Huiping Zhou

Differential signalling by muscarinic receptors in smooth muscle: m2-mediated inactivation of myosin light chain kinase via Gi3, Cdc42/Rac1 and p21-activated kinase 1 pathway, and m3-mediated MLC20 (20 kDa regulatory light chain of myosin II) phosphorylation via Rho-associated kinase/myosin phosphatase targeting subunit 1 and protein kinase C/CPI-17 pathway

Signalling via m3 and m2 receptors in smooth muscles involved activation of two G-protein-dependent pathways by each receptor. m2 receptors were coupled via Gbetagammai3 with activation of phospholipase C-beta3, phosphoinositide 3-kinase and Cdc42/Rac1 (where Cdc stands for cell division cycle) and p21-activated kinase 1 (PAK1), resulting in phosphorylation and inactivation of myosin light chain kinase (MLCK). Each step was inhibited by methoctramine and pertussis toxin. PAK1 activity was abolished in cells expressing both Cdc42-DN (where DN stands for dominant negative) and Rac1-DN. MLCK phosphorylation was inhibited by PAK1 antibody, and in cells expressing Cdc42-DN and Rac1-DN. m3 receptors were coupled via Galpha(q/11) with activation of phospholipase C-beta1 and via RhoA with activation of Rho-associated kinase (Rho kinase), phospholipase D and protein kinase C (PKC). Rho kinase and phospholipase D activities were inhibited by C3 exoenzyme and in cells expressing RhoA-DN. PKC activity was inhibited by bisindolylmaleimide, and in cells expressing RhoA-DN; PKC activity was also inhibited partly by Y27632 (44+/-5%). PKC-induced phosphorylation of PKC-activated 17 kDa inhibitor protein of type 1 phosphatase (CPI-17) at Thr38 was abolished by bisindolylmaleimide and inhibited partly by Y27632 (28+/-3%). Rho-kinase-induced phosphorylation of myosin phosphatase targeting subunit (MYPT1) and was abolished by Y27632. Sustained phosphorylation of 20 kDa regulatory light chain of myosin II (MLC20) and contraction were abolished by bisindolylmaleimide Y27632 and C3 exoenzyme and in cells expressing RhoA-DN. The results suggest that Rho-kinase-dependent phosphorylation of MYPT1 and PKC-dependent phosphorylation and enhancement of CPI-17 binding to the catalytic subunit of MLC phosphatase (MLCP) act co-operatively to inhibit MLCP activity, leading to sustained stimulation of MLC20 phosphorylation and contraction. Because Y27632 inhibited both Rho kinase and PKC activities, it could not be used to ascertain the contribution of MYPT1 to inhibition of MLCP activity. m2-dependent phosphorylation and inactivation of MLCK precluded its involvement in sustained MLC20 phosphorylation and contraction.

Gq-dependent signalling by the lysophosphatidic acid receptor LPA3 in gastric smooth muscle: reciprocal regulation of MYPT1 phosphorylation by Rho kinase and cAMP-independent PKA

The present study characterized the signalling pathways initiated by the bioactive lipid, LPA (lysophosphatidic acid) in smooth muscle. Expression of LPA(3) receptors, but not LPA(1) and LPA(2), receptors was demonstrated by Western blot analysis. LPA stimulated phosphoinositide hydrolysis, PKC (protein kinase C) and Rho kinase (Rho-associated kinase) activities: stimulation of all three enzymes was inhibited by expression of the G(alphaq), but not the G(alphai), minigene. Initial contraction and MLC(20) (20 kDa regulatory light chain of myosin II) phosphorylation induced by LPA were abolished by inhibitors of PLC (phospholipase C)-beta (U73122) or MLCK (myosin light-chain kinase; ML-9), but were not affected by inhibitors of PKC (bisindolylmaleimide) or Rho kinase (Y27632). In contrast, sustained contraction, and phosphorylation of MLC(20) and CPI-17 (PKC-potentiated inhibitor 17 kDa protein) induced by LPA were abolished selectively by bisindolylmaleimide. LPA-induced activation of IKK2 {IkappaB [inhibitor of NF-kappaB (nuclear factor kappaB)] kinase 2} and PKA (protein kinase A; cAMP-dependent protein kinase), and degradation of IkappaBalpha were blocked by the RhoA inhibitor (C3 exoenzyme) and in cells expressing dominant-negative mutants of IKK2(K44A) or RhoA(N19RhoA). Phosphorylation by Rho kinase of MYPT1 (myosin phosphatase targeting subunit 1) at Thr(696) was masked by phosphorylation of MYPT1 at Ser(695) by PKA derived from IkappaB degradation via RhoA, but unmasked in the presence of PKI (PKA inhibitor) or C3 exoenzyme and in cells expressing IKK2(K44A). We conclude that LPA induces initial contraction which involves activation of PLC-beta and MLCK and phosphorylation of MLC(20), and sustained contraction which involves activation of PKC and phosphorylation of CPI-17 and MLC(20). Although Rho kinase was activated, phosphorylation of MYPT1 at Thr(696) by Rho kinase was masked by phosphorylation of MYPT1 at Ser(695) via cAMP-independent PKA derived from the NF-kappaB pathway.

Strain differences in the neural, behavioral, and molecular correlates of sweet and salty taste in naive, ethanol- and sucrose-exposed P and NP rats

Strain differences between naive, sucrose- and ethanol-exposed alcohol-preferring (P) and alcohol-nonpreferring (NP) rats were investigated in their consumption of ethanol, sucrose, and NaCl; chorda tympani (CT) nerve responses to sweet and salty stimuli; and gene expression in the anterior tongue of T1R3 and TRPV1/TRPV1t. Preference for 5% ethanol and 10% sucrose, CT responses to sweet stimuli, and T1R3 expression were greater in naive P rats than NP rats. The enhancement of the CT response to 0.5 M sucrose in the presence of varying ethanol concentrations (0.5-40%) in naive P rats was higher and shifted to lower ethanol concentrations than NP rats. Chronic ingestion of 5% sucrose or 5% ethanol decreased T1R3 mRNA in NP and P rats. Naive P rats also demonstrated bigger CT responses to NaCl+benzamil and greater TRPV1/TRPV1t expression. TRPV1t agonists produced biphasic effects on NaCl+benzamil CT responses, enhancing the response at low concentrations and inhibiting it at high concentrations. The concentration of a TRPV1/TRPV1t agonist (Maillard reacted peptides conjugated with galacturonic acid) that produced a maximum enhancement in the NaCl+benzamil CT response induced a decrease in NaCl intake and preference in P rats. In naive P rats and NP rats exposed to 5% ethanol in a no-choice paradigm, the biphasic TRPV1t agonist vs. NaCl+benzamil CT response profiles were higher and shifted to lower agonist concentrations than in naive NP rats. TRPV1/TRPV1t mRNA expression increased in NP rats but not in P rats exposed to 5% ethanol in a no-choice paradigm. We conclude that P and NP rats differ in T1R3 and TRPV1/TRPV1t expression and neural and behavioral responses to sweet and salty stimuli and to chronic sucrose and ethanol exposure.

Inhibitory phosphorylation of IP3 receptor type I (IP3R-I) In vivo is selectively mediated by cyclic GMP-dependent protein kinase-1α (PKG-1α) in gastric smooth muscle

We have recently shown that PKG-Ia and PKG-I[3 are expressed in smooth muscle cells together with IP3 receptor-associated PKG substrate (IRAG). IRAG binds selectively to the N-terminal domain of PKG-I[3 and its phosphorylation at Ser696 by PKG-I~ is postulated to be essential for inhibition of [P3-induced Ca 2+ release by PKG. Both IP3Rol and IP3R-III were expressed in gastric smooth muscle cells, but only IP3R-I was phosphorylated by PKG in vivo leading to inhibition of IP~-dependent Ca 2+ release. In vitro phosphorylation of IP3RI in microsomal membranes by purified PKG-Ia holoenzyme inhibited IP3-induced Ca 2. release. Aim: To identify the PKG-I isoform responsible for inhibitory phosphorylation of IP3R-1 in vivo. Methods: Dominant negative PKG-In (T515A, T517A) and PKG-I~ (K405A) were expressed separately in cultured rabbit gastric smooth muscle cells. The ability of PKG to pbosphorylate IP3R-I and inhibit IP3-induced Ca 2§ release was determined in cells expressing both PKG-I isoforms and in cells expressing dominant negative PKG-Ia or PKG-IB. Results: Stimulation of PKG with sodium nitroprnsside (SNP I~M) or the nonhydrolyzable PKG activator, 8-pCPT--cGMP (10 g.M), caused phospboryiation (327 +_ 35% to 436 -+ 39% above basal level) of IP3R-I in muscle cells expressing both wild type PKG-I isoforms or in cells expressing dominant negative PKG-I[~, but not in cells expressing dominant negative PKG-Ia. IP3 stimulated Ca 2 + release in permeabilized muscle cells expressing wild type and mutant PKG-I isoforms to the same extent (29 -+ 3% to 33 + 4% decrease in steady-state ~5Ca2+ cell content). Treatment of the cells with SNP or 8-pCPT-cGMP inhibited IP3-induced Ca 2+ release in cells expressing both wild type PKG-I isoforms and in ceils expressing dominant negative PKG-I[3, but not in cells expressing dominant negative ?KG~Ia. The inhibition of lP3-indnced Ca 2+ release in cells expressing wild type and PKGI~ mutant was completely reversed by the PKG inhibitor, KT5823 (1 ~M). The results provide direct evidence that selective phosphorylation of IP~R-I by the PKG-Ia isoform mediates inhibition of IP3-induced Ca 2+ release in gastric smooth muscle cells. PKG-IB did not mediate phosphorylation of IP3R-I despite co-expression of IRAG in these cells. Conclusion: PKG-I~x mediates pbosphorylation of IP3R-1 and inhibition of IP3-induced Ca 2+ release.