Abstract-It has been known for some time that agonist-induced contractions of vascular smooth muscle are often associated with a sensitization of the contractile apparatus to intracellular Ca 2ϩ . One mechanism that has been suggested to explain Ca 2ϩ sensitization is inhibition of myosin phosphatase activity. In the present study, we tested the hypothesis that differential localization of the phosphatase might be associated with its inhibition. Quantitative confocal microscopy of freshly dissociated, fully contractile smooth muscle cells was used in parallel with measurements of myosin light chain and myosin phosphatase phosphorylation. The results indicate that, in the smooth muscle cells, the catalytic and targeting subunits of the phosphatase are dissociated from each other in an agonist-specific manner and that the dissociation is accompanied by a slower rate of myosin phosphorylation. Targeting of myosin phosphatase to the cell membrane precedes the dissociation of subunits and is associated with phosphorylation of the targeting subunit at a Rho-associated kinase (ROK) phosphorylation site. The phosphorylation and membrane translocation of the targeting subunit are inhibited by a ROK inhibitor. This dissociation of subunits may provide a mechanism for the decreased phosphatase activity of phosphorylated myosin phosphatase. (Circ Res. 2002;90:546-553.)

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Heat shock has been known to change cellular responses to noxious stimuli by inducing heat-shock proteins (Hsps). We hypothesized that a heat-shock response modulates cytokine production in murine aortic vascular smooth muscle cells (VSMCs). VSMCs were exposed to 44 degrees C for 15-60 min, and subjected to interleukin-1beta (IL-1beta) or tumor necrosis factor alpha (TNFalpha), which induced interleukin-6 (IL-6) production. Expression of Hsps was examined with immunoblots, immunocytochemistry, or enzyme-linked immunosorbent assay (ELISA), and that of IL-6 with reverse transcription-polymerase chain reaction (RT-PCR) or ELISA. Heat shock (44 degrees C for 45 min) induced Hsp72 in VSMCs at 4 h and elicited its maximal expression at 8 h after the end of heat shock. Treatment with IL-1beta increased IL-6 transcription in VSMCs up to 24 h in an incubation time-dependent manner. Treatment with IL-1beta or TNFalpha caused a concentration-dependent increase in IL-6 production in culture medium, which was attenuated by heat shock. Although treatment with Hsp72 or Hsp60 alone did not significantly affect basal IL-6 release into culture medium statistically, cotreatment with IL-1beta and Hsp72, but not Hsp60 or boiled Hsp72, decreased IL-1beta-induced IL-6 production in culture medium. Introduction of Hsp72, but not Hsp60, into VSMCs decreased IL-1beta-induced IL-6 production in culture medium. These results indicate that the heat-shock response transcriptionally attenuated production of IL-6 in murine aortic VSMCs.

1. Flavonoids modulate vascular tone through an endothelium-dependent or -independent mechanism. Although a few mechanisms for endothelium-independent relaxation have been suggested, such as interference with protein kinase C or cAMP or cGMP phosphodiesterase, the inhibition of Ca(2+) release from intracellular stores or Ca(2+) influx from extracellular fluids, the mode of action of flavonoids remains elusive. 2. We hypothesized that treatment with flavone inhibits vascular smooth muscle contraction by decreasing the phosphorylation of the myosin phosphatase target subunit (MYPT1). 3. Rat aortic rings were denuded of endothelium, mounted in organ baths and contracted with U46619, a thromboxane A(2) analogue. 4. Flavone dose-dependently inhibited the U46619-induced contractile response and myosin light chain (MLC(20)) phosphorylation. At 10(-7) mol/L, U46619 induced vascular contraction with the concomitant phosphorylation of MYPT1 at Thr855, but not at Thr697. Incubation with flavone (100 or 300 micromol/L) for 30 min attenuated the phosphorylation of MYPT1(Thr855), but not MYPT1(Thr697). 5. It is concluded that treatment with flavone inhibits vascular smooth muscle contraction by decreasing the phosphorylation of the MYPT1. These results suggest that flavone causes endothelium-independent relaxation through, at least in part, the inhibition of p160 Rho-associated coiled-coil-containing protein kinase (ROCK) signalling.

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