Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension

Abstract

Abnormal smooth-muscle contractility may be a major cause of disease states such as hypertension, and a smooth-muscle relaxant that modulates this process would be useful therapeutically. Smooth-muscle contraction is regulated by the cytosolic Ca2+ concentration and by the Ca2+ sensitivity of myofilaments: the former activates myosin light-chain kinase and the latter is achieved partly by inhibition of myosin phosphatase. The small GTPase Rho and its target, Rho-associated kinase, participate in this latter mechanism in vitro, but their participation has not been demonstrated in intact muscles. Here we show that a pyridine derivative, Y-27632, selectively inhibits smooth-muscle contraction by inhibiting Ca2+ sensitization. We identified the Y-27632 target as a Rho-associated protein kinase, p160ROCK. Y-27632 consistently suppresses Rho-induced, p160ROCK-mediated formation of stress fibres in cultured cells and dramatically corrects hypertension in several hypertensive rat models. Our findings indicate that p160ROCK-mediated Ca2+ sensitization is involved in the pathophysiology of hypertension and suggest that compounds that inhibit this process might be useful therapeutically.

TGF-β-mediated activation of RhoA signalling is required for efficient V12HaRas and V600EBRAF transformation

“…We observed a rapid concentration-dependent decrease of PO 4 -MLC2 in response to SB-431542 in REF52 cells (Figure 1d). Inhibition of PO 4 -MLC2 levels was readily observable with 5 mM SB-431542 treatment, and this further declined with 10 and 20 mM SB-431542 treatments and approached the level of inhibition reached after treatment with the ROCK inhibitor Y27632 (Uehata et al, 1997) (Figure 1d). …”

Molecular mechanisms of ATP secretion during immunogenic cell death

“…Thus, U2OS cells were treated with 1530 distinct chemicals from the ICCB Known Bioactives Library or the US-Drug Collection (encompassing most US Food and Drug Administration-approved and some experimental drugs), alone or in combination with MTX, for 18 h, stained with quinacrine and then analyzed by automated, quantitative epifluorescence microscopy ( Figure 2a). This screen led to the identification of several compounds that are capable of preventing the MTX-triggered loss of quinacrine-dependent fluorescence (Figure 2b): monensin, which blocks intracellular trafficking and exocytosis; 38 blebbistatin, which inhibits myosin II, thereby blocking the apoptosis-associated blebbing of the plasma membrane; 39 Y-27632, which inhibits ROCK1, 40 a protein that -upon cleavage by caspase-3 -participates in apoptotic blebbing; 41,42 and mefloquine, which inhibits PANX1 channels. 43 Subsequent validation experiments confirmed that monensin, blebbistatin and several chemically-unrelated inhibitors of ROCK1 (i.e., Y-27632, bearing with a central aminoethyl group, and H-1152, characterized by a central sulfonyl junction) and PANX1 (i.e., the synthetic quinine analogue mefloquine and the disulfonic stilbene derivatives 4,4 0 -diisothiocyano-2,2 0 -stilbenedisulfonic acid (DIDS) and 4-acetamido-4-isothiocyano-stilbene-2,2-disulfonic acid (SITS)) blunt the loss of quinacrine fluorescence as triggered in U2OS cells by MTX (Figure 2c).…”

Neuropeptide-stimulated cell migration in prostate cancer cells is mediated by RhoA kinase signaling and inhibited by neutral endopeptidase

“…The Rho-associated coiled-coil forming protein kinases p160ROCK/ROCK-1 (Ishizaki et al, 1996 and ROCK-a/ROCK-2 (Leung et al, 1996;Matsui et al, 1996) are direct downstream effectors of RhoA that regulate actin stress fiber formation. They are specifically inhibited by the synthetic pyridine derivative, Y-27632 (Uehata et al, 1997;Ishizaki et al, 2000). Pretreatment of PC-3 cells for 60 min with 10 mM Y-27632 abolished actin stress fiber formation stimulated by bombesin (panel d)) and ET-1 (panel f), demonstrating that bombesin and ET-1 stimulate the RhoA effector, ROCK, to direct cytoskeletal reorganization.…”