LC20 Phosphorylation Research Articles

Myofilament calcium sensitization in non-human primate lower esophageal sphincter and esophagus

Background/Objectives: Peristalsis in the esophagus (ESO) moves ingested contents into the stomach. The lower esophageal sphincter (LES) is located at the distal end of the ESO and generates tone to prevent reflux of gastric contents. Previous studies suggest that the expression and/or phosphorylation of proteins involved in myofilament Ca2+ sensitization differs between phasic and tonic smooth muscles (Brozovich et al., 2016; PMID: 27037223). We hypothesize that differences in expression and/or phosphorylation of these proteins contributes to differences in contractile activity between LES and ESO. The aims were to 1) characterize contractile activity in Cynomolgus monkey LES and ESO, 2) evaluate myofilament Ca2+ sensitization protein expression and phosphorylation, 3) examine changes in phosphorylation when contractile activity is enhanced or inhibited pharmacologically. Methods: Contraction was assessed in strips of LES and ESO by isometric tension recordings. Carbachol (CCh, 1 μM) was used to enhance contractions. ROCK2 (SR3677; 2 μM) or CavL (nifedipine; 1 μM) antagonists were used to inhibit contractions. Tissues were snap frozen in liquid nitrogen after peak response and prepared for protein expression analysis using Jess immunoelectrophoresis. Results: Under basal conditions, the LES exhibited tone whereas the ESO had phasic activity. CCh significantly increased contractions. Nifedipine and SR3677 significantly decreased basal and CCh-induced contractions with nifedipine having the greater effect. MLCK and MYPT1 expression was significantly greater in LES than ESO while LC20 and CPI-17 expression was greater in ESO. ROCK2 expression was similar in LES and ESO. Under basal conditions MYPT1 and LC20 phosphorylation was greater in LES. MYPT1 and LC20 phosphorylation was decreased by SR3677 but not nifedipine in both tissues. CCh increased MYPT1 and LC20 phosphorylation in both tissues though was greater in LES. SR3677 inhibited phosphorylation of both proteins in LES and ESO. Discussion/Conclusions: The differences in expression and phosphorylation of key proteins involved in myofilament Ca2+ sensitization likely contribute to differences in contractile activity. Notably, there is greater LC20 phosphorylation in LES than ESO under basal conditions and with CCh. Furthermore, there is an important role for ROCK2 in regulating basal contractile activity in LES and ESO as both contraction and phosphorylation of MYPT1 and LC20 are reduced by SR3677. While nifedipine significantly reduced contractile activity in LES and ESO, it did not reduce phosphorylation of MYPT1 indicating that the ROCK2 pathway is independent of Ca2+ influx. These findings may improve understanding of the pathophysiology associated with esophageal dysmotility. NIH DK129528 to CAC. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The interplay between alterations in esophageal microbiota associated with Th17 immune response and impaired LC20 phosphorylation in achalasia.

Achalasia is an esophageal motility disorder with an unknown etiology. We aimed to determine the pathogenesis of achalasia by studying alterations in esophageal smooth muscle contraction and the associated inflammatory response, and evaluate the role of esophageal microbiota in achalasia development. We analyzed esophageal mucosa and lower esophageal sphincter (LES) samples, obtained from patients with type II achalasia who underwent peroral endoscopic myotomy. Esophageal conditioned media obtained from patients were transferred into the mouse esophagus to determine whether the esophageal intraluminal environment is associated with achalasia. Approximately 30% of 20-kDa myosin light chains (LC20) was phosphorylated in LES from the control group under resting and stimulated conditions, whereas less than 10% of LC20 phosphorylation was detected in achalasia under all conditions. The hypophosphorylation of LC20 in achalasia was associated with the downregulation of the myosin phosphatase-inhibitor protein CPI-17. Th17-related cytokines, including IL-17A, IL-17F, IL-22, and IL-23A, were significantly upregulated in achalasia. α-Diversity index of esophageal microbiota and the proportion of several microbes, including Actinomyces and Dialister, increased in achalasia. Actinomyces levels positively correlated with IL-23A levels, whereas Dialister levels were positively associated with IL-17A, IL-17F, and IL-22 levels. Esophageal IL-17F levels increased in mice after oral administration of the conditioned media. In LES of patients with achalasia, hypophosphorylation of LC20, a possible cause of impaired contractility, was associated with CPI-17 downregulation and an increased Th17-related immune response. The esophageal intraluminal environment, represented by the esophageal microbiota, could be associated with the development and exacerbation of achalasia.

Death-associated protein kinase 3 regulates the myogenic reactivity of cerebral arteries.

What is the central question of this study? DAPK3 contributes to the Ca2+ -sensitization of vascular smooth muscle contraction: does this protein kinase participate in the myogenic response of cerebral arteries? What is the main finding and its importance? Small molecule inhibitors of DAPK3 effectively block the myogenic responses of cerebral arteries. HS38-dependent changes to vessel constriction occur independent of LC20 phosphorylation, and therefore DAPK3 appears to operate via the actin cytoskeleton. A role for DAPK3 in the myogenic response was not previously reported, and the results support a potential new therapeutic target in the cerebrovascular system. The vascular smooth muscle (VSM) of resistance blood vessels is a target of intrinsic autoregulatory responses to increased intraluminal pressure, the myogenic response. In the brain, the myogenic reactivity of cerebral arteries is critical to homeostatic blood flow regulation. Here we provide the first evidence to link the death-associated protein kinase 3 (DAPK3) to the myogenic response of rat and human cerebral arteries. DAPK3 is a Ser/Thr kinase involved in Ca2+ -sensitization mechanisms of smooth muscle contraction. Ex vivo administration of a specific DAPK3 inhibitor (i.e., HS38) could attenuate vessel constrictions invoked by serotonin as well as intraluminal pressure elevation. The HS38-dependent dilatation was not associated with any change in myosin light chain (LC20) phosphorylation. The results suggest that DAPK3 does not regulate Ca2+ sensitization pathways during the myogenic response of cerebral vessels but rather operates to control the actin cytoskeleton. A slow return of myogenic tone was observed during the sustained ex vivo exposure of cerebral arteries to HS38. Recovery of tone was associated with greater LC20 phosphorylation that suggests intrinsic signalling compensation in response to attenuation of DAPK3 activity. Additional experiments with VSM cells revealed HS38- and siDAPK-dependent effects on the actin cytoskeleton and focal adhesion kinase phosphorylation status. The translational importance of DAPK3 to the human cerebral vasculature was noted, with robust expression of the protein kinase and significant HS38-dependent attenuation of myogenic reactivity found for human pial vessels.

A temporal Ca2+ desensitization of myosin light chain kinase in phasic smooth muscles induced by CaMKKβ/PP2A pathways.

Cell signaling pathways regulating myosin regulatory light chain (LC20) phosphorylation contribute to determining contractile responses in smooth muscles. Following excitation and contraction, phasic smooth muscles, such as the digestive tract and urinary bladder, undergo relaxation due to a decline of cellular Ca2+ concentration and decreased Ca2+ sensitivity of LC20 phosphorylation, named Ca2+ desensitization. Here, we determined the mechanisms underlying the temporal Ca2+ desensitization of LC20 phosphorylation in phasic smooth muscles using permeabilized strips of the mouse ileum and urinary bladder. Upon stimulation with pCa6.0 at 20°C, contraction and LC20 phosphorylation peaked within 30 s and then declined to about 50% of the peak force at 2 min after stimulation. During the relaxation phase after the contraction, LC20 kinase [myosin light chain kinase (MLCK)] was inactivated, but no fluctuation in LC20 phosphatase activity occurred, suggesting that MLCK inactivation is a cause of the Ca2+-induced Ca2+ desensitization of LC20 phosphorylation. MLCK inactivation was associated with phosphorylation at the calmodulin-binding domain of the kinase. Treatment with STO-609 and TIM-063 antagonists for Ca2+/calmodulin (CaM)-dependent protein kinase kinase-β (CaMKKβ) attenuated both the phasic response of the contraction and MLCK phosphorylation, whereas neither CaM kinase II, AMP-activated protein kinase, nor p21-activated kinase induced MLCK inactivation in phasic smooth muscles. Conversely, protein phosphatase 2A inhibition amplified the phasic response. Signaling pathways through CaMKKβ and protein phosphatase 2A may contribute to regulating the phasic response of smooth muscle contraction.

Mfge8 attenuates human gastric antrum smooth muscle contractions

Coordinated gastric smooth muscle contraction is critical for proper digestion and is adversely affected by a number of gastric motility disorders. In this study we report that the secreted protein Mfge8 (milk fat globule-EGF factor 8) inhibits the contractile responses of human gastric antrum muscles to cholinergic stimuli by reducing the inhibitory phosphorylation of the MYPT1 (myosin phosphatase-targeting subunit (1) subunit of MLCP (myosin light chain phosphatase), resulting in reduced LC20 (smooth muscle myosin regulatory light chain (2) phosphorylation. Mfge8 reduced the agonist-induced increase in the F-actin/G-actin ratios of β-actin and γ-actin1. We show that endogenous Mfge8 is bound to its receptor, α8β1 integrin, in human gastric antrum muscles, suggesting that human gastric antrum muscle mechanical responses are regulated by Mfge8. The regulation of gastric antrum smooth muscles by Mfge8 and α8 integrin functions as a brake on gastric antrum mechanical activities. Further studies of the role of Mfge8 and α8 integrin in regulating gastric antrum function will likely reveal additional novel aspects of gastric smooth muscle motility mechanisms.

Small GTPase Cdc42 and p130 Crk‐associated substrate (p130Cas) contribute to actin cytoskeleton dynamics in the myogenic response of rat cerebral arteries to intraluminal pressure

The constriction and relaxation of cerebral resistance arteries/arterioles in response to changes in intraluminal pressure, referred to as the myogenic response, is a key physiological mechanism for the regulation of blood pressure and flow in the brain. Actin cytoskeleton dynamics in vascular smooth muscle cells involving increased actin polymerization contribute to myogenic tone development in response to pressure elevation, but our understanding of the cellular mechanisms involved is limited. Members of the Rho family of small GTPases including RhoA, Rac1 and Cdc42 are established regulators of actin cytoskeleton dynamics. RhoA‐Rho‐associated coiled‐coil kinase (ROCK)‐mediated signaling contributes to actin polymerization in the cerebral arterial myogenic response, but whether Rac1 and/or Cdc42 play a role is not known. Here, the contribution of Rac1 and Cdc42 to myogenic vasoconstriction of endothelium‐denuded posterior cerebral arteries of adult rats was assessed by pressure myography combined with ultra‐sensitive immunoblotting to quantify G‐actin content, and level of phosphorylation of myosin regulatory light chain (LC20), myosin phosphatase targeting subunit 1 (MYPT1), p130Cas, N‐WASP, Arp2, cofilin and HSP27 in the absence and presence of GTPase inhibition. Treatment of vessels with Rac1 inhibitor Ehop‐016 (2–4 μM) before or after pressurization from 10 to 80 mmHg had no effect, whereas inhibition of Cdc42 with ML‐141 (1–10 μM) suppressed myogenic tone development. Significantly, ML‐141 (10 μM) did not affect the increase in LC20 or MYPT1‐T855 phosphorylation in response to pressurization from 10 to 80 mmHg, but it completely blocked the 50% decline in G‐actin content. Pressurization increased the level of p130Cas (Y165, Y249, Y410), N‐WASP (Y256), Arp2 (T237/238), HSP27 (S82) and cofilin (S3) phosphorylation. ML‐141 blocked the increase in p130Cas, N‐WASP, Arp2 and HSP27, but not cofilin phosphorylation. In contrast, inhibition of ROCK activity with H1152 (0.3 μM), which is known to suppress MYPT1, LC20 and cofilin phosphorylation, had no effect on the level of p130Cas, N‐WASP or Arp2 phosphorylation. Pharmacological suppression of alpha5‐integrins‐, focal adhesion kinase‐, or Src kinase‐dependent signaling, that inhibit MYPT1 and LC20 phosphorylation, G‐actin reduction and myogenic tone development, also prevented the pressure‐dependent increase in p130Cas, N‐WASP, Arp2, HSP27 and cofilin phosphorylation. Taken together, these data suggest that the myogenic response of rat cerebral arteries involves Cdc42 signaling that stimulates actin cytoskeleton dynamics via a p130Cas‐, N‐WASP‐ and Arp2‐dependent mechanism. This pathway is distinct from RhoA‐ROCK signaling that increases crossbridge cycling via increased MYPT1 and LC20 phosphorylation, as well as actin dynamics via cofilin. Activation of both RhoA and Cdc42 signaling in response to pressure elevation is dependent on integrin, focal adhesion kinase and Src kinase activity.Support or Funding InformationThis work was funded by a research grant from the Canadian Institutes of Health Research MOP‐97988 (WCC).

Rho-associated kinase and zipper-interacting protein kinase, but not myosin light chain kinase, are involved in the regulation of myosin phosphorylation in serum-stimulated human arterial smooth muscle cells.

Myosin regulatory light chain (LC20) phosphorylation plays an important role in vascular smooth muscle contraction and cell migration. Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) phosphorylates LC20 (its only known substrate) exclusively at S19. Rho-associated kinase (ROCK) and zipper-interacting protein kinase (ZIPK) have been implicated in the regulation of LC20 phosphorylation via direct phosphorylation of LC20 at T18 and S19 and indirectly via phosphorylation of MYPT1 (the myosin targeting subunit of myosin light chain phosphatase, MLCP) and Par-4 (prostate-apoptosis response-4). Phosphorylation of MYPT1 at T696 and T853 inhibits MLCP activity whereas phosphorylation of Par-4 at T163 disrupts its interaction with MYPT1, exposing the sites of phosphorylation in MYPT1 and leading to MLCP inhibition. To evaluate the roles of MLCK, ROCK and ZIPK in these phosphorylation events, we investigated the time courses of phosphorylation of LC20, MYPT1 and Par-4 in serum-stimulated human vascular smooth muscle cells (from coronary and umbilical arteries), and examined the effects of siRNA-mediated MLCK, ROCK and ZIPK knockdown and pharmacological inhibition on these phosphorylation events. Serum stimulation induced rapid phosphorylation of LC20 at T18 and S19, MYPT1 at T696 and T853, and Par-4 at T163, peaking within 30–120 s. MLCK knockdown or inhibition, or Ca2+ chelation with EGTA, had no effect on serum-induced LC20 phosphorylation. ROCK knockdown decreased the levels of phosphorylation of LC20 at T18 and S19, of MYPT1 at T696 and T853, and of Par-4 at T163, whereas ZIPK knockdown decreased LC20 diphosphorylation, but increased phosphorylation of MYPT1 at T696 and T853 and of Par-4 at T163. ROCK inhibition with GSK429286A reduced serum-induced phosphorylation of LC20 at T18 and S19, MYPT1 at T853 and Par-4 at T163, while ZIPK inhibition by HS38 reduced only LC20 diphosphorylation. We also demonstrated that serum stimulation induced phosphorylation (activation) of ZIPK, which was inhibited by ROCK and ZIPK down-regulation and inhibition. Finally, basal phosphorylation of LC20 in the absence of serum stimulation was unaffected by MLCK, ROCK or ZIPK knockdown or inhibition. We conclude that: (i) serum stimulation of cultured human arterial smooth muscle cells results in rapid phosphorylation of LC20, MYPT1, Par-4 and ZIPK, in contrast to the slower phosphorylation of kinases and other proteins involved in other signaling pathways (Akt, ERK1/2, p38 MAPK and HSP27), (ii) ROCK and ZIPK, but not MLCK, are involved in serum-induced phosphorylation of LC20, (iii) ROCK, but not ZIPK, directly phosphorylates MYPT1 at T853 and Par-4 at T163 in response to serum stimulation, (iv) ZIPK phosphorylation is enhanced by serum stimulation and involves phosphorylation by ROCK and autophosphorylation, and (v) basal phosphorylation of LC20 under serum-free conditions is not attributable to MLCK, ROCK or ZIPK.

Quantitation of myosin regulatory light chain phosphorylation in biological samples with multiple reaction monitoring mass spectrometry

The 20-kDa regulatory light chain of myosin II plays an important role in regulating smooth muscle contractile force. LC20 is phosphorylated canonically by myosin light chain kinase in a Ca2+/calmodulin-dependent manner at S19. The diphosphorylation of LC20 at T18 and S19 has been observed in smooth muscle tissues. Given that the phosphorylation of LC20 is positively correlated with tension development, the molar stoichiometry of LC20 phosphorylation is commonly profiled as a measure of smooth muscle contractility. Herein, we describe a novel multiple reaction monitoring (MRM)-mass spectrometry (MS) approach for the quantification of LC20 phosphorylation at T18 and S19. Unique precursor as well as y- and b-ion transitions were identified for unphosphorylated LC20-(TS), monophosphorylated LC20-(TpS) and diphosphorylated LC20-(pTpS) peptides. The MRM-MS assay could accurately define molar phosphorylation stoichiometries of S19 and T18 over a broad range (i.e., 0–2 mol P/mol LC20). Correlations of the results for two quantification techniques indicate that the MRM-MS assay performs equally to Phos-tag SDS-PAGE for the determination of LC20 phosphorylation stoichiometry in arterial tissue samples. The MRM-MS technique provides a robust alternative to antibody-based detection systems for the quantification of LC20 phosphorylation.

Tools and protocol for quantification of myosin phosphorylation with MRM-MS

The phosphorylation of myosin regulatory light chain (LC20) at Thr18 and Ser19 is positively correlated with tension development in smooth muscle tissue, and the molar stoichiometry of LC20 phosphorylation is commonly profiled as a measure of smooth muscle contractility. We provide details for a newly applied multiple reaction monitoring (MRM)-mass spectrometry (MS) method for the quantification of LC20 phosphorylation at Thr18 and Ser19. This MRM-MS method provides a robust alternative to antibody-based detection systems (such as Phos-Tag SDS-PAGE) for the quantification of LC20 phosphorylation.

Abnormal myosin phosphatase targeting subunit 1 phosphorylation and actin polymerization contribute to impaired myogenic regulation of cerebral arterial diameter in the type 2 diabetic Goto-Kakizaki rat

The myogenic response of cerebral resistance arterial smooth muscle to intraluminal pressure elevation is a key physiological mechanism regulating blood flow to the brain. Rho-associated kinase plays a critical role in the myogenic response by activating Ca2+ sensitization mechanisms: (i) Rho-associated kinase inhibits myosin light chain phosphatase by phosphorylating its targeting subunit myosin phosphatase targeting subunit 1 (at T855), augmenting 20 kDa myosin regulatory light chain (LC20) phosphorylation and force generation; and (ii) Rho-associated kinase stimulates cytoskeletal actin polymerization, enhancing force transmission to the cell membrane. Here, we tested the hypothesis that abnormal Rho-associated kinase-mediated myosin light chain phosphatase regulation underlies the dysfunctional cerebral myogenic response of the Goto-Kakizaki rat model of type 2 diabetes. Basal levels of myogenic tone, LC20, and MYPT1-T855 phosphorylation were elevated and G-actin content was reduced in arteries of pre-diabetic 8–10 weeks Goto-Kakizaki rats with normal serum insulin and glucose levels. Pressure-dependent myogenic constriction, LC20, and myosin phosphatase targeting subunit 1 phosphorylation and actin polymerization were suppressed in both pre-diabetic Goto-Kakizaki and diabetic (18–20 weeks) Goto-Kakizaki rats, whereas RhoA, ROK2, and MYPT1 expression were unaffected. We conclude that abnormal Rho-associated kinase-mediated Ca2+ sensitization contributes to the dysfunctional cerebral myogenic response in the Goto-Kakizaki model of type 2 diabetes.

PKC-mediated cerebral vasoconstriction: Role of myosin light chain phosphorylation versus actin cytoskeleton reorganization

Defective protein kinase C (PKC) signaling has been suggested to contribute to abnormal vascular contraction in disease conditions including hypertension and diabetes. Our previous work on agonist and pressure-induced cerebral vasoconstriction implicated PKC as a major contributor to force production in a myosin light chain (LC20) phosphorylation-independent manner. Here, we used phorbol dibutyrate to selectively induce a PKC-dependent constriction in rat middle cerebral arteries and delineate the relative contribution of different contractile mechanisms involved. Specifically, we employed an ultra-sensitive 3-step western blotting approach to detect changes in the content of phosphoproteins that regulate myosin light chain phosphatase (MLCP) activity, thin filament activation, and actin cytoskeleton reorganization. Data indicate that PKC activation evoked a greater constriction at a similar level of LC20 phosphorylation achieved by 5-HT. PDBu-evoked constriction persisted in the presence of Gö6976, a selective inhibitor of Ca2+-dependent PKC, and in the absence of extracellular Ca2+. Biochemical evidence indicates that either + or − extracellular Ca2+, PDBu (i) inhibits MLCP activity via the phosphorylation of myosin targeting subunit of myosin phosphatase (MYPT1) and C-kinase potentiated protein phosphatase-1 inhibitor (CPI-17), (ii) increases the phosphorylation of paxillin and heat shock protein 27 (HSP27), and reduces G-actin content, and (iii) does not change the phospho-content of the thin filament proteins, calponin and caldesmon. PDBu-induced constriction was more sensitive to disruption of actin cytoskeleton compared to inhibition of cross-bridge cycling. In conclusion, this study provided evidence for the pivotal contribution of cytoskeletal actin polymerization in force generation following PKC activation in cerebral resistance arteries.

A Role for the Tyrosine Kinase Pyk2 in Depolarization-induced Contraction of Vascular Smooth Muscle

Depolarization of the vascular smooth muscle cell membrane evokes a rapid (phasic) contractile response followed by a sustained (tonic) contraction. We showed previously that the sustained contraction involves genistein-sensitive tyrosine phosphorylation upstream of the RhoA/Rho-associated kinase (ROK) pathway leading to phosphorylation of MYPT1 (the myosin-targeting subunit of myosin light chain phosphatase (MLCP)) and myosin regulatory light chains (LC20). In this study, we addressed the hypothesis that membrane depolarization elicits activation of the Ca(2+)-dependent tyrosine kinase Pyk2 (proline-rich tyrosine kinase 2). Pyk2 was identified as the major tyrosine-phosphorylated protein in response to membrane depolarization. The tonic phase of K(+)-induced contraction was inhibited by the Pyk2 inhibitor sodium salicylate, which abolished the sustained elevation of LC20 phosphorylation. Membrane depolarization induced autophosphorylation (activation) of Pyk2 with a time course that correlated with the sustained contractile response. The Pyk2/focal adhesion kinase (FAK) inhibitor PF-431396 inhibited both phasic and tonic components of the contractile response to K(+), Pyk2 autophosphorylation, and LC20 phosphorylation but had no effect on the calyculin A (MLCP inhibitor)-induced contraction. Ionomycin, in the presence of extracellular Ca(2+), elicited a slow, sustained contraction and Pyk2 autophosphorylation, which were blocked by pre-treatment with PF-431396. Furthermore, the Ca(2+) channel blocker nifedipine inhibited peak and sustained K(+)-induced force and Pyk2 autophosphorylation. Inhibition of Pyk2 abolished the K(+)-induced translocation of RhoA to the particulate fraction and the phosphorylation of MYPT1 at Thr-697 and Thr-855. We conclude that depolarization-induced entry of Ca(2+) activates Pyk2 upstream of the RhoA/ROK pathway, leading to MYPT1 phosphorylation and MLCP inhibition. The resulting sustained elevation of LC20 phosphorylation then accounts for the tonic contractile response to membrane depolarization.

Endothelin-1, but not angiotensin II, induces afferent arteriolar myosin diphosphorylation as a potential contributor to prolonged vasoconstriction

Bolus administration of endothelin-1 elicits long-lasting renal afferent arteriolar vasoconstriction, in contrast to transient constriction induced by angiotensin II. Vasoconstriction is generally evoked by myosin regulatory light chain (LC20) phosphorylation at Ser19 by myosin light chain kinase (MLCK), which is enhanced by Rho-associated kinase (ROCK)-mediated inhibition of myosin light chain phosphatase (MLCP). LC20 can be diphosphorylated at Ser19 and Thr18, resulting in reduced rates of dephosphorylation and relaxation. Here we tested whether LC20 diphosphorylation contributes to sustained endothelin-1 but not transient angiotensin II-induced vasoconstriction. Endothelin-1 treatment of isolated arterioles elicited a concentration- and time-dependent increase in LC20 diphosphorylation at Thr18 and Ser19. Inhibition of MLCK or ROCK reduced endothelin-1-evoked LC20 mono- and diphosphorylation. Pretreatment with an ETB but not an ETA receptor antagonist abolished LC20 diphosphorylation, and an ETB receptor agonist induced LC20 diphosphorylation. In contrast, angiotensin II caused phosphorylation exclusively at Ser19. Thus, endothelin-1 and angiotensin II induce afferent arteriolar constriction via LC20 phosphorylation at Ser19 due to calcium activation of MLCK and ROCK-mediated inhibition of MLCP. Endothelin-1, but not angiotensin II, induces phosphorylation of LC20 at Thr18. This could contribute to the prolonged vasoconstrictor response to endothelin-1.

A tale of two threonines: myosin phosphatase inhibition and calcium sensitization of smooth muscle.

A tale of two threonines: myosin phosphatase inhibition and calcium sensitization of smooth muscle.

ERK and p38MAPK pathways regulate myosin light chain phosphatase and contribute to Ca2+ sensitization of intestinal smooth muscle contraction

Mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated protein kinase (ERK) and p38MAPK, are known regulators of smooth muscle contractility. The contraction of smooth muscle is mainly regulated by the phosphorylation of regulatory light chains of myosin II (LC20), which is driven by the balance between myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). We hypothesized that one possible mechanism for MAPK-dependent modulation of intestinal smooth muscle contractility is via the regulation of MLCP activity. Contractile responses to carbachol (CCh) and effects of MAPK inhibitors on CCh-induced contractions were assessed with isolated rat ileal longitudinal smooth muscle strips. Biochemical assessments of MLCP activity and myosin phosphatse targeting subunit (MYPT1) and CPI-17 phosphorylations were completed. Treatment of ileal smooth muscle with PD98059 (10 μM; MEK inhibitor) or SB203580 (10 μM; p38MAPK inhibitor) significantly inhibited CCh-induced contractile force. Decreased MLCP activity was observed during sustained contractions induced by CCh; the MLCP activity was recovered by treatment with PD98059 and SB203580. However, MYPT1 (Thr697 and Thr855) and CPI-17 (Thr38) phosphorylations were not affected. Application of ML-7 (MLCK inhibitor) during CCh-induced sustained contraction elicited an MLCP-dependent relaxation, the rate of which was accelerated by application of PD98059 and SB203580 with proportional changes in LC20 phosphorylation levels but not MYPT1 phosphorylation (Thr697 or Thr855). ERK and p38MAPK contribute to CCh-induced sustained contraction in a LC20 phosphorylation dependent manner. Moreover, both kinases inhibit MLCP activity possibly by a novel mechanism.

ABNORMAL PHOSPHORYLATION OF MYPT1 AT T855 & T697 SITES IS ASSOCIATED WITH DYSFUNCTIONAL MYOGENIC CONSTRICTION TO INTRAVASCULAR PRESSURE IN CEREBRAL ARTERIOLES OF 8-10 WEEK GOTO-KAKIZAKI RATS

The myogenic response of cerebral resistance arterioles is responsible for intravascular pressure-dependent regulation of blood flow in the brain. Myogenic constriction in response to pressure elevation is dependent on mechanisms intrinsic to vascular smooth muscles, including: 1) Ca2+ influx, activation of myosin light chain kinase, and phosphorylation of 20 kDa regulatory light chains (LC20); and 2) phosphorylation of myosin light chain targeting subunit 1 (MYPT1) at T855 and suppression of myosin light chain phosphatase activity. Phosphorylation at MYPT1 at T697 does not contribute to the myogenic response at physiological pressures, but may be relevant at elevated pressures associated with hypertension (i.e >120 mmHg at the cerebral artery level). Inappropriate regulation of these mechanisms leading to dysfunctional control of cerebral diameter may contribute to stroke in type 2 diabetes (T2D). We employed the Goto-Kakizaki (GK) T2D rat model to test whether alterations in LC20 and MYPT1 phosphorylation are associated with dysfunctional myogenic regulation of cerebral arterial diameter. Endothelium-denuded vessels of 8-10 wk GK and Wistar (WR) control rats were studied by pressurized arterial myography to characterize abnormal myogenic control of diameter, and by ultra-high-sensitivity western blotting to quantify LC20, and MYPT1-T855 and -T697 phosphorylation levels at 10, 60 and 120 mmHg. A pressure-dependent increase in phospho-LC20 and -MYPT1-T855 content was detected in vessels of WR that exhibited normal myogenic constriction between 60 and 120 mmHg, but not in arterioles of age-matched GK rats that exhibited increased basal tone and a loss of myogenic control of diameter. The levels of phospho-LC20 and -MYPT1-T855 were elevated at 10 mmHg in GK compared to WR arteries, and did not increase with pressure elevation to 60 or 120 mmHg. Moreover, no change in phospho-MYPT1-T697 content with pressure elevation was apparent in WR vessels, but the levels in arterioles of GKs were significantly elevated. Inhibition of Rho-associated kinase (ROK) with H1152 (0.5 μM) abolished the pressure-dependent increase in phosphorylation of MYPT1-T855 and LC20, as well as the myogenic response of WR vessels. The enhanced basal tone and elevated phosphorylation of MYPT1-T855 and LC20 in GK arterioles at < 60mmHg were suppressed by H1152, but the elevated phospho-MYPT1-T697 content was unaffected suggesting the involvement of another kinase. Our findings suggest that dysfunctional control of ROK activity leading to inappropriate MYPT1 and LC20 phosphorylations, as well as the phosphorylation of MYPT1-T697 by an unidentified kinase, underlie the abnormal myogenic regulation of cerebral arterial diameter in T2D rats. AI-HS, Killam trusts, CIHR

Cytoskeletal Reorganization Evoked by Rho-associated kinase- and Protein Kinase C-catalyzed Phosphorylation of Cofilin and Heat Shock Protein 27, Respectively, Contributes to Myogenic Constriction of Rat Cerebral Arteries

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 LC(20), 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 mm Hg to between 40 and 120 mm Hg 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 and 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 Rho-associated kinase and PKC signaling contributes significantly to force generation in myogenic constriction of cerebral resistance arteries.

A calcium‐independent PKC isoform mediates cerebral vasoconstriction through actin cytoskeleton reorganization (841.2)

The role of PKC in force production and vasoconstriction in resistance arterioles is hypothesized to be through several mechanisms including: increased calcium influx, calcium sensitization, and thin filament regulation. PKC involvement in these pathways was proposed based on pharmacological experiments without direct biochemical measurement of the phosphoproteins. We studied the effect of phorbol dibutyrate (PDBu, 1 microM) on rat middle cerebral artery in a pressure myography setup. A slow profound constriction was obtained as a result of PDBu treatment. The constriction was associated with increased myosin light chain (LC20) phosphorylation that was similar to that evoked by a maximal dose of 5‐HT producing a smaller constriction. PDBu constrictions were abolished by GF109302X, but neither with a calcium‐dependent PKC inhibitor, nor with calcium‐free Krebs’, though the latter reduced LC20 phosphorylation. There was no evidence of increased phosphorylation of caldesmon and calponin, ruling out a role for thin filament regulation. G‐actin content was greatly reduced in both regular and calcium‐free Krebs’ solution together with an increase in paxillin phosphorylation in both conditions implicating actin cytoskeleton reorganization as a potential mechanism contributing to force generation independent of LC20 phosphorylation following PKC activation.Grant Funding Source: Supported by CIHR MOP‐97988

Novel Contributions of the Smoothelin‐like 1 Protein in Vascular Smooth Muscle Contraction and its Potential Involvement in Myogenic Tone

Vascular smooth muscle contraction and the myogenic response regulate blood flow in the resistance vascular and contribute to systemic blood pressure. Three pathways are currently known to contribute to the development of the myogenic response: (i) Ca(2+) -dependent phosphorylation of LC20; (ii) Ca(2+) sensitization through inhibition of myosin phosphatase; and (iii) cortical actin polymerization. A number of regulatory smooth muscle proteins are integrated with these pathways to fine tune the response and facilitate adaptations to vascular (patho)physiologies. Of particular interest is the SMTN family of proteins, consisting of SMTN-A, SMTN-B, and the SMTN-like protein, SMTNL1. The SMTN-B and SMTNL1 proteins are both implicated in regulating smooth muscle contractility and contributing to vascular adaptations associated with hypertension, pregnancy, and exercise training. In the case of SMTNL1, the protein plays multiple roles in regulating contraction through functional interactions with contractile regulators as well as transcriptional control of the contractile phenotype and Ca(2+) -sensitizing capacity. For the first time, preliminary results suggest SMTNL1 is involved in the myogenic response of the cerebral resistance vasculature. In this regard, global SMTNL1 deletion is associated with greater myogenic reactivity of cerebral arterioles, although the precise mechanism accounting for this finding remains to be defined.

Ca2+ sensitization due to myosin light chain phosphatase inhibition and cytoskeletal reorganization in the myogenic response of skeletal muscle resistance arteries

Abstract The myogenic response of resistance arteries to intravascular pressure elevation is a fundamental physiological mechanism of crucial importance for blood pressure regulation and organ-specific control of blood flow. The importance of Ca(2+) entry via voltage-gated Ca(2+) channels leading to phosphorylation of the 20 kDa myosin regulatory light chains (LC20) in the myogenic response is well established. Recent studies, however, have suggested a role for Ca(2+) sensitization via activation of the RhoA/Rho-associated kinase (ROK) pathway in the myogenic response. The possibility that enhanced actin polymerization is also involved in myogenic vasoconstriction has been suggested. Here, we have used pressurized resistance arteries from rat gracilis and cremaster skeletal muscles to assess the contribution to myogenic constriction of Ca(2+) sensitization due to: (1) phosphorylation of the myosin targeting subunit of myosin light chain phosphatase (MYPT1) by ROK; (2) phosphorylation of the 17 kDa protein kinase C (PKC)-potentiated protein phosphatase 1 inhibitor protein (CPI-17) by PKC; and (3) dynamic reorganization of the actin cytoskeleton evoked by ROK and PKC. Arterial diameter, MYPT1, CPI-17 and LC20 phosphorylation, and G-actin content were determined at varied intraluminal pressures ± H1152, GF109203X or latrunculin B to suppress ROK, PKC and actin polymerization, respectively. The myogenic response was associated with an increase in MYPT1 and LC20 phosphorylation that was blocked by H1152. No change in phospho-CPI-17 content was detected although the PKC inhibitor, GF109203X, suppressed myogenic constriction. Basal LC20 phosphorylation at 10 mmHg was high at ∼40%, increased to a maximal level of ∼55% at 80 mmHg, and exhibited no additional change on further pressurization to 120 and 140 mmHg. Myogenic constriction at 80 mmHg was associated with a decline in G-actin content by ∼65% that was blocked by inhibition of ROK or PKC. Taken together, our findings indicate that two mechanisms of Ca(2+) sensitization (ROK-mediated phosphorylation of MYPT1-T855 with augmentation of LC20 phosphorylation, and a ROK- and PKC-evoked increase in actin polymerization) contribute to force generation in the myogenic response of skeletal muscle arterioles.