Gα12 or Gα13 minigene, respectively. Both PARl and PAR2 agonists induced a transient initial contraction that was selectively blocked by the inhibition of PI hydrolysis with U73122 and MLC kinase activity with ML-9. PAR1-induced sustained contraction was preferentially inhibited by the PKC inhibitor bisindolylmaleimide and to a minor extent by the Rho kinase inhibitor Y27632, whereas PAR2-induced sustained contraction was preferentially inhibited by Y27632. Activation of both PARl and PAR2 induced MLC20 phosphorylation, whereas phosphorylation of MYPTl and CPI-17 are receptor-specific: only PARl induced CPI-17 phosphorylation and only PAR2 induced MYPTl phosphorylation.Activation of PARl and PAR2 also induced IκBα degradation and NF-κB activation; the effects were abolished by the blockade of RhoA activity by Clostridium botulinum C3 exoenzyme suggesting NF-κB is downstream of RhoA. PAR1- and PAR2-stimulated Rho kinase activity was significantly augmented by the inhibitors of PKA (PKI), IKK2 (IKKIV), or NF-κB (MG132), and in cells expressing dominant negative mutants of IKK (IKK(K44A), IκBα (IκBα (S32A/S36A)), or phosphorylation-deficient RhoA (RhoA(S188A)). In addition, activation of PARl induced Gα12 phosphorylation, which was abolished by bisindolylmaleimide, suggests that phosphorylation was mediated by PKC derived from the activation of RhoA. Only PAR1-stimulated Rho kinase activity was significantly augmented by the PKC inhibitor. The effect of PKC inhibitor was additive to that of the PKA inhibitor