Nicotine exposure alters human vascular smooth muscle cell phenotype from a contractile to a synthetic type.

OBJECTIVE: Cigarette smoking is a known risk factor for arteriosclerosis. In atheromatous plaques, vascular smooth muscle cells (VSMCs) display a phenotype that is different from the contractile type under normal conditions. Nicotine is the major pharmacological agent in cigarette smoke. However, any direct effect of nicotine on VSMCs remains uncertain. Because nicotine promotes VSMC migration, its phenotype may change due to nicotine. APPROACH AND RESULTS: We used human aorta primary smooth muscle cells (HuAoSMCs), differentiated with transforming growth factor-ß, to investigate changes in the protein levels of differentiation markers and in the activity of mitogen-activated protein kinases (MAPKs) after exposure to 0.1 µM of nicotine for 48 h. After nicotine exposure, the protein levels of myosin II 10 (2.93-fold) and ß-actin (1.66-fold), synthetic type markers, were increased. In contrast, the levels of the contractile type markers, myosin II 11 (0.63-fold), high-molecular-weight caldesmon (0.40-fold) and SM22 (0.66-fold), which concern differentiated VSMC, were decreased. Moreover, nicotine exposure induced enhanced activation of p38 MAPK (1.30-fold) and extracellular signal-regulated kinase (1.91-fold). These results indicated that the phenotype of HuAoSMCs had changed to a synthetic-like type because of nicotine exposure. Thus, nicotine is one factor that can alter protein expression of differentiation markers in VSMCs. Besides, the increase of intracellular Ca(2+) levels suggested that these effects of nicotine were mediated through nicotinic acetylcholine receptors. CONCLUSION: Nicotine has already been reported to promote VSMC migration from the tunica media to atheromatous plaques in the vascular intima. This phenomenon may occur because nicotine directly induces VSMC transformation from contractile type to synthetic-like type via nicotinic acetylcholine receptors and G protein-coupled receptors.

Role of non-kinase activity of myosin light-chain kinase in regulating smooth muscle contraction, a review dedicated to Dr. Setsuro Ebashi.

Myosin light-chain kinase (MLCK) of smooth muscle consists of an actin-binding domain at the N-terminal, the catalytic domain in the central portion, and the myosin-binding domain at the C-terminal. The kinase activity is mediated by the catalytic domain that phosphorylates the myosin light-chain of 20kDa (MLC20), activating smooth muscle myosin to interact with actin. Although the regulatory role of the kinase activity is well established, the role of non-kinase activity derived from actin-binding and myosin-binding domains remains unknown. This review is dedicated to Dr. Setsuro Ebashi, who devoted himself to elucidating the non-kinase activity of MLCK after establishing calcium regulation through troponin in skeletal and cardiac muscles. He proposed that the actin-myosin interaction of smooth muscle could be activated by the non-kinase activity of MLCK, a mechanism that is quite independent of MLC20 phosphorylation. The authors will extend his proposal for the role of non-kinase activity. In this review, we express MLCK and its fragments as recombinant proteins to examine their effects on the actin-myosin interaction in vitro. We also down-regulate MLCK in the cultured smooth muscle cells, and propose that MLC20 phosphorylation is not obligatory for the smooth muscle to contract.

Insight into the mechanism of fast movement of myosin from Chara corallina.

Chara myosin, two-headed plant myosin belonging to class XI, slides F-actin at maximally 60 microm s(-1). To elucidate the mechanism of this fast sliding, we extensively investigated its mechanochemical properties. The maximum actin activated ATPase activity, Vmax, was 21.3 s(-1) head(-1) in a solution, but when myosin was immobilized on the surface, its activity was 57.6 s(-1) head(-1) at 2 mg ml(-1) of F-actin. The sliding velocity and the actin activated ATPase activity were greatly inhibited by ADP, suggesting that ADP dissociation was the rate limiting step. With the extensive assay of motility by varying the surface density, the duty ratio of Chara myosin was found to be 0.49-0.44 from velocity measurements and 0.34 from the landing rate analysis. At the surface density of 10 molecules microm(-2), Chara myosin exhibited pivot movement under physiological conditions. Based on the results obtained, we will discuss the sliding mechanism of Chara myosin according to the working stroke model in terms of its physiological aspects. aspects.

Biochemical properties of ordinary and dark muscle myosin from carp skeletal muscle.

Two types of myosin isolated from ordinary (fast) and dark (slow) muscles of carp were examined by ATPase and in vitro motility assays. Vmax of the ATPase activity and sliding velocity of ordinary myosin showed 1.6 and 1.5 times higher activities than those of dark myosin, whereas those of mammalian fast myosin were much higher, 3 to 10 times, than those of slow myosin. Although ordinary myosin had almost identical activities to those of mammalian fast myosin, activities of dark myosin was twice of those of mammalian slow myosin. This high motile activity of dark myosin can account for the physiological role of dark muscle in cruising of fish. By comparing Km of the actin-activated ATPase activity, ordinary myosin was appeared to have higher affinity to F-actin than dark myosin, and this was confirmed by the binding assay of HMM or S-1 of carp myosin to F-actin. Investigation of myosin assembly by electron microscopy and the centrifugation assay revealed that ordinary myosin assembled much poorly than dark myosin or mammalian fast myosin. This phenomenon may reflect characteristic cellular function of fish skeletal muscle.