Cheng-Hai Zhang

Myosin light chain kinase is central to smooth muscle contraction and required for gastrointestinal motility in mice

BACKGROUND & AIMS Smooth muscle is essential for maintaining homeostasis for many body functions and provides adaptive responses to stresses imposed by pathologic disorders. Identified cell signaling networks have defined many potential mechanisms for initiating smooth muscle contraction with or without myosin regulatory light chain (RLC) phosphorylation by myosin light chain kinase (MLCK). We generated tamoxifen-inducible and smooth muscle-specific MLCK knockout (KO) mice and provide direct loss-of-function evidence that shows the primary importance of MLCK in phasic smooth muscle contractions. METHODS We used the Cre-loxP system to establish Mlck floxed mice in which exons 23, 24, and 25 were flanked by 2 loxP sites. Smooth muscle-specific MLCK KO mice were generated by crossing Mlck floxed mice with SM-CreER(T2) (ki) mice followed by tamoxifen treatment. The phenotype was assessed by histologic, biochemical, molecular, cell biological, and physiologic analyses. RESULTS Targeted deletion of MLCK in adult mouse smooth muscle resulted in severe gut dysmotility characterized by weak peristalsis, dilation of the digestive tract, and reduction of feces excretion and food intake. There was also abnormal urinary bladder function and lower blood pressure. Isolated muscles showed a loss of RLC phosphorylation and force development induced by K(+)-depolarization. The kinase knockout also markedly reduced RLC phosphorylation and force development with acetylcholine which activates Ca(2+)-sensitizing signaling pathways. CONCLUSIONS MLCK and its phosphorylation of RLC are required physiologically for smooth muscle contraction and are essential for normal gastrointestinal motility.

The cellular and molecular basis of bitter tastant-induced bronchodilation.

Bitter tastants can activate bitter taste receptors on constricted smooth muscle cells to inhibit L-type calcium channels and induce bronchodilation.

Myosin Light Chain Kinase Is Necessary for Tonic Airway Smooth Muscle Contraction

Different interacting signaling modules involving Ca2+/calmodulin-dependent myosin light chain kinase, Ca2+-independent regulatory light chain phosphorylation, myosin phosphatase inhibition, and actin filament-based proteins are proposed as specific cellular mechanisms involved in the regulation of smooth muscle contraction. However, the relative importance of specific modules is not well defined. By using tamoxifen-activated and smooth muscle-specific knock-out of myosin light chain kinase in mice, we analyzed its role in tonic airway smooth muscle contraction. Knock-out of the kinase in both tracheal and bronchial smooth muscle significantly reduced contraction and myosin phosphorylation responses to K+-depolarization and acetylcholine. Kinase-deficient mice lacked bronchial constrictions in normal and asthmatic airways, whereas the asthmatic inflammation response was not affected. These results indicate that myosin light chain kinase acts as a central participant in the contractile signaling module of tonic smooth muscle. Importantly, contractile airway smooth muscles are necessary for physiological and asthmatic airway resistance.

Trio Is a Key Guanine Nucleotide Exchange Factor Coordinating Regulation of the Migration and Morphogenesis of Granule Cells in the Developing Cerebellum

Orchestrated regulation of neuronal migration and morphogenesis is critical for neuronal development and establishment of functional circuits, but its regulatory mechanism is incompletely defined. We established and analyzed mice with neural-specific knock-out of Trio, a guanine nucleotide exchange factor with multiple guanine nucleotide exchange factor domains. Knock-out mice showed defective cerebella and severe signs of ataxia. Mutant cerebella had no granule cells in the internal granule cell layer due to aberrant granule cell migration as well as abnormal neurite growth. Trio-deficient granule cells showed reduced extension of neurites and highly branched and misguided processes with perturbed stabilization of actin and microtubules. Trio deletion caused down-regulation of the activation of Rac1, RhoA, and Cdc42, and mutant granule cells appeared to be unresponsive to neurite growth-promoting molecules such as Netrin-1 and Semaphorin 6A. These results suggest that Trio may be a key signal module for the orchestrated regulation of neuronal migration and morphogenesis during cerebellar development. Trio may serve as a signal integrator decoding extrinsic signals to Rho GTPases for cytoskeleton organization.

Role of myosin light chain kinase in regulation of basal blood pressure and maintenance of salt-induced hypertension.

Vascular tone, an important determinant of systemic vascular resistance and thus blood pressure, is affected by vascular smooth muscle (VSM) contraction. Key signaling pathways for VSM contraction converge on phosphorylation of the regulatory light chain (RLC) of smooth muscle myosin. This phosphorylation is mediated by Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) but Ca(2+)-independent kinases may also contribute, particularly in sustained contractions. Signaling through MLCK has been indirectly implicated in maintenance of basal blood pressure, whereas signaling through RhoA has been implicated in salt-induced hypertension. In this report, we analyzed mice with smooth muscle-specific knockout of MLCK. Mesenteric artery segments isolated from smooth muscle-specific MLCK knockout mice (MLCK(SMKO)) had a significantly reduced contractile response to KCl and vasoconstrictors. The kinase knockout also markedly reduced RLC phosphorylation and developed force. We suggest that MLCK and its phosphorylation of RLC are required for tonic VSM contraction. MLCK(SMKO) mice exhibit significantly lower basal blood pressure and weaker responses to vasopressors. The elevated blood pressure in salt-induced hypertension is reduced below normotensive levels after MLCK attenuation. These results suggest that MLCK is necessary for both physiological and pathological blood pressure. MLCK(SMKO) mice may be a useful model of vascular failure and hypotension.

The Transmembrane Protein 16A Ca2+-activated Cl− Channel in Airway Smooth Muscle Contributes to Airway Hyperresponsiveness

RATIONALE Asthma is a chronic inflammatory disorder with a characteristic of airway hyperresponsiveness (AHR). Ca(2+)-activated Cl(-) [Cl((Ca))] channels are inferred to be involved in AHR, yet their molecular nature and the cell type they act within to mediate this response remain unknown. OBJECTIVES Transmembrane protein 16A (TMEM16A) and TMEM16B are Cl((Ca)) channels, and activation of Cl((Ca)) channels in airway smooth muscle (ASM) contributes to agonist-induced airway contraction. We hypothesized that Tmem16a and/or Tmem16b encode Cl((Ca)) channels in ASM and mediate AHR. METHODS We assessed the expression of the TMEM16 family, and the effects of niflumic acid and benzbromarone on AHR and airway contraction, in an ovalbumin-sensitized mouse model of chronic asthma. We also cloned TMEM16A from ASM and examined the Cl(-) currents it produced in HEK293 cells. We further studied the impacts of TMEM16A deletion on Ca(2+) agonist-induced cell shortening, and on Cl((Ca)) currents activated by Ca(2+) sparks (localized, short-lived Ca(2+) transients due to the opening of ryanodine receptors) in mouse ASM cells. MEASUREMENTS AND MAIN RESULTS TMEM16A, but not TMEM16B, is expressed in ASM cells and its expression in these cells is up-regulated in ovalbumin-sensitized mice. Niflumic acid and benzbromarone prevent AHR and contraction evoked by methacholine in ovalbumin-sensitized mice. TMEM16A produces Cl((Ca)) currents with kinetics similar to native Cl((Ca)) currents. TMEM16A deletion renders Ca(2+) sparks unable to activate Cl((Ca)) currents, and weakens caffeine- and methacholine-induced cell shortening. CONCLUSIONS Tmem16a encodes Cl((Ca)) channels in ASM and contributes to Ca(2+) agonist-induced contraction. In addition, up-regulation of TMEM16A and its augmented activation contribute to AHR in an ovalbumin-sensitized mouse model of chronic asthma. TMEM16A may represent a potential therapeutic target for asthma.

Altered Contractile Phenotypes of Intestinal Smooth Muscle in Mice Deficient in Myosin Phosphatase Target Subunit 1

BACKGROUND & AIMS The regulatory subunit of myosin light chain phosphatase, MYPT1, has been proposed to control smooth muscle contractility by regulating phosphorylation of the Ca(2+)-dependent myosin regulatory light chain. We generated mice with a smooth muscle-specific deletion of MYPT1 to investigate its physiologic role in intestinal smooth muscle contraction. METHODS We used the Cre-loxP system to establish Mypt1-floxed mice, with the promoter region and exon 1 of Mypt1 flanked by 2 loxP sites. These mice were crossed with SMA-Cre transgenic mice to generate mice with smooth muscle-specific deletion of MYPT1 (Mypt1(SMKO) mice). The phenotype was assessed by histologic, biochemical, molecular, and physiologic analyses. RESULTS Young adult Mypt1(SMKO) mice had normal intestinal motility in vivo, with no histologic abnormalities. On stimulation with KCl or acetylcholine, intestinal smooth muscles isolated from Mypt1(SMKO) mice produced robust and increased sustained force due to increased phosphorylation of the myosin regulatory light chain compared with muscle from control mice. Additional analyses of contractile properties showed reduced rates of force development and relaxation, and decreased shortening velocity, compared with muscle from control mice. Permeable smooth muscle fibers from Mypt1(SMKO) mice had increased sensitivity and contraction in response to Ca(2+). CONCLUSIONS MYPT1 is not essential for smooth muscle function in mice but regulates the Ca(2+) sensitivity of force development and contributes to intestinal phasic contractile phenotype. Altered contractile responses in isolated tissues could be compensated by adaptive physiologic responses in vivo, where gut motility is affected by lower intensities of smooth muscle stimulation for myosin phosphorylation and force development.

Myosin phosphatase target subunit 1 (MYPT1) regulates the contraction and relaxation of vascular smooth muscle and maintains blood pressure

Background: MYPT1 is a regulatory subunit of myosin phosphatase. Results: Deleting MYPT1 in vascular smooth muscle enhances myosin phosphorylation, contractility, and blood pressure. Conclusion: Genetic evidence shows MYPT1 plays a role in modulating vascular smooth muscle contractility. Significance: Although MYPT1 is not essential for vascular smooth muscle contractility, it contributes to blood pressure maintenance in vivo through signaling to myosin phosphorylation. Myosin light chain phosphatase with its regulatory subunit, myosin phosphatase target subunit 1 (MYPT1) modulates Ca2+-dependent phosphorylation of myosin light chain by myosin light chain kinase, which is essential for smooth muscle contraction. The role of MYPT1 in vascular smooth muscle was investigated in adult MYPT1 smooth muscle specific knock-out mice. MYPT1 deletion enhanced phosphorylation of myosin regulatory light chain and contractile force in isolated mesenteric arteries treated with KCl and various vascular agonists. The contractile responses of arteries from knock-out mice to norepinephrine were inhibited by Rho-associated kinase (ROCK) and protein kinase C inhibitors and were associated with inhibition of phosphorylation of the myosin light chain phosphatase inhibitor CPI-17. Additionally, stimulation of the NO/cGMP/protein kinase G (PKG) signaling pathway still resulted in relaxation of MYPT1-deficient mesenteric arteries, indicating phosphorylation of MYPT1 by PKG is not a major contributor to the relaxation response. Thus, MYPT1 enhances myosin light chain phosphatase activity sufficient for blood pressure maintenance. Rho-associated kinase phosphorylation of CPI-17 plays a significant role in enhancing vascular contractile responses, whereas phosphorylation of MYPT1 in the NO/cGMP/PKG signaling module is not necessary for relaxation.

Activation of BK channels may not be required for bitter tastant-induced bronchodilation

To the Editor: Deshpande et al.1 reported that bitter tastants increase intracellular Ca2+ concentration (to similar levels produced by the bronchoconstrictive agonists histamine and bradykinin) yet cause marked bronchodilation. This implies that elevated Ca2+ concentration inhibits contraction, challenging the classic Ca2+-dependent mechanism underlying smooth muscle contraction2,3. To resolve this apparent paradox, the authors showed that bitter tastants can generate localized Ca2+ events, and that bitter tastantinduced relaxation and hyperpolarization can be inhibited by the largeconductance Ca2+-activated K+ (BK) channel antagonist iberiotoxin1; thus, they propose that bitter tastant–induced bronchodilation results from its ability to generate localized Ca2+ signals, which in turn open BK channels and hyperpolarize the membrane. However, their assertion of the involvement of BK channel activation was solely based on the effect of iberiotoxin on bitter tastant–induced relaxation and change in membrane potential as assessed by voltage-sensitive dyes. We are concerned that the association of BK channel activity with relaxation has not been directly examined, raising questions about the proposed relaxation mechanism. We therefore directly studied the effect of bitter tastants on the activity of BK channels and examined the relaxation effect of multiple BK channel inhibitors in mouse airway smooth muscle, the same type of smooth muscle tissue used in Deshpande et al.1. To directly investigate the effect of bitter tastants on BK channel function, we used patch-clamp technology (see Supplementary Methods), an unequivocal methodology for studying ion channel activity. We first examined the effects of the bitter tastant chloroquine on spontaneous transient outward currents (STOCs). These currents result from the opening of BK channels in response to local, short-lived Ca2+ events, that is, Ca2+ sparks, thus representing reliable readouts of the BK channel activity in these cells4,5. If bitter tastants generate localized Ca2+ events as Deshpande et al.1 have suggested, it is plausible that they would increase the activity of STOCs. However, we found that chloroquine at 1 mM, a dose that causes total relaxation (Fig. 3c in Deshpande et al.1), did not increase STOC amplitude or frequency within the first minute or so of application (Fig. 1a,b; n = 9). To our surprise, about 2 min after application chloroquine began to inhibit and then completely block STOCs (Fig. 1a,b). The inhibition was reversible (Fig. 1a), indicating that chloroquine at this concentration does not cause appreciable damage to the cells. BK channels are gated by both Ca2+ and membrane potential, and hence we investigated the effect of chloroquine on depolarization-evoked K+ currents. Upon depolarization from –70 mV to –15 mV, mouse airway smooth muscle cells produced a peak current of 104 ± 8 pA (Fig. 1c; n = 14). Chloroquine (1 mM) inhibited this current by 35% 2 min after application (Fig. 1c; P < 0.05 with paired t test, n = 8). Similarly, iberiotoxin (100 nM) suppressed this current by 30% (data not shown; P < 0.05 with paired t test, n = 6). These results prompted us to reexamine bitter tastant–induced relaxation and the effect of BK channel blockers on this relaxation in isolated mouse airway. The bitter tastants quinine, chloroquine and denatonium all relaxed methacholine-induced contraction and did so in a concentrationdependent manner (Fig. 2a,b), consistent with the results of Deshpande et al.1. At 1 mM, within 4.5 ± 0.4 min (n = 29), chloroquine relaxed methacholineinduced contraction by 91.3 ± 2.4%. The extent of this relaxation in intrapulmonary mainstem bronchi (Fig. 2b,c) was comparable to that in trachea and extrapulmonary mainstem bronchi (Supplementary Fig. 1a,b). However, chloroquine (1 mM) still fully reversed methacholineinduced contraction in the presence of 100 nM iberiotoxin (Fig. 2d). This is in contrast to the result of Deshpande et al.1, who showed that chloroquine only partially reverses the contraction under the same conditions. The reasons for this discrepancy are not known. However, because of this discrepancy, we also examined the effect of iberiotoxin at 300 nM. We found that at this high concentration iberotoxin still exerted no effect on chloroquine-induced relaxation (Fig. 2d,e). These results indicate that bitter tastants do not activate BK chan100 s Chloro 1 mM Wash

In vivo roles for myosin phosphatase targeting subunit-1 phosphorylation sites T694 and T852 in bladder smooth muscle contraction.

Force production and maintenance in smooth muscle is largely controlled by myosin regulatory light chain (RLC) phosphorylation, which relies on a balance between Ca2+/calmodulin‐dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. MYPT1 is the regulatory subunit of MLCP that biochemically inhibits MLCP activity via T694 or T852 phosphorylation in vitro. Here we separately investigated the contribution of these two phosphorylation sites in bladder smooth muscles by establishing two single point mutation mouse lines, T694A and T852A, and found that phosphorylation of MYPT1 T694, but not T852, mediates force maintenance via inhibition of MLCP activity and enhancement of RLC phosphorylation in vivo. Our findings reveal the role of MYPT1 T694/T852 phosphorylation in vivo in regulation of smooth muscle contraction.