We examined whether CB1 receptors in smooth muscle conform to the signaling pattern observed with other Gi-coupled receptors that stimulate contraction via two G??-dependent pathways (PLC-?3 and phosphatidylinositol 3-kinase/integrin-linked kinase). Here we show that the anticipated G??-dependent signaling was abrogated. Except for inhibition of adenylyl cyclase via G?i, signaling resulted from G??-independent phosphorylation of CB1 receptors by GRK5, recruitment of ?-arrestin1/2, and activation of ERK1/2 and Src kinase. Neither uncoupling of CB1 receptors from Gi by pertussis toxin (PTx) or Gi minigene nor expression of a G??-scavenging peptide had any effect on ERK1/2 activity. The latter was abolished in muscle cells expressing ?-arrestin1/2 siRNA. CB1 receptor internalization and both ERK1/2 and Src kinase activities were abolished in cells expressing kinase-deficient GRK5(K215R). Activation of ERK1/2 and Src kinase endowed CB1 receptors with the ability to inhibit concurrent contractile activity. We identified a consensus sequence (102KSPSKLSP109) for phosphorylation of RGS4 by ERK1/2 and showed that expression of a RGS4 mutant lacking Ser103/Ser108 blocked the ability of anandamide to inhibit acetylcholine-mediated phosphoinositide hydrolysis or enhance G?q:RGS4 association and inactivation of G?q. Activation of Src kinase by anandamide enhanced both myosin phosphatase RhoA-interacting protein (M-RIP):RhoA and M-RIP:MYPT1 association and inhibited Rho kinase activity, leading to increase of myosin light chain (MLC) phosphatase activity and inhibition of sustained muscle contraction. Thus, unlike other Gi-coupled receptors in smooth muscle, CB1 receptors did not engage G?? but signaled via GRK5/?-arrestin activation of ERK1/2 and Src kinase: ERK1/2 accelerated inactivation of G?q by RGS4, and Src kinase enhanced MLC phosphatase activity, leading to inhibition of ACh-stimulated contraction.
Previous studies have identified differences in the expression of proteins that regulate myosin light chain phosphorylation and contraction in tonic and phasic smooth muscle. cGMP plays a critical role in smooth muscle relaxation and is important for optimal function of phasic and tonic smooth muscle. The intracellular cGMP levels are regulated by its hydrolysis via phosphodiesterase 5 (PDE5) and efflux via novel multidrug resistance protein 5 (MRP5). In the present study we tested the hypothesis that the differences in the phasic and tonic behavior of smooth muscles may be related to differences in mechanisms that terminate cGMP signaling. Expression of PDE5 and MRP5 was significantly (more than 2-fold) higher in fundus compared with antrum. The NO donor S-nitrosoglutathione (GSNO) caused an increase in PDE5 activity and intra- and extracellular cGMP levels in both fundus and antrum. Stimulation of PDE5 activity and increase in extracellular cGMP were significantly higher in fundus, whereas increase in intracellular cGMP was significantly higher in antrum. GSNO-induced increase in extracellular cGMP was blocked in dispersed cells by the cyclic nucleotide export blocker probenecid and in cultured muscle cells by depletion of ATP or suppression of MRP5 by siRNA, providing evidence that cGMP efflux was mediated by ATP-dependent export via MRP5. Consistent with the higher expression and activity levels of PDE5 and MRP5, GSNO-induced PKG activity and muscle relaxation were significantly lower in muscle cells from fundus compared with antrum. Thus higher expression of PDE5 and MRP5 in muscle cells from fundus correlates with tonic phenotype of muscle.
We examined expression of protease-activated receptors 2 (PAR2) and characterized their signaling pathways in rabbit gastric muscle cells. The PAR2 activating peptide SLIGRL (PAR2-AP) stimulated Gq, G13, Gi1, PI hydrolysis, and Rho kinase activity, and inhibited cAMP formation. Stimulation of PI hydrolysis was partly inhibited in cells expressing PAR2 siRNA, Gaq or Gai minigene and in cells treated with pertussis toxin, and augmented by expression of dominant negative regulator of G protein signaling (RGS4(N88S)). Stimulation of Rho kinase activity was abolished by PAR-2 or Ga13 siRNA, and by Ga13 minigene. PAR2-AP induced a biphasic contraction; initial contraction was selectively blocked by the inhibitor of PI hydrolysis (U73122) or MLC kinase (ML-9), whereas sustained contraction was selectively blocked by the Rho kinase inhibitor (Y27632). PAR2-AP induced phosphorylation of MLC20, MYPT1 but not CPI-17. PAR2-AP also caused a decrease in the association of NF-kB and PKA catalytic subunit: the effect of PAR2-AP was blocked by PAR2 siRNA or phosphorylation-deficient RhoA (RhoA(S188A)). PAR2-AP-induced degradation of IkBa and activation of NF-kB were abolished by the blockade of RhoA activity by Clostridium botulinum C3 exoenzyme suggesting RhoA-dependent activation of NF-kB. PAR2-AP-stimulated Rho kinase activity was significantly augmented by the inhibitors of PKA (myristoylated PKI), IKK2 (IKKIV) or NF-kB (MG132), and in cells expressing dominant negative mutants of IKK (IKK(K44A), IkBa (IkBa (S32A/S36A)) or RhoA(S188A), suggesting feedback inhibition of Rho kinase activity via PKA derived from NF-kB pathway. PAR2-AP induced phosphorylation of RhoA and the phosphorylation was attenuated in cells expressing phosphorylation-deficient RhoA(S188A). Our results identified signaling pathways activated by PAR2 to mediate smooth muscle contraction and a novel pathway for feedback inhibition of PAR2-stimulated RhoA. The pathway involves activation of the NF-kB to release catalytic subunit of PKA from its binding to IkBa and phosphorylation of RhoA at Ser(188).
The Jk(a-b-) phenotype is rare in most populations and often detected after transfusion or pregnancy. After immunisation, anti-Jk3 forms and it can be difficult to find compatible Jk(a-b-) donors. Using anti-Jk(a) and anti-Jk(b) in a conventional tube method is unsuitable for identifying Jk(a-b-) in mass screening of blood donors. Jk(a-b-) phenotypes are associated with the absence of urea transporters on erythrocytes, making red blood cells (RBC) resistant to lysis by 2M urea, while Jk(a+b-), Jk(a-b+) and Jk(a+b+) phenotypes are susceptible to lysis.
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