Ca2+ sensitivity of smooth muscle and nonmuscle myosin II reflects the ratio of activities of myosin light-chain kinase (MLCK) to myosin light-chain phosphatase (MLCP) and is a major, regulated determinant of numerous cellular processes. We conclude that the majority of phenotypes attributed to the monomeric G protein RhoA and mediated by its effector, Rho-kinase (ROK), reflect Ca2+ sensitization: inhibition of myosin II dephosphorylation in the presence of basal (Ca2+ dependent or independent) or increased MLCK activity. We outline the pathway from receptors through trimeric G proteins (Galphaq, Galpha12, Galpha13) to activation, by guanine nucleotide exchange factors (GEFs), from GDP. RhoA. GDI to GTP. RhoA and hence to ROK through a mechanism involving association of GEF, RhoA, and ROK in multimolecular complexes at the lipid cell membrane. Specific domains of GEFs interact with trimeric G proteins, and some GEFs are activated by Tyr kinases whose inhibition can inhibit Rho signaling. Inhibition of MLCP, directly by ROK or by phosphorylation of the phosphatase inhibitor CPI-17, increases phosphorylation of the myosin II regulatory light chain and thus the activity of smooth muscle and nonmuscle actomyosin ATPase and motility. We summarize relevant effects of p21-activated kinase, LIM-kinase, and focal adhesion kinase. Mechanisms of Ca2+ desensitization are outlined with emphasis on the antagonism between cGMP-activated kinase and the RhoA/ROK pathway. We suggest that the RhoA/ROK pathway is constitutively active in a number of organs under physiological conditions; its aberrations play major roles in several disease states, particularly impacting on Ca2+ sensitization of smooth muscle in hypertension and possibly asthma and on cancer neoangiogenesis and cancer progression. It is a potentially important therapeutic target and a subject for translational research.

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We here review mechanisms that can regulate the activity of myosin II, in smooth muscle and non‐muscle cells, by modulating the Ca2+ sensitivity of myosin regulatory light chain (RLC) phosphorylation. The major mechanism of Ca2+ sensitization of smooth muscle contraction and non‐muscle cell motility is through inhibition of the smooth muscle myosin phosphatase (MLCP) that dephosphorylates the RLC in smooth muscle and non‐muscle. The active, GTP‐bound form of the small GTPase RhoA activates a serine/threonine kinase, Rho‐kinase, that phosphorylates the regulatory subunit of MLCP and inhibits phosphatase activity. G‐protein‐coupled release of arachidonic acid may also contribute to inhibition of MLCP acting, at least in part, through the Rho/Rho‐kinase pathway. Protein kinase C(s) activated by phorbol esters and diacylglycerol can also inhibit MLCP by phosphorylating and thereby activating CPI‐17, an inhibitor of its catalytic subunit; this mechanism is independent of the Rho/Rho‐kinase pathway and plays only a minor, transient role in the G‐protein‐coupled mechanism of Ca2+ sensitization. Ca2+ sensitization by the Rho/Rho‐kinase pathway contributes to the tonic phase of agonist‐induced contraction in smooth muscle, and abnormally increased activation of myosin II by this mechanism is thought to play a role in diseases such as high blood pressure and cancer cell metastasis.

Smooth muscle cell (SMC) differentiation is regulated by a complex array of local environmental cues, but the intracellular signaling pathways and the transcription mechanisms that regulate this process are largely unknown. We and others have shown that serum response factor (SRF) contributes to SMC-specific gene transcription, and because the small GTPase RhoA has been shown to regulate SRF, the goal of the present study was to test the hypothesis that RhoA signaling is a critical mechanism for regulating SMC differentiation. Coexpression of constitutively active RhoA in rat aortic SMC cultures significantly increased the activity of the SMCspecific promoters, SM22 and SM ␣-actin, whereas coexpression of C3 transferase abolished the activity of these promoters. Inhibition of either stress fiber formation with the Rho kinase inhibitor Y-27632 (10 M) or actin polymerization with latrunculin B (0.5 M) significantly decreased the activity of SM22 and SM ␣-actin promoters. In contrast, increasing actin polymerization with jasplakinolide (0.5 M) increased SM22 and SM ␣-actin promoter activity by 22-fold and 13-fold, respectively. The above interventions had little or no effect on the transcription of an SRF-dependent c-fos promoter or on a minimal thymidine kinase promoter that is not SRFdependent. Taken together, the results of these studies indicate that in SMC, RhoA-dependent regulation of the actin cytoskeleton selectively regulates SMC differentiation marker gene expression by modulating SRF-dependent transcription. The results also suggest that RhoA signaling may serve as a convergence point for the multiple signaling pathways that regulate SMC differentiation. Vascular smooth muscle cell (SMC)1 differentiation is an important process during vasculogenesis and angiogenesis, and it is recognized that alterations in SMC phenotype play a role in the progression of several prominent cardiovascular disease states including atherosclerosis, hypertension, and restenosis (1-3). It is well established that SMC do not terminally differentiate and that SMC phenotype is regulated by a complex array of local environmental cues including humoral factors, cell-cell and cell-matrix interactions, inflammatory stimuli, and mechanical stresses (reviewed in Refs. 4 and 5). However, the mechanisms by which these diverse signals regulate SMC phenotype and the transcription mechanisms that ultimately regulate SMC differentiation are largely unknown. It is clear though that the identification of the signaling and transcription pathways that control SMC-specific gene expression will be an important step toward our understanding of blood vessel development and the role played by SMC during the development of cardiovascular disease.To date, no transcription factors have been identified that specify SMC lineage or by themselves can explain SMC-specific gene expression. However, serum response factor (SRF), a MADS box transcription factor, has been shown to contribute to the regulation of most SMC differentiation marker genes and may be a ...

The sarcoplasmic reticulum (SR) was studied in the smooth muscles of rabbit main pulmonary artery, mesenteric vein, aorta, mesenteric artery, taenia coli, guinea pig mesenteric artery, and human uterus, and correlated with contractions of the smooth muscles in Cafree media . S R volumes, were determined in main pulmonary artery (5 .1 %), aorta (5%0), portal-anterior mesenteric vein (2 .2%), taenia coli (2%), and mesenteric artery (1 .8%) : because of tangentially sectioned membranes these estimates are subject to a correction factor of up to +50% of the values measured . Smooth muscles that contained a relatively large volume of SR maintained significant contractile responses to drugs in the virtual absence of extracellular calcium at room temperatures, while smooth muscles that had less SR did not . The unequal maximal contractions of main pulmonary artery elicited by different drugs were also observed in Ca-free, high potassium-depolarizing solution, indicating that they were secondary to some mechanism independent of changes in membrane potential or calcium influx . Longitudinal tubules of SR run between and are fenestrated about groups of surface vesicles separated from each other by intervening dense bodies . Extracellular markers (ferritin and lanthanum) entered the surface vesicles, but not the SR. The peripheral SR formed couplings with the surface membrane : the two membranes were separated by gaps of approximately 10 nm traversed by electron-opaque connections suggestive of a periodicity of approximately 20-25 nm . These couplings are considered to be the probable sites of electromechanical coupling in twitch smooth muscles . Close contacts between the SR and the surface vesicles may have a similar function, or represent sites of calcium extrusion . The presence of both thick and thin myofilaments and of rough SR in smooth muscles supports the dual, contractile and morphogenetic, function of smooth muscle .