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: Previous studies have shown that enhanced accumulation of contractile proteins such as smooth muscle myosin light chain kinase (smMLCK) plays a major role in human airway smooth muscle cells (HASM) cell hypercontractility and hypertrophy. Furthermore, serum IgE levels play an important role in smooth muscle hyperreactivity. However, the effect of IgE on smMLCK expression has not been investigated. In this study, we demonstrate that IgE increases the expression of smMLCK at mRNA and protein levels. This effect was inhibited significantly with neutralizing abs directed against FcεRI but not with anti-FcεRII/CD23. Furthermore, Syk knock down and pharmacological inhibition of mitogen activated protein kinases (MAPK) (ERK1/2, p38, and JNK) and phosphatidylinositol 3-kinase (PI3K) significantly diminished the IgE-mediated upregulation of smMLCK expression in HASM cells. Taken together, our data suggest a role of IgE in regulating smMLCK in HASM cells. Therefore, targeting the FcεRI activation on HASM cells may offer a novel approach in controlling the bronchomotor tone in allergic asthma.PLoS ONE 04/2014; 9(4):e93946. · 3.53 Impact Factor
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ABSTRACT: We used two-photon (2-p) FRET microscopy to provide serial, non-invasive measurements of [Ca(2+)] in arterioles of living 'biosensor' mice. These express a genetically encoded Ca(2+) indicator (GECI), either FRET-based exMLCK or intensity-based GCaMP2. FRET ratios, Rmin and Rmax, required for in vivo Ca(2+) calibration of exMLCK were obtained in isolated arteries. For in vivo experiments, mice were anesthetized (1.5% isoflurane) and arterioles within a depilated ear were visualized through the intact skin (i.e. non-invasively), by 2-p excitation of exMLCK (at 820nm) or GCaMP2 (at 920nm). Spontaneous or agonist-evoked [Ca(2+)] transients in arteriolar smooth muscle cells were imaged (at 2 Hz) with both exMLCK and GCaMP2. To examine changes in arteriolar [Ca(2+)] that might accompany hypertension, 5 exMLCK mice were implanted with telemetric blood pressure transducers and osmotic mini-pumps containing Angiotensin II (350 ng/kg/min) and fed a high (6%) salt diet for 9 days. [Ca(2+)] was measured every other day in 5 smooth muscle cells of 2-3 arterioles in each animal. Prior to AngII/salt, [Ca(2+)] was 246 ± 42nM. [Ca(2+)] increased transiently to 599 ± 0.0nM on day 2 after beginning AngII/salt, then remained elevated at 331 ± 42nM for 4 more days, before returning to 265 ± 47nM 6 days after removal of AngII/salt. In summary, two-photon excitation of exMLCK and GCaMP2 provides a method for non-invasive, longitudinal quantification of [Ca(2+)] dynamics and vascular structure in individual arterioles of a particular animal over an extended period of time, a capability that should enhance future studies of hypertension and vascular function.AJP Heart and Circulatory Physiology 05/2014; · 4.01 Impact Factor
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ABSTRACT: Our understanding of the molecular events contributing to myogenic control of diameter in cerebral resistance arteries in response to changes in intravascular pressure, a fundamental mechanism regulating blood flow to the brain, is incomplete. Myosin light chain kinase and phosphatase activities are known to be increased and decreased, respectively, to augment phosphorylation of the 20 kDa regulatory light chain subunits (LC20) of myosin II which permits cross-bridge cycling and force development. Here, we assessed the contribution of dynamic reorganization of the actin cytoskeleton and thin filament regulation to the myogenic response and serotonin-evoked constriction of pressurized rat middle cerebral arteries. Arterial diameter and the levels of phosphorylated LC20, calponin, caldesmon, cofilin and HSP27, as well as G-actin content were determined. A decline in G-actin content was observed following pressurization from 10 mmHg to between 40 and 120 mmHg, and in three conditions in which myogenic or agonist-evoked constriction occurred in the absence of a detectable change in LC20 phosphorylation. No changes in thin filament protein phosphorylation were evident. Pressurization reduced G-actin content and elevated the levels of cofilin and HSP27 phosphorylation. Inhibitors of Rho-associated kinase (ROK) and protein kinase C (PKC) prevented the decline in G-actin, reduced cofilin and HSP27 phosphoprotein content, respectively, and blocked the myogenic response. Furthermore, phosphorylation modulators of HSP27 and cofilin induced significant changes in arterial diameter and G-actin content of myogenically active arteries. Taken together, our findings suggest that dynamic reorganization of the cytoskeleton involving increased actin polymerization in response to ROK and PKC signaling contributes significantly to force generation in myogenic constriction of cerebral resistance arteries.Journal of Biological Chemistry 06/2014; · 4.60 Impact Factor