Role of guanine nucleotide-binding proteins--ras-family or trimeric proteins or both--in Ca2+ sensitization of smooth muscle.

Abstract

The purpose of this study was to identify guanine nucleotide-binding proteins (G proteins) MgCl2/1 mM dithiothreitol/1 mM phenylmethylsulfonyl fluoride/20 ,tM GTP (buffer A), disrupted in a glass homogenizer, and centrifuged at 640 x g for 15 min, and the supernatant was decanted. The residue was twice resuspended in 70 ml of buffer A and recentrifuged. The combined supernatants containing the cell membrane fragments were centrifuged at 186,000 x g for 90 min, and the pellet was resuspended in 50 ml of buffer A containing 2% octyl glucoside, homogenized, and gently shaken for 45 min. The solution was centrifuged at 142,000 x g for 90 min, and the supematant was filtered through a 0.45-,tm Minisart cellulose acetate filter (Sartorius). The solution (-0.4 mg of protein per ml) was loaded into a 10-ml MonoS column equili-

Pertussis toxin‐sensitive Gi‐proteins and intracellular calcium sensitivity of vasoconstriction in the intact rat tail artery

“…PTX lowered vasoconstriction induced by the agonist, NA, con®rming results previously published by several laboratories (Abebe et al, 1995) but not by all (Gong et al, 1996).…”

Cardiovascular effects of a novel potent and highly selective azaindole‐based inhibitor of Rho‐kinase

“…One effector of Rho is Rho-kinase (ROCK), a serine/threonine kinase, which is comprised of two isoenzymes, ROCK-1 (or ROKb) and ROCK-2 (or ROKa). It is well established that ROCK plays a pivotal role in the regulation of the tonic phase of smooth muscle cell contraction (Gong et al, 1996;Christ and Wingard, 2005). The contractile effect results from ROCK-mediated phosphorylation of the myosin-binding subunit (MBS), the regulatory subunit of myosin light chain (MLC) phosphatase.…”

Rho-associated Kinase Directly Induces Smooth Muscle Contraction through Myosin Light Chain Phosphorylation

“…Direct activation of G-proteins by the application of GTP␥S (8,9), agonists (1,(5)(6)(7)(8), and GTP-activated Rho (10 -12) could exert Ca 2ϩ -sensitizing effects on saponin-or ␤-escin-permeabilized smooth muscle. However, the activated Rho failed to induce Ca 2ϩ sensitization of extensively Triton X-100-permeabilized smooth muscle (11). Considering that extensive Triton X-100-permeabilization allows higher molecular weight compounds to diffuse from the cytosol of smooth muscle of the rabbit portal vein (13), important diffusible factor(s) for the Ca 2ϩ sensitization of smooth muscle might be lost during extensive permeabilization by Triton X-100, an event that would result in no response to activated Rho.…”