Y27632, a Rho‐activated kinase inhibitor, normalizes dysregulation in alpha1‐adrenergic receptor‐induced contraction of Lyon hypertensive rat artery smooth muscle

Abstract

RhoA-activated kinase (ROK) is involved in disorders of smooth muscle contraction found in hypertension model animals and patients. We examined whether the α1-adrenergic receptor agonistinduced ROK signal is perturbed in resistance small mesentery artery (SMA) of Lyon genetically hypertensive (LH) rats, using a ROK antagonist, Y27632. Smooth muscle strips of SMA and aorta were isolated from LH and Lyon normotensive (LN) rats. After Ca 2+ -depletion and pre-treatment with phenylephrine (PE), smooth muscle contraction was induced by serial additions of CaCl 2 . In LH SMA Ca 2+ permeated cells to a lesser extent as compared to LN SMA, while CaCl 2 -induced contraction of LH SMA was greater than that of LN SMA, indicating a higher ratio of force to Ca 2+ in LH SMA contraction (Ca 2+ sensitization). No hyper-contraction was observed in LH aorta tissues. Treatment of LH SMA with Y27632 restored both Ca 2+ permeability and Ca 2+ -force relationship to levels seen for LN SMA. In response to PE stimulation, phosphorylation of CPI-17, a phosphorylation-dependent myosin phosphatase inhibitor protein, and MYPT1 at Thr853, the inhibitory phosphorylation site of the myosin phosphatase regulatory subunit, was increased in LN SMA, but remained unchanged in LH SMA. These results suggest that the disorder in ROKdependent Ca 2+ permeability and Ca 2+ -force relationship is responsible for LH SMA hypercontraction. Unlike other hypertensive models, the ROK-induced hyper-contractility of LH SMA is independent of MYPT1 and CPI-17 phosphorylation, which suggests that ROK-mediated inhibition of myosin phosphatase does not affect SMA hyper-contractility in LH SMA cells.

Pressure-dependent contribution of Rho kinase-mediated calcium sensitization in serotonin-evoked vasoconstriction of rat cerebral arteries

“…Neppl et al 2009; versus only MYTPT1‐T855, e.g. Freitas et al 2009; Tsai & Jiang, 2006), despite the fact that synthetic MYPT1‐T697 phosphopeptide is a ∼30‐fold more potent inhibitor of PP1C activity in vitro compared to phosphorylated MYPT1‐T855 peptide (Khromov et al 2009). An explanation for the varied presence of MYPT1‐T697 phosphorylation has not been forthcoming, prompting speculation that MYPT1‐T697 may not be a ROK phosphorylation site in vivo , but perhaps phosphorylated by another kinase, such as zipper‐interacting kinase or integrin‐linked kinase (MacDonald et al 2001; Murányi et al 2002).…”

Diversity and plasticity in signaling pathways that regulate smooth muscle responsiveness: Paradigms and paradoxes for the myosin phosphatase, the master regulator of smooth muscle contraction

“…The functional pleiotropy of CPI-17 may explain why CPI-17 signaling is silenced in the larger arteries, smooth muscle from genetically hypertensive rats and from other organs and models (90, 109,110,111,112). The multi-functionality may be a common feature of the endogenous regulator proteins for kinases/phosphatases, such as DARPP32, phosphatase inhibitor-1, inhibitor-2, a Raf kinase inhibitor protein (RKIP), and cyclin-dependent kinase inhibitors (p21CIP/WAF, p27KIP1, and p57) (113,114,115,116,117,118,119,120,121,122,123).…”

The importance of Rho-associated kinase-induced Ca2+ sensitization as a component of electromechanical and pharmacomechanical coupling in rat ureteric smooth muscle

“…There is abundant evidence that phosphorylation of T855 catalyzed by ROK occurs in a variety of smooth muscle tissues in response to diverse stimuli; however, the physiological importance of T697 phosphorylation by ROK remains uncertain. Thus, while there are several examples of T697 phosphorylation occurring in some types of smooth muscles [18][19][20][21][22][23][24][25][26][27][28][29][30], there are many examples of T855 phosphorylation occurring without T697 phosphorylation in smooth muscle tissues in response to a variety of contractile stimuli [30][31][32][33][34][35][36][37][38][39]. MLCP activity can also be inhibited via the G q/11 -protein kinase C (PKC) pathway (for review see [10]) leading to phosphorylation at T38 of the 17-kDa cytosolic phosphatase inhibitor protein, CPI-17, which, in the phosphorylated state, binds to the catalytic subunit of MLCP, inhibiting the phosphatase activity and resulting in an increase in LC 20 phosphorylation and force [40][41][42].…”