Question: 

Vascular endothelium is known to be involved in autoregulation of brain circulatory system under physiological and pathophysiological conditions. We have previously reported that in rat brain vasculature 8-Br-cGMP inhibits translocation of myosin phosphatase targeting subunit, MYPT1, phosphorylated at Thr853 (pMYPT1^Thr853), which is induced by the stable thromboxane analogue, U46619. This site is phosphorylated by Rho-kinase and considered to be responsible for inhibition of smooth muscle myosin phosphatase. Here we investigated the effect of inhibition of the endogenous NO-production on U46619 induced contractile response, pMYPT1^Thr853 -phosphorylation and -translocation in isolated mouse A. basilaris.

Methodology: 

Contraction of mouse A. basilaris was measured using a wire myorgaphy. Phosphorylation and translocation of pMYPT1^Thr853 were studied with Western blotting and immunohistochemistry using a phosphospecific antibody.

Results and Conclusions: 

In isolated rings from mouse A. basilaris, cumulative application U46619 induced small contraction only at 1 mM (0.15 N/m) and had no effect on pMYPT1^Thr853-phosphorylation. By contrast, application of 100 mM L-NAME increased the basal and U46619 induced tone (1.56 N/m) and phosphorylation of pMYPT1^Thr853 (n = 3). Under basal conditions and in presence of 1 mM U46619 the immunoreactive signal of pMYPT1^Thr853 showed homogenous cytosolic distribution. Pretreatment with 100 mM L-NAME, followed by stimulation with U46619, induced marked change in smooth muscle cell shape and increased colocalization of pMYPT1^Thr853 with phalloidin stained actin. Our results suggest that in mouse A. basilaris endogenous NO-production is a major regulator of vascular tone, and is modulated by phosphorylation and redistribution of pMYPT1^Thr853.