Agonist- and depolarization-induced signals for myosin light chain phosphorylation and force generation of cultured vascular smooth muscle cells.

Phosphorylation of myosin light chain (MLC) and contraction of differentiated smooth muscle cells in vascular walls are regulated by Ca2+ -dependent activation of MLC kinase, and by Rho-kinase- or protein-kinases-C-dependent inhibition of MLC phosphatase (MLCP). We examined regulatory pathways for MLC kinase and MLCP in cultured vascular smooth muscle cells (VSMCs), and for isometric force generation of VSMCs reconstituted in collagen fibers. Protein levels of RhoA, Rho-kinase and MYPT1 (a regulatory subunit of MLCP) were upregulated in cultured VSMCs, whereas a MLCP inhibitor protein, CPI-17, was downregulated. Endothelin-1 evoked a steady rise in levels of Ca2+, MLC phosphorylation and the contractile force of VSMCs, whereas angiotensin-II induced transient signals. Also, Thr853 phosphorylation of MYPT1 occurred in response to stimuli, but neither agonist induced phosphorylation of MYPT1 at Thr696. Unlike fresh aortic tissues, removal of Ca2+ or addition of voltage-dependent Ca2+ -channel blocker did not inhibit contractions of reconstituted VSMC fibers induced by agonists or even high concentrations of extracellular K+ ions. Inhibitors of Ins(1,4,5)P3-receptor and Rho-kinase antagonized agonist-induced or high-K+ -induced contraction in both reconstituted fibers and fresh tissues. These results indicate that both Ins(1,4,5)P3-induced Ca2+ release and Rho-kinase-induced MYPT1 phosphorylation at Thr853 play pivotal roles in MLC phosphorylation of cultured VSMCs where either Ca2+ -influx or CPI-17-MLCP signaling is downregulated.

CPI-17-deficient smooth muscle of chicken.

Ca(2+) sensitivity of arterial contractility is governed by regulating myosin phosphatase activity in response to agonist stimuli. CPI-17, a myosin phosphatase inhibitor phosphoprotein, is phosphorylated concomitantly with agonist-induced contractile Ca(2+) sensitization in mammalian artery. CPI-17 has not been detected in chicken artery, but is readily detectable in pigeon artery. To evaluate a role of CPI-17, we compared contractility of the arteries of 'CPI-17-deficient' chicken with those of CPI-17-rich rabbit and pigeon, and studied the effect of CPI-17-reconstitution in chicken artery. Other major regulatory/contractile proteins for Ca(2+) sensitization are expressed in both chicken and rabbit arteries. Agonists, such as an alpha(1)-agonist and endothelin-1, produced significant contraction in arteries of all species under physiological Ca(2+)-containing conditions. Depletion of Ca(2+) abolished these contractions in chicken but partially inhibited them in rabbit and pigeon arteries. Unlike CPI-17-rich tissues, chicken arteries exerted little Ca(2+) sensitization in response to alpha(1)-agonist or endothelin-1. GTPgammaS produced a slight Ca(2+) sensitizing effect in chicken artery, but this was significantly smaller compared with CPI-17-rich tissues. A PKC activator (PDBu) did not generate but rather reduced a contraction in both intact and alpha-toxin-permeabilized chicken artery in contrast to a large contraction in CPI-17-rich arteries. Myosin light chain phosphorylation was reduced by PDBu in chicken but elevated in rabbit artery. Addition of recombinant CPI-17 into beta-escin-permeabilized chicken artery restored PDBu-induced and enhanced GTPgammaS-induced Ca(2+) sensitization. Thus, CPI-17 is essential for G protein/PKC-mediated Ca(2+) sensitization in smooth muscle.

Ral-GTPase influences the regulation of the readily releasable pool of synaptic vesicles.

The Ral proteins are members of the Ras superfamily of GTPases. Because they reside in synaptic vesicles, we used transgenic mice expressing a dominant inhibitory form of Ral to investigate the role of Ral in neurosecretion. Using a synaptosomal secretion assay, we found that while K(+)-evoked secretion of glutamate was normal, protein kinase C-mediated enhancement of glutamate secretion was suppressed in the mutant mice. Since protein kinase C effects on secretion have been shown to be due to enhancement of the size of the readily releasable pool of synaptic vesicles docked at the plasma membrane, we directly measured the refilling of this readily releasable pool of synaptic vesicles after Ca(2+)-triggered exocytosis. Refilling of the readily releasable pool was suppressed in synaptosomes from mice expressing dominant inhibitory Ral. Moreover, we found that protein kinase C and calcium-induced phosphorylation of proteins thought to influence synaptic vesicle function, such as MARCKS, synapsin, and SNAP-25, were all reduced in synaptosomes from these transgenic mice. Concomitant with these studies, we searched for new functions of Ral by detecting proteins that specifically bind to it in cells. Consistent with the phenotype of the transgenic mice described above, we found that active but not inactive RalA binds to the Sec6/8 (exocyst) complex, whose yeast counterpart is essential for targeting exocytic vesicles to specific docking sites on the plasma membrane. These findings demonstrate a role for Ral-GTPase signaling in the modulation of the readily releasable pool of synaptic vesicles and suggest the possible involvement of Ral-Sec6/8 (exocyst) binding in modulation of synaptic strength.