Signaling processes for initiating smooth muscle contraction upon neural stimulation.
ABSTRACT Relationships among biochemical signaling processes involved in Ca2+/calmodulin (CaM)-dependent phosphorylation of smooth muscle myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK) were determined. A genetically-encoded biosensor MLCK for measuring Ca(2+)-dependent CaM binding and activation was expressed in smooth muscles of transgenic mice. We performed real-time evaluations of the relationships among [Ca2+](i), MLCK activation, and contraction in urinary bladder smooth muscle strips neurally stimulated for 3 s. Latencies for the onset of [Ca2+](i) and kinase activation were 55 +/- 8 and 65 +/- 6 ms, respectively. Both increased with RLC phosphorylation at 100 ms, whereas force latency was 109 +/- 3 ms. [Ca2+](i), kinase activation, and RLC phosphorylation responses were maximal by 1.2 s, whereas force increased more slowly to a maximal value at 3 s. A delayed temporal response between RLC phosphorylation and force is probably due to mechanical effects associated with elastic elements in the tissue. MLCK activation partially declined at 3 s of stimulation with no change in [Ca2+](i) and also declined more rapidly than [Ca2+](i) during relaxation. The apparent desensitization of MLCK to Ca2+ activation appears to be due to phosphorylation in its calmodulin binding segment. Phosphorylation of two myosin light chain phosphatase regulatory proteins (MYPT1 and CPI-17) or a protein implicated in strengthening membrane adhesion complexes for force transmission (paxillin) did not change during force development. Thus, neural stimulation leads to rapid increases in [Ca2+](i), MLCK activation, and RLC phosphorylation in phasic smooth muscle, showing a tightly coupled Ca2+ signaling complex as an elementary mechanism initiating contraction.
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ABSTRACT: Key points • Parasympathetic nerves release the neurotransmitters ATP and acetylcholine that activate purinergic and muscarinic receptors, respectively, to initiate contraction of urinary bladder smooth muscle. • Although both receptors mediate Ca(2+) influx for myosin regulatory light chain (RLC) phosphorylation necessary for contraction, the muscarinic receptor may also recruit cellular mechanisms affecting the Ca(2+) sensitivity of RLC phosphorylation. • Using transgenic mice expressing Ca(2+)/calmodulin sensor myosin light chain kinase (MLCK) in smooth muscles, the effects of selective purinergic or muscarinic receptor inhibition were examined on neurally stimulated tissues in relation to signalling pathways converging on RLC phosphorylation. • Purinergic-mediated Ca(2+) signals provide the initial Ca(2+)/calmodulin activation of MLCK with muscarinic receptors supporting sustained responses. • Activation of muscarinic receptors leads phosphorylation of myosin light chain phosphatase inhibitor CPR-17 to enhance Ca(2+) sensitivity while also initiating phosphorylation-dependent Ca(2+) desensitization of MLCK. The interplay between Ca(2+) sensitization and desensitization mechanisms fine tunes the contractile signalling module for force development.The Journal of Physiology 08/2012; 590(Pt 20):5107-21. DOI:10.1113/jphysiol.2012.235424 · 4.54 Impact Factor
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ABSTRACT: Myosin phosphatase (MP) is a key regulator of myosin light chain (LC20) phosphorylation, a process essential for motility, apoptosis, and smooth muscle contractility. Although MP inhibition is well studied, little is known about MP activation. We have recently demonstrated that prostate apoptosis response (Par)-4 modulates vascular smooth muscle contractility. Here, we test the hypothesis that Par-4 regulates MP activity directly. We show, by proximity ligation assays, surface plasmon resonance and coimmunoprecipitation, that Par-4 interacts with the targeting subunit of MP, MYPT1. Binding is mediated by the leucine zippers of MYPT1 and Par-4 and reduced by Par-4 phosphorylation. Overexpression of Par-4 leads to increased phosphatase activity of immunoprecipitated MP, whereas small interfering RNA knockdown of endogenous Par-4 significantly decreases MP activity and increases MYPT1 phosphorylation. LC20 phosphorylation assays demonstrate that overexpression of Par-4 reduces LC20 phosphorylation. In contrast, a phosphorylation site mutant, but not wild-type Par-4, interferes with zipper-interacting protein kinase (ZIPK)-mediated MP inhibition. We conclude from our results Par-4 operates through a "padlock" model in which binding of Par-4 to MYPT1 activates MP by blocking access to the inhibitory phosphorylation sites, and inhibitory phosphorylation of MYPT1 by ZIPK requires "unlocking" of Par-4 by phosphorylation and displacement of Par-4 from the MP complex.Molecular biology of the cell 04/2010; 21(7):1214-24. DOI:10.1091/mbc.E09-08-0711 · 5.98 Impact Factor
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ABSTRACT: The corneal endothelium maintains stromal deturgescence, which is a prerequisite for corneal transparency. The principal challenge to stromal deturgescence is the swelling pressure associated with the hydrophilic glycosaminoglycans in the stroma. This negative pressure induces fluid leak into the stroma from the anterior chamber, but the rate of leak is restrained by the tight junctions of the endothelium. This role of the endothelium represents its barrier function. In healthy cornea, the fluid leak is counterbalanced by an active fluid pump mechanism associated with the endothelium itself. Although this pump-leak hypothesis was postulated several decades ago, the mechanisms underlying regulation of the balance between the pump and leak functions remain largely unknown. In the last couple of decades, the ion transport systems that support the fluid pump activity have been discovered. In contrast, despite significant evidence for corneal edema secondary to endothelial barrier dysfunction, the molecular aspects underlying its regulation are relatively unknown. Recent findings in our laboratory, however, indicate that barrier integrity (i.e., structural and functional integrity of the tight junctions) of the endothelium is sensitive to remodeling of its peri-junctional actomyosin ring, which is located at the apical junctional complex. This review provides a focused perspective on dynamic regulation of the barrier integrity of endothelium vis-à-vis plasticity of the peri-junctional actomyosin ring and its association with cell signaling downstream of small GTPases of the Rho family. Based on findings to date, it appears that development of specific pharmacological strategies to treat corneal edema in response to inflammatory stress would be possible in the near future.Optometry and vision science: official publication of the American Academy of Optometry 02/2010; 87(4):E239-54. DOI:10.1097/OPX.0b013e3181d39464 · 2.04 Impact Factor