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The present study determined the changes in the expression levels of MYPT1, CPI-17 and MLC20 in the ileum of mice with neonatal induced necrotizing enterocolitis (NEC) to provide a basis for a pathogenesis model that includes smooth muscle changes during NEC. A group of 7-day-old BALB/c mice were fed with formula (40 μl/g, 5 times/day) and given hypoxia treatments (5% O2 and 95% N2 for 10 min, twice daily) for 4 days to induce NEC and establish a mouse model. A control group of 7-day-old BALB/c mice were left with their mother for the duration of the treatment. After establishing the model, the two groups of mice were sacrificed, and the terminal ileum tissue was collected and subjected to western blot analysis and immunohistochemistry. The results showed the expression levels of MYPT1 and pMYPT1 in the ileum of the mice in the NEC group were lower than those in the control group (P<0.01). The levels of CPI17 and pCPI17 were higher in the NEC group compared with those in the control group. The expression level of MLC20 in NEC group was lower than that in the control group (P<0.01), but the level of pMLC20 in the NEC group was higher (P<0.05). The results of immunohistochemistry showed that the staining intensities of MYPT1, CPI-17 and MLC20 in the NEC group were lighter than those in the control group, and the proportion of positive cells was also lower in the NEC group (P<0.01). Taken together our results suggest that establishment of NEC is accompanied by changes in the protein levels of MYPT1 and pCPI-17, which can regulate smooth muscle contraction in the ileum. [ABSTRACT FROM AUTHOR]

Gomisin A (GA) is an active ingredient of the fruits of Schisandra chinensis which has been widely used as a tonic in traditional Korean medicine. GA induces not only endothelium-dependent but also endothelium-independent relaxation in an isolated rat's thoracic aorta. This study was aimed to investigate the molecular mechanism by which GA induces endothelium-independent vasorelaxation. Rat aortic rings were denuded of endothelium, mounted in organ baths, and subjected to contraction or relaxation. We measured the amount of GTP RhoA as well as the phosphorylation level of 20 kDa myosin light chains (MLC), myosin phosphatase-targeting subunit 1 (MYPT1) and protein kinase C-potentiated inhibitory protein for heterotrimeric myosin light-chain phosphatase of 17 kDa (CPI17). Pretreatment with GA dose-dependently inhibited the concentration-response curves in response to sodium fluoride (NaF) or thromboxane A agonist U46619, but not to phorbol 12, 13-dibutyrate (PDBu). GA decreased the activation of RhoA as well as the phosphorylation level of MLC, MYPT1, and CPI17 induced by 8.0 mM NaF or 30 nM U46619. However, K channel blockers such as glibenclamide, apamin, or charybdotoxin did not affect the vascular relaxation induced by GA. Furthermore, GA did not affect the level of phosphorylation of CPI17 induced by PDBu. GA reduces vascular contraction through inhibition of RhoA/Rho-kinase pathway in endothelium-denuded rat aorta. [ABSTRACT FROM AUTHOR]

The present study was undertaken to investigate the molecular mechanisms by which glyceollin I inhibits vascular contraction in rat aorta. Rat aortic rings were treated with either glyceollin I or vehicle when vascular contraction reached plateaus. We measured the activity of GTP-RhoA and Rho GTPase-activating protein (RhoGAP) and the phosphorylation level of the myosin light chain (MLC20), myosin phosphatase targeting subunit 1 (MYPT1), and PKC-potentiated inhibitory protein for heterotrimeric MLCP of 17 kDa (CPI17). Glyceollin I reduced vascular contraction whether endothelium is present or denuded. Glyceollin I reduced vascular contraction induced by NaF, U46619, phenylephrine, or PDBu. Blockers of K+ channels did not affect the vasorelaxation induced by glyceollin I. Glyceollin I reduced activation of RhoA as well as phosphorylation level of MLC20. Glyceollin I also reduced phosphorylation of MYPT1 and CPI17 induced by NaF but not PDBu. However, glyceollin I had no direct effect on RhoGAP activation in vitro. Glyceollin I reduced vascular contraction, at least in part, through inhibition of the RhoA/Rho-kinase signaling pathway. [ABSTRACT FROM AUTHOR]

Abstract: We hypothesized that fluoride induces vascular contraction through activation of the RhoA/Rho kinase pathway in isolated rat aortas. Rat aortic rings were mounted in organ baths and contracted with sodium fluoride (NaF). We measured the amount of GTP-RhoA as well as vascular tension. We also determined the level of phosphorylation of the myosin light chain (MLC20), myosin phosphatase targeting subunit 1 (MYPT1) and PKC-potentiated inhibitory protein for heterotrimeric MLCP of 17kDa (CPI17). In both physiological salt solution and Ca2+-free solution, NaF increased vascular tension and MLC20 phosphorylation in dose-dependent manners. NaF increased not only phosphorylation level of MYPT1Thr855 and CPI17Thr38, but also the amount of GTP-RhoA. Both H1152 and Y27632, inhibitors of Rho kinase, but not Ro31-8220, an inhibitor of PKC, attenuated NaF-induced contraction and phosphorylation level of MLC20, MYPT1Thr855 and CPI17Thr38. In conclusion, fluoride induces vascular contraction through activation of the RhoA/Rho kinase pathway. [Copyright &y& Elsevier]

Abstract: Two major physiological roles have been defined for zipper interacting protein kinase (ZIPK), regulation of apoptosis in non-muscle cells and regulation of Ca2+ sensitization in smooth muscle. Although much attention has focused on the role of ZIPK in the regulation of apoptotic events, its roles in smooth muscle are likely to have equal if not greater physiological relevance. We first identified ZIPK as a major protein kinase controlling the phosphorylation of myosin phosphatase (SMPP-1M) and the inhibitor protein CPI17 in smooth muscle. Phosphorylation of SMPP-1M and CPI17 by ZIPK inhibits phosphatase activity towards myosin and causes profound Ca2+ sensitization and contraction in smooth muscle. ZIPK will also directly phosphorylate both muscle and non-muscle myosin. The highly selective actions of ZIPK in the control of myosin phosphorylation potentially make the enzyme an ideal candidate for the development of novel therapeutics to treat smooth muscle related disorders such as hypertension or asthma. [Copyright &y& Elsevier]

CPI17 and myosin binding subunit of type 1 protein phosphatase (MBS) are the regulators of myosin light chain phosphatase (MLCP). The function of both regulators is controlled by phosphorylation. The phosphorylation of CPI17 at Thr38 significantly enhances the inhibitory activity of CPI17 and the phosphorylation at Thr641 of MBS decreases the MLCP activity. Here, we found that p21-activated protein kinase (PAK) phosphorylates both CPI17 at Thr38 and MBS at Thr641. For CPI17, PAK specifically phosphorylated at Thr38, since the mutation of Thr38 to Ala completely abolished the phosphorylation. On the other hand, PAK phosphorylated Thr641 but not Thr799 of MBS, the site phosphorylated by Rho kinase. Because PAK phosphorylates MBS more than 1 mol/mol, it is anticipated that PAK also phosphorylates other sites in addition to Thr641. CPI17 phosphorylation induced by PAK significantly enhanced the inhibitory activity of CPI17. On the other hand, the phosphorylation of MBS by PAK also decreased the MLCP activity. These results raise the possibility that the PAK pathway plays a role in MLCP regulation. [Copyright &y& Elsevier]

Dephosphorylation of the two key regulatory factors of myosin light chain phosphatase (MLCP), CPI17 and MBS (myosin binding subunit) of MLCP was studied. While Thr38 phosphorylated CPI17 is quite susceptible to protein phosphatases, phosphorylated MBS was highly resistant to dephosphorylation. Type 2A, 2B and 2C protein phosphatases (PP2A, PP2B and PP2C), but not type 1 (PP1), dephosphorylated CPI17. The majority of the CPI17 phosphatase activity in smooth muscle was attributed to PP2A and PP2C. Phospholipids inhibited dephosphorylation of MBS and arachidonic acid (AA) inhibited PP2A activity against both MBS and CPI17, raising the possibility that AA favors the preservation of active MLCP. Consistently, while the phosphorylation of CPI17 was promptly decreased when the agonist was removed, the phosphorylation of MBS was unchanged in intact smooth muscle fiber. The results suggest that MBS phosphorylation mediated regulation of MLCP is not suitable for regulating rapid change in myosin phosphorylation. On the other hand, phosphorylated CPI17 is readily dephosphorylated thus likely to play a role in regulating fast phosphorylation–dephosphorylation cycle in cells. [Copyright &y& Elsevier]