Major sites for Rho-kinase on the myosin phosphatase target subunit (MYPT1) are Thr695 and Thr850. Phosphorylation of Thr695 inhibits phosphatase activity but the role of phosphorylation at Thr850 is not clear and is evaluated here. Phosphorylation of both Thr695 and Thr850 by Rho-kinase inhibited activity of the type 1 phosphatase catalytic subunit. Rates of phosphorylation of the two sites were similar and efficacy of inhibition following phosphorylation was equivalent for each site. Phosphorylation of each site on MYPT1 was detected in A7r5 cells, but Thr850 was preferred by Rho-kinase and Thr695 was phosphorylated by an unidentified kinase(s).

1 Using a method employing front-surface fura-2¯uorometry to measure the cytosolic Ca 2+ concentration, [Ca 2+ ] i , the mechanism of endothelium-dependent regulation of vascular tone by thrombin was studied in porcine renal interlobar arterial strips. 2 At concentrations lower than 3 u ml 71, thrombin evoked only early transient relaxation, while at 3 u ml 71 and higher concentrations, thrombin caused an early relaxation and a subsequent transient contraction. Both thrombin-induced relaxation and contraction were abolished by removing the endothelium. Similar biphasic responses were observed with a protease-activated receptor-1-activating peptide. 3 Early relaxation was associated with a decrease in [Ca 2+ ] i , while the transient contraction was not associated with a change in [Ca 2+ ] i of smooth muscle cells. 4 A thromboxane A 2 (TXA 2 )/prostaglandin H 2 (PGH 2 ) receptor antagonist (10 75 M ONO-3708) completely inhibited the thrombin-induced contraction, whereas a thromboxane A 2 synthase inhibitor (10 75 M OKY-046) only partly inhibited it. 5 When the thrombin-induced contraction was inhibited by ONO-3708, either pretreatment with N o -nitro-L-arginine methylester (L-NAME) or an increase in the amount of external K + to 40 mM did not abolish thrombin-induced relaxation during phenylephrine-induced sustained contraction. However, the combination of pretreatment with L-NAME and an elevation of external K + to 40 mM completely abolished the relaxation. 6 There was no signi®cant dierence in the concentration-dependent eects of thrombin on the initial early relaxation between conditions in which the contractile components either were or were not inhibited. 7 Thrombin is thus considered to mainly activate protease-activated receptor-1 and cause a biphasic response, early relaxation and a transient contraction, in the porcine renal interlobar artery in an endothelium-dependent manner. The thrombin-induced endothelium-dependent relaxation was mediated by nitric oxide and hyperpolarizing factors, while the contraction was mediated by TXA 2 and PGH 2 .

1 The mechanism of endothelium-dependent regulation of vascular tone of bradykinin was investigated by simultaneously monitoring the changes in the cytosolic Ca 2+ concentration and the force of smooth muscle in fura-2-loaded strips of the porcine renal artery with endothelium. 2 During phenylephrine-induced sustained contraction, bradykinin (43610 79 M) caused endothelium-dependent triphasic changes in the force of the strips, composed of an initial relaxation, a subsequent transient contraction and a late sustained relaxation. 3 At low concentrations (10 710 ± 10 79 M), bradykinin caused an endothelium-dependent biphasic relaxation with no contraction. 4 A thromboxane A 2 (TXA 2 )/prostaglandin H 2 (PGH 2 ) receptor antagonist (10 75 M ONO-3708) completely inhibited, while a TXA 2 synthase inhibitor (10 75 M OKY-046) only partially inhibited, the transient contraction induced by bradykinin. 5 Under conditions where the bradykinin-induced contraction was inhibited by ONO-3708 during the phenylephrine-induced contraction, bradykinin induced only a transient relaxation in the presence of N o -nitro-L-arginine methyl ester (L-NAME). This transient relaxation was inhibited when the precontraction was initiated by phenylephrine plus 40 mM extracellular K + . The removal of L-NAME from this condition caused a partial reappearance of the initial relaxation and a complete reappearance of the sustained relaxation. 6 In conclusion, bradykinin caused the endothelium-dependent triphasic regulation of vascular tone in the porcine renal artery. The concentrations of bradykinin required to induce a contraction was higher than that required to induce relaxation. Both TXA 2 and PGH 2 were involved in the bradykinin-induced contraction. The initial relaxation was mediated by nitric oxide and hyperpolarizing factors while the sustained relaxation depended on nitric oxide.

Objective-The region of the 110 kDa regulatory subunit (MYPT1) of smooth muscle myosin phosphatase involved in the regulation of contraction was determined under physiological conditions. Methods and Results-Using HIV Tat protein-mediated protein transduction, the N-terminal fragments of MYPT1 were introduced to the intact porcine coronary arterial strips. Pre-incubation with 3 mol/L TAT-MYPT1

The contraction of smooth muscle is regulated primarily by intracellular Ca2+ signal. It is well established that the elevation of the cytosolic Ca2+ level activates myosin light chain kinase, which phosphorylates 20 kDa regulatory myosin light chain and activates myosin ATPase. The simultaneous measurement of cytosolic Ca2+ concentration and force development revealed that the alteration of the Ca2+-sensitivity of the contractile apparatus as well as the Ca2+ signal plays a critical role in the regulation of smooth muscle contraction. The fluctuation of an extent of myosin phosphorylation for a given change in Ca2+ concentration is considered to contribute to the major mechanisms regulating the Ca2+-sensitivity. The level of myosin phosphorylation is determined by the balance between phosphorylation and dephosphorylation. The phosphorylation level for a given Ca2+ elevation is increased either by Ca2+-independent activation of phosphorylation process or inhibition of dephosphorylation. In the last decade, the isolation and cloning of myosin phosphatase facilitated the understanding of regulatory mechanism of dephosphorylation process at the molecular level. The inhibition of myosin phosphatase can be achieved by (1) alteration of hetrotrimeric structure, (2) phosphorylation of 110 kDa regulatory subunit MYPT1 at the specific site and (3) inhibitory protein CPI-17 upon its phosphorylation. Rho-kinase was first identified to phosphorylate MYPT1, and later many kinases were found to phosphorylate MYPT1 and inhibit dephosphorylation of myosin. Similarly, the phosphorylation of CPI-17 can be catalysed by multiple kinases. Moreover, the myosin light chain can be phosphorylated by not only authentic myosin light chain kinase in a Ca2+-dependent manner but also by multiple kinases in a Ca2+-independent manner, thus adding a novel mechanism to the regulation of the Ca2+-sensitivity by regulating the phosphorylation process. It is now clarified that the protein kinase network is involved in the regulation of myosin phosphorylation and dephosphorylation. However, the physiological role of each component remains to be determined. One approach to accomplish this purpose is to investigate the effects of the dominant negative mutants of the signalling molecule on the smooth muscle contraction. In this regards, a protein transduction technique utilizing the cell-penetrating peptides would provide a useful tool. In the preliminary study, we succeeded in introducing a fragment of MYPT1 into the arterial strips, and found enhancement of contraction.

To determine the mechanism of bradykinin‐induced production of endothelium‐derived contracting factors, we monitored the changes in cytosolic Ca2+ concentration ([Ca2+]i) in in situ endothelial cells in porcine aortic valvular strips and the changes in [Ca2+]i of smooth muscle cells and force in porcine interlobar renal arterial strips using front‐surface fluorometry of fura‐2. In the presence of Nω‐nitro‐l‐arginine methyl ester, bradykinin caused an endothelium‐dependent transient elevation of [Ca2+]i and contraction in smooth muscle in the interlobar renal artery. This contraction was completely inhibited by a prostaglandin H2/thromboxane A2 receptor antagonist. In the absence of extracellular Ca2+, bradykinin failed to induce contraction. However, replenishing extracellular Ca2+ to 0.75 mm and higher induced an instantaneous contraction. However, replenishing Ca2+per se did not induce any contraction in the absence of bradykinin. Pretreatment with either 10−5m 1‐(β‐(3‐(4‐methoxyphenyl)propoxy)‐4‐methoxyphenethyl)‐1H‐imidazole hydrochloride (SKF96365) or 0.2 mm Ni2+ abolished the contraction induced by bradykinin in the presence of extracellular Ca2+. Treatment with 10−5m indomethacin completely inhibited the contractile response induced by Ca2+ replenishment, regardless of the timing of its application, before or after the application of bradykinin. In endothelial cells in the valvular strips, bradykinin caused a transient [Ca2+]i elevation in the presence of 1.25 mm extracellular Ca2+, but [Ca2+]i returned to the resting level within 10 min. Neither 10−5m SKF96365 nor 0.2 mm Ni2+ had any effect on the peak [Ca2+]i elevation, but decreased [Ca2+]i in the declining phase. In the absence of extracellular Ca2+, bradykinin induced a transient [Ca2+]i elevation to a level similar to that seen in the presence of 1.25 mm extracellular Ca2+. However, [Ca2+]i then rapidly returned to the prestimulation level within 5 min. Subsequent Ca2+ replenishment to 0.75 mm and higher in the presence of bradykinin elevated [Ca2+]i to significantly higher levels than the resting level seen in the media containing 1.25 mm Ca2+. In conclusion, Ca2+ influx in the endothelial cells is essential for bradykinin to induce endothelium‐dependent contraction in the porcine interlobar renal artery.

In carcinogenesis, intercellular interactions within and between cell types are critical but remain poorly understood. We present a study on intercellular interactions between normal and premalignant epithelial cells and their functional relevance in the context of premalignant to malignant progression in Barrett’s esophagus. Using whole transcriptome profiling we found that in the presence of normal epithelial cells, dysplastic cells but not normal cells, exhibit marked down-regulation of a number of key signaling pathways, including the transforming growth factor beta (TGFβ) and epithelial growth factor (EGF). Functional assays revealed both cell types showed repressed proliferation and significant changes in motility (speed, displacement and directionality) as a result of interactions between the two cell types. Cellular interactions appear to be mediated through both direct cell-cell contact and secreted ligands. The findings of this study are important in that they reveal, for the first time, the effects of cellular communication on gene expression and cellular function between premalignant (dysplastic) epithelial cells and their normal counterparts.

No abstract