Objective-We have recently demonstrated that protein kinase C (PKC) and Rho-kinase play important roles in coronary vasospasm in a porcine model. However, it remains to be examined whether there is an interaction between the two molecules to cause the spasm. Methods and Results-A segment of left porcine coronary artery was chronically treated with IL-1␤-bound microbeads in vivo. Two weeks after the operation, phorbol ester caused coronary spasm in vivo and coronary hypercontractions in vitro at the IL-1␤-treated segment; both were significantly inhibited by hydroxyfasudil, a specific Rho-kinase inhibitor. Guanosine 5Ј-[␥-thio]triphosphate (GTP␥S), which activates Rho with a resultant activation of Rho-kinase, enhanced Ca 2ϩ sensitization of permeabilized vascular smooth muscle cells, which were resistant to the blockade of PKC by calphostin C. The GTP␥S-induced Ca 2ϩ sensitization was greater in the spastic segment than in the control segment. Western blot analysis revealed that only PKC␦ isoform was activated during the hypercontraction.Conclusions-These results demonstrate that PKC and Rho-kinase coexist on the same intracellular signaling pathway, with PKC located upstream on Rho-kinase, and that among the PKC isoforms, only PKC␦ may be involved. Thus, the strategy to inhibit Rho-kinase rather than PKC may be a more specific and useful treatment for coronary spasm. Key Words: arteriosclerosis Ⅲ coronary disease Ⅲ smooth muscle Ⅲ signal transduction C oronary artery spasm plays an important role in the pathogenesis of a variety of ischemic heart diseases, including not only variant angina but also other forms of angina, myocardial infarction, ventricular arrhythmias, and sudden death. 1,2 However, the intracellular mechanism for the spasm remains to be elucidated.We previously developed a porcine model of coronary spasm in which the spasm was repeatedly induced by autacoids, such as serotonin and histamine, at atherosclerotic lesions made by a combination of balloon endothelial removal and high-cholesterol feeding. [3][4][5] We subsequently developed a porcine model in which long-term adventitial treatment with interleukin-1␤ (IL-1␤), one of the major inflammatory cytokines, induces arteriosclerotic changes and vasospastic responses of the coronary artery. 6 -15 Since the histological changes and vasospastic responses in our porcine models are similar to those observed in humans, our models may be useful to elucidate the mechanisms of the spasm in humans.We recently demonstrated that in our porcine models, coronary artery spasm induced by serotonin is mediated by protein kinase C (PKC) 5,7 and Rho-kinase. 9,11-15 Rho-kinase, one of the major target proteins of small Rho GTPase, 16,17 was upregulated in spastic coronary segment, leading to an increased phosphorylation of myosin-binding subunit of myosin phosphatase (MLCPh) and the resultant suppression of MLCPh. 11-15 Indeed, we recently demonstrated that Rho-kinase is substantially involved in hypercontractions of isolated arteriosclerotic human arteries in v...

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Inhibitory effects of docosahexaenoic acid (DHA) on blood vessel contractions induced by various constrictor stimulants were investigated in the rat thoracic aorta. The inhibitory effects of DHA were also compared with those of eicosapentaenoic acid (EPA) and linoleic acid (LA). DHA exhibited a strong inhibitory effect on the sustained contractions induced by U46619, a TXA2 mimetic. This inhibitory effect of DHA was not affected by removal of the endothelium or by treatment with either indomethacin or Nω-nitro-l-arginine. DHA also significantly diminished PGF2α-induced contraction but did not show any appreciable inhibitory effects on the contractions to both phenylephrine (PE) and high-KCl. Similarly, EPA exhibited significant inhibitory effects against the contractions induced by both U46619 and PGF2α without substantially affecting either PE- or high-KCl-induced contractions. However, both DHA and EPA generated more potent inhibitions against contractions induced by U46619 than those by PGF2α. In contrast, LA did not show significant inhibitory effects against any contractions, including those induced by U46619. The present findings suggest that DHA and EPA elicit more selective inhibition against blood vessel contractions that are mediated through stimulation of prostanoid receptors than those through α-adrenoceptor stimulation or membrane depolarization. Although DHA and EPA have similar inhibitory potencies against prostanoid receptor-mediated contractions, they had a more potent inhibition against TXA2 receptor (TP receptor)-mediated contractions than against PGF2α receptor (FP receptor)-mediated responses. Selective inhibition by either DHA or EPA of prostanoid receptor-mediated blood vessel contractions may partly underlie the mechanisms by which these ω-3 polyunsaturated fatty acids exert their circulatory-protective effects.

SignificanceWhich brain area drives hand/arm movements after learning or brain injury? When does motor cortical activity generate appropriate hand/arm movements? To address these issues, it is necessary to manipulate motor cortical activity in a controlled manner. Optogenetic tools allow neuronal activity to be manipulated in a variety of animals, but forelimb movements in nonhuman primates have not previously been optogenetically induced or modulated. Here, we improved a method of optogenetic cortical stimulation and induced overt forelimb movements in the common marmoset, a New World monkey. Photostimulation also modulated voluntary forelimb movements, with the modulated movement trajectories depending on the stimulation site and timing. Our results open doors for noninvasive interrogation of motor circuits in behaving nonhuman primates.

Calponin, a thin filament-associated protein, inhibits actomyosin adenosinetriphosphatase in solution and has been suggested to modulate smooth muscle contractility. We used permeabilized guinea pig taenia coli smooth muscle to investigate whether calponin can modulate actin-myosin interaction in a more organized contractile system. Fibers were permeabilized with Triton X-100 and glycerol, which permit access of large macromolecules to the contractile apparatus. For contractures elicited by Ca2+ (6.6 microM + 0.1 microM calmodulin), the recombinant alpha-isoform of chicken gizzard calponin (CaP) decreased isometric force (Fo) and unloaded shortening velocity (Vus) in a dose-dependent manner; 1 microM CaP had minimal effects on force (< 10%) but reduced Vus by approximately 50% and 10 microM CaP reduced Fo to 27% of control and Vus to near zero levels. To eliminate any effects of the binding of calmodulin by CaP and consequent inhibition of myosin light chain kinase activity, we also studied fibers activated by thiophosphorylation of the myosin regulatory light chain. Fo was only moderately inhibited, remaining at approximately 75% of control in the presence of CaP (10 microM), whereas Vus was reduced to 32% of control. A similar inhibition was obtained with a mutant (CaPcys175) that retains the ability to bind to actin. CaP phosphorylated by protein kinase C and CaPcys175 mutant labeled with 1,5-IAEDANS, which bind actin poorly, were not effective inhibitors. Our results indicate that 1) CaP more strongly inhibits Vus (approximately cross-bridge cycle rate) than Fo (approximately number of activated cross bridges) and 2) the effects of CaP are related to its binding to actin. Thus the function of CaP in regulation of smooth muscle contractility may be more strongly related to its function as a modulator of velocity, as related to the "latch state," than as an "on-off" switch.

Muscle contraction is accompanied by passive stretching or deformation of cells and tissues. The present study aims to clarify whether or not acute passive stretching evokes glucose transporter 4 (GLUT4) translocation and glucose uptake in skeletal muscles of mice. Passive stretching mainly induced GLUT4 translocation from an intracellular membrane-rich fraction (PF5) to a plasma membrane-rich fraction (F2) and accelerated glucose uptake in hindlimb muscles; whereas electrical stimulation, which mimics physical exercise in vivo, and insulin, each induced GLUT4 translocation from an intracellular membrane-rich fraction (PF5) to a fraction rich in plasma membrane (F2), and to one rich in transverse tubules (PF3), along with subsequent glucose uptake. Mechanical stretching increased phosphorylation of Akt and p38 mitogen-activated protein kinase (p38 MAPK), but it had no apparent effect on the activity of AMP-activated protein kinase (AMPK). Electrical stimulation augmented the activity of not only AMPK but also phosphorylation of Akt and p38 MAPK. Our results suggest that passive stretching produces translocation of GLUT4 mainly from the fraction rich in intracellular membrane to that rich in plasma membrane, and that the glucose uptake could be Akt- and p38 MAPK-dependent, but AMPK-independent manners.

Background: We previously reported that protein kinase C (PKC)-δ was initially translocated from the cytosol to the membrane fraction (on day 4), followed by PKC-α, with the progression of cerebral vasospasm after subarachnoid hemorrhage (SAH) on day 7. Rho/Rho-kinase pathways have also been proposed to be involved in the vasospasm. Thus we investigated the interactive role of Rho-kinase and PKC in the development of cerebral vasospasm after SAH. Methods: The cerebral vasospasm was produced using a ‘two-hemorrhage’ canine model. The animals were treated with Y-27632, a Rho-kinase inhibitor, and rottlerin, a PKC-δ inhibitor, both injected into the cisterna magna. Results: Y-27632 inhibited the vasospasm, 20-kDa myosin light chain (MLC₂₀) phosphorylation, and PKC-δ translocation after the second injection of autologous blood on day 4. In contrast, Y-27632 did not affect the vasospasm on day 7. Rottlerin also inhibited the vasospasm on day 4, but had no effect on MLC₂₀ phosphorylation and RhoA translocation. The vasospasm was accompanied with the phosphorylation of caldesmon (CaD), an actin-linked regulatory protein, which was strongly attenuated by Y-27632 and rottlerin. The application of PKC-δ to skinned strips of isolated canine basilar arteries caused a contraction and an increase in CaD phosphorylation. Conclusion: The development of cerebral vasospasm after SAH (on day 4) is caused by at least two mechanisms: one involves MLC₂₀ phosphorylation mediated by the inhibition of MLC₂₀ phosphatase by Rho-kinase, and the other CaD phosphorylation mediated by the activation of PKC-δ by Rho-kinase, which results in the alleviation of the inhibition by CaD of myosin Mg²⁺-ATPase activity.