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A persistent basal tone in the internal anal sphincter (IAS) is essential for keeping the anal canal closed and fecal continence. Its inhibition via the rectoanal inhibitory reflex (RAIR) is required for successful defecation. However, cellular signals underlying the IAS basal tone remain enigmatic. Here we report the origin and molecular mechanisms of calcium signals that control the IAS basal tone, using a combination approach including a novel IAS slice preparation that retains cell arrangement and architecture as in vivo, 2-photon imaging, and cell-specific gene-modified mice. We found that IAS smooth muscle cells generate two forms of contractions (i.e., phasic and sustained contraction) and Ca(2+) signals (i.e., synchronized Ca(2+) oscillations (SCaOs) and asynchronized Ca(2+) oscillations (ACaOs)) that last for hours. RyRs, TMEM16A, L-type Ca(2+) channels, and gap junctions are required for SCaOs, which account for phasic contraction and 75% of sustained contraction. Nevertheless, only RyRs are required for ACaOs, which contribute 25% of sustained contraction. Nitric oxide, the primary neurotransmitter mediating the RAIR, blocks both types of Ca(2+) signals, leading to IAS’s full relaxation. Our results show that the oscillating nature of Ca(2+) signals generates and maintains the basal tone without causing cytotoxicity to IAS. Our study provides insight into fecal continence and normal defecation.

The paracellular gap formed by endothelial cell (EC) contraction is fundamental for endothelium permeability, but the mechanism underlying EC contraction has yet to be determined. Here, we identified the zipper-interacting protein kinase (ZIPK) as the kinase for EC contraction and myosin light chain (MLC) phosphorylation. Inhibition of ZIPK activity by pharmacological inhibitors and small interfering RNAs led to a significant decrease in the mono- and diphosphorylation of MLCs along with a contractile response to thrombin, suggesting an essential role of ZIPK in EC paracellular permeability. To assess the role of ZIPK in vivo, we established mouse lines with conditional deletion of Zipk gene. The endothelium-specific deletion of Zipk led to embryonic lethality, whereas the UBC-Cre(ERT2)–mediated deletion of Zipk by tamoxifen induction at adulthood caused no apparent phenotype. The induced deletion of Zipk significantly inhibited ischemia-reperfusion–induced blood-brain barrier dysfunction and neuronal injuries from middle cerebral artery occlusion and reperfusion, as evidenced by reduced infarct and edema volume, attenuated Evans blue dye leakage, and improved neuronal behavior. We thus concluded that ZIPK and its phosphorylation of MLC were required for EC contraction and ischemic neuronal injuries. ZIPK may be a prospective therapeutic target for stroke.—Zhang, Y., Zhang, C., Zhang, H., Zeng, W., Li, S., Chen, C., Song, X., Sun, J., Sun, Z., Cui, C., Cao, X., Zheng, L., Wang, P., Zhao, W., Zhang, Z., Xu, Y., Zhu, M., Chen, H. ZIPK mediates endothelial cell contraction through myosin light chain phosphorylation and is required for ischemic-reperfusion injury.

Allergic inflammation has long been implicated in asthmatic hyperresponsiveness of airway smooth muscle (ASM), but its underlying mechanism remains incompletely understood. Serving as G protein-coupled receptor agonists, several inflammatory mediators can induce membrane depolarization, contract ASM, and augment cholinergic contractile response. We hypothesized that the signal cascade integrating on membrane depolarization by the mediators might involve asthmatic hyperresponsiveness.We sought to investigate the signaling transduction of inflammatory mediators in ASM contraction and assess its contribution in the genesis of hyperresponsiveness.We assessed the capacity of inflammatory mediators to induce depolarization currents by electrophysiological analysis. We analyzed the phenotypes of transmembrane protein 16A (TMEM16A) knockout mice, applied pharmacological reagents, and measured the CaInflammatory mediators, such as 5-hydroxytryptamin, histamine, U46619, and leukotriene DA G protein-coupled receptor-TMEM16A-voltage-dependent Ca

Myosin light chain phosphatase with its regulatory subunit, myosin phosphatase target subunit 1 (MYPT1) modulates Ca(2+)-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.

Smooth muscle sphincters exhibit basal tone and control passage of contents through organs such as the gastrointestinal tract; loss of this tone leads to disorders such as faecal incontinence. However, the molecular mechanisms underlying this tone remain unknown. Here, we show that deletion of myosin light-chain kinases (MLCK) in the smooth muscle cells from internal anal sphincter (IAS-SMCs) abolishes basal tone, impairing defecation. Pharmacological regulation of ryanodine receptors (RyRs), L-type voltage-dependent Ca2+ channels (VDCCs) or TMEM16A Ca2+-activated Cl− channels significantly changes global cytosolic Ca2+ concentration ([Ca2+]i) and the tone. TMEM16A deletion in IAS-SMCs abolishes the effects of modulators for TMEM16A or VDCCs on a RyR-mediated rise in global [Ca2+]i and impairs the tone and defecation. Hence, MLCK activation in IAS-SMCs caused by a global rise in [Ca2+]i via a RyR-TMEM16A-VDCC signalling module sets the basal tone. Targeting this module may lead to new treatments for diseases like faecal incontinence., The molecular basis of the basal tone generated by internal anal sphincters (IAS) is largely unknown. Here, the authors show that the tone arises from a global rise in intracellular Ca2+ in smooth muscle cells via a Ryanodine receptor-TMEM16A-L-type Ca2+ channel-MLC kinase pathway, suggesting a potential therapy for IAS motility disorders.

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.

Bitter tastants can activate bitter taste receptors on constricted smooth muscle cells to inhibit L-type calcium channels and induce bronchodilation., Bronchodilators are a standard medicine for treating airway obstructive diseases, and β2 adrenergic receptor agonists have been the most commonly used bronchodilators since their discovery. Strikingly, activation of G-protein-coupled bitter taste receptors (TAS2Rs) in airway smooth muscle (ASM) causes a stronger bronchodilation in vitro and in vivo than β2 agonists, implying that new and better bronchodilators could be developed. A critical step towards realizing this potential is to understand the mechanisms underlying this bronchodilation, which remain ill-defined. An influential hypothesis argues that bitter tastants generate localized Ca2+ signals, as revealed in cultured ASM cells, to activate large-conductance Ca2+-activated K+ channels, which in turn hyperpolarize the membrane, leading to relaxation. Here we report that in mouse primary ASM cells bitter tastants neither evoke localized Ca2+ events nor alter spontaneous local Ca2+ transients. Interestingly, they increase global intracellular [Ca2+]i, although to a much lower level than bronchoconstrictors. We show that these Ca2+ changes in cells at rest are mediated via activation of the canonical bitter taste signaling cascade (i.e., TAS2R-gustducin-phospholipase Cβ [PLCβ]- inositol 1,4,5-triphosphate receptor [IP3R]), and are not sufficient to impact airway contractility. But activation of TAS2Rs fully reverses the increase in [Ca2+]i induced by bronchoconstrictors, and this lowering of the [Ca2+]i is necessary for bitter tastant-induced ASM cell relaxation. We further show that bitter tastants inhibit L-type voltage-dependent Ca2+ channels (VDCCs), resulting in reversal in [Ca2+]i, and this inhibition can be prevented by pertussis toxin and G-protein βγ subunit inhibitors, but not by the blockers of PLCβ and IP3R. Together, we suggest that TAS2R stimulation activates two opposing Ca2+ signaling pathways via Gβγ to increase [Ca2+]i at rest while blocking activated L-type VDCCs to induce bronchodilation of contracted ASM. We propose that the large decrease in [Ca2+]i caused by effective tastant bronchodilators provides an efficient cell-based screening method for identifying potent dilators from among the many thousands of available bitter tastants., Author Summary Bitter taste receptors (TAS2Rs), a G-protein-coupled receptor family long thought to be solely expressed in taste buds on the tongue, have recently been detected in airways. Bitter substances can activate TAS2Rs in airway smooth muscle to cause greater bronchodilation than β2 adrenergic receptor agonists, the most commonly used bronchodilators. However, the mechanisms underlying this bronchodilation remain elusive. Here we show that, in resting primary airway smooth muscle cells, bitter tastants activate a TAS2R-dependent signaling pathway that results in an increase in intracellular calcium levels, albeit to a level much lower than that produced by bronchoconstrictors. In bronchoconstricted cells, however, bitter tastants reverse the bronchoconstrictor-induced increase in calcium levels, which leads to the relaxation of smooth muscle cells. We find that this reversal is due to inhibition of L-type calcium channels. Our results suggest that under normal conditions, bitter tastants can activate TAS2Rs to modestly increase calcium levels, but that when smooth muscle cells are constricted, they can block L-type calcium channels to induce bronchodilation. We postulate that this novel mechanism could operate in other extraoral cells expressing TAS2Rs.

Rationale: Asthma is a chronic inflammatory disorder with a characteristic of airway hyperresponsiveness (AHR). Ca2+-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 Ca2+ agonist–induced cell shortening, and on Cl(Ca) currents activated by Ca2+ sparks (localized, short-lived Ca2+ 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 Ca2+ 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 Ca2+ 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.

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.

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 Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) but Ca2+-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 (MLCKSMKO) 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. MLCKSMKO 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. MLCKSMKO mice may be a useful model of vascular failure and hypotension.

Different interacting signaling modules involving Ca(2+)/calmodulin-dependent myosin light chain kinase, Ca(2+)-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.