Unchanged myosin kinase activity in hypertrophied rat heart

The decrease in myosin ATPase activity observed in cardiac hypertrophy induced by cardiac overload has been related to an isoenzymic redistribution of myosin. To test the hypothesis of an additional regulation of myosin ATPase through light chain phosphorylation, we measured the myosin kinase activity together in sham-operated and 50% to 100% hypertrophied rat hearts. The myosin kinase were purified approximately 600 fold with 6% yield by ion exchange chromatography and calmodulin-affinity chromatography. The presence of very important levels of proteolytic activity in the rat heart resulted in a partial loss of the myosin kinase calmodulin-dependency. The major component from both myosin kinase purified fractions was a 63 kdaltons protein. The protein content was identical in myosin kinase purified fractions from sham-operated and hypertrophied hearts. The calmodulin-dependent activity of myosin kinase, assayed in the presence of 0.1 mM Ca2+ and 10(-6) M calmodulin (about 6.6 nmol P X min-1 X mg-1), was identical in sham-operated and 50% to 100% hypertrophied hearts. Thus, myosin kinase specific activity, in these conditions, was unchanged in rat heart chronic hypertrophy. This result suggests that no direct functional relationship exists between the enzymatic properties of myosin and myosin kinase during the chronic phase of cardiac hypertrophy.     

Effects of extracellular calcium concentrations on myosin P light chain phosphorylation in hearts from running-trained rats

Certain biochemical responses to clevated perfusate calcium concentrations were studied in the isolated hearts of sedentary and running-trained rats from which natural actomyosin was prepared. Both the V_max and Ca²⁺ sensitivity of the Ca²⁺-stimulated, Mg²⁺-dependent actomyosin adenosine triphosphatase (ATPase) were similar in the hearts of trained and sedentary animals, and were not altered when the concentration of extracellular Ca²⁺ was increased. Enhanced myosin Ca²⁺-ATPase activities and phosphate contents of myosin P light chains were found in the hearts of trained animals compared with controls, and these differences were still maintained when perfusate Ca²⁺ concentrations were increased. Although treatment of the perfused hearts with isoproterenol also increased both parameters in the trained and sedentary series, the V_max for myosin Ca²⁺-ATPase and the alkali-labile phosphate contents of myosin P light chains remained, throughout, significantly greater in the hearts of trained rats than in their sedentary counterparts. These differences were not eliminated by the combined use of isoproterenol and high perfusate Ca²⁺. The results suggest that an enhanced capacity for trans-sarcolemmal Ca²⁺ flux in the hearts of trained animals may be responsible for enhanced Ca²⁺-dependent phosphorylation of myosin P light chains, and thus improving cardiac function.     

Dendroaspis natriuretic peptide relaxes gastric antral circular smooth muscle of guinea-pig through the cGIvlP/cGMP-dependent protein kinase pathway

AIM: To systematically investigate if cGMP/cGMPdependent protein kinase G (PKG) signaling pathway may participate in dendroaspis natriuretic peptide (DNP)-induced relaxation of gastric circular smooth muscle.METHODS: The content of cGMP in guinea pig gastric antral smooth muscle tissue and perfusion solution were measured using radioimmunoassay,spontaneous contraction of gastric antral circular muscles recorded using a 4-channel physiograph; and Ca2 -activated K currents (Ik(Ca)and spontaneous transient outward currents (STOCs) in isolated gastric antral myocytes were recorded using the whole-cell patch clamp technique.]RESULTS: DNP markedly enhanced cGMP levels in gastric antral smooth muscle tissue and in the perfusion medium.DNP induced relaxation in gastric antral circular smooth muscle,which was inhibited by KT5823,a cGMP-dependent PKG inhibitor.DNP increased IK(Ca)* This effect was almost completely blocked by KT5823,and partially blocked by LY83583,an inhibitor of guanylate cyclase to change the production of cGMP.DNP also increased STOCs.The effect of DNP on STOCs was abolished in the presence of KT5823,but not affected by KT-5720,a PKA-specific inhibitor.CONCLUSION: DNP activates IK(Ca) and relaxes guinea-pig gastric antral circular smooth muscle via the cGMP/PKG-dependent singling axis instead of cAMP/PKA pathway.     

牛磺酸对支气管哮喘大鼠气道MLCK和血清IL-5表达的影响

目的 探讨牛磺酸对支气管哮喘(简称哮喘)大鼠气道肌球蛋白轻链激酶(MLCK)和血清IL-5表达的影响.方法 32只清洁级健康SD大鼠随机等分为正常对照组、哮喘模型组、牛磺酸干预组、地塞米松干预组.以卵蛋白和氢氧化铝混悬液诱发大鼠哮喘模型.牛磺酸干预组和地塞米松干预组于雾化前1h分别给予腹腔注射0.5 g/kg牛磺酸和0.5 mg/kg地塞米松.计数BALF中白细胞总数和嗜酸粒细胞数,采用免疫组织化学法检测大鼠气道MLCK的表达,采用双抗体夹心法检测大鼠血清IL-5的含量.结果 ①与正常对照组比较,哮喘模型组MLCK和IL-5水平均较高(t=4.324,P＜0.05;t=3.984,P＜0.05);与哮喘模型组比较,牛磺酸干预组MLCK和IL-5水平均较低(￡=3.987,P＜0.05;t=4.375,P＜0.05);与地塞米松组比较,牛磺酸干预组MLCK和IL-5水平变化不大,差异无统计学意义(t=0.780,P＞0.05;t=0.674,P＞0.05).②牛磺酸干预组和地塞米松干预组的大鼠哮喘症状较哮喘模型组明显减轻,但牛磺酸干预组没有出现体质量减轻,食欲减退等不良反应.结论 牛磺酸可能具有通过降低哮喘大鼠气道MLCK水平参与气道舒张以及减轻气道炎症的作用.     

维生素E与地塞米松对小鼠急性肺损伤炎症及肌球蛋白轻链激酶表达的影响

目的 观察维生素E联煤系厝姿啥孕∈蠹毙苑嗡鹕搜字⒓凹∏虻鞍浊崃醇っ父(MLCK)表达的影响.方法 将40只8周龄Balb/c雌性小鼠按随机数字表法分为4组:生理盐水对照组(1.5 ml/kg)、细菌脂多糖组(1 mg/kg)、维生素E联合地塞米松治疗组(联合治疗组)、维生素E治疗组(维生素E组).联合治疗组给予腹腔注射维生素E 50 mg/kg和地塞米松1 mg/kg,1 h后再给予脂多糖1 mg/kg滴鼻,维生素E组仅给予维生素E脂质体50 mg/kg,观察各组小鼠肺组织病理改变.采用免疫组织化学SABC法检测肺组织MLCK免疫反应细胞,用逆转录-PCR反应检测肺组织中MLCK mRNA的表达,Western blot法检测肺组织中MLCK蛋白的表达.各组间均数比较采用单因素方差分析,均数两两比较采用SNK检验.结果 脂多糖组小鼠的肺部炎症反应显著,以中性粒细胞浸润为主,伴明显的肺泡充血、水肿.两治疗组肺组织炎症、充血明显改善;对照组、脂多糖组、联合治疗组、维生素E组BALF中细胞总数分别为(1.1±0.4)、(5.6±2.1)、(4.0±1.0)、(4.2±1.3)×109/L,脂多糖组与其他3组比较,差异有统计学意义(F=14.53,均P<0.05).脂多糖组MLCK免疫反应细胞较对照组明显增加,主要在上皮和内皮,联合治疗组和维生素E组MLCK免疫反应细胞明显减少;脂多糖组与两治疗组肺组织MLCK mRNA吸光度值比较,差异无统计学意义(F=2.76,均P>0.05);脂多糖组与两治疗组肺组织MLCK蛋白吸光度值比较,差异有统计学意义(F=12.06,均P<0.01).结论 维生素E联合地塞米松可抑制脂多糖诱导的小鼠急性肺损伤肺部炎症及肺组织MLCK蛋白的表达,其作用可能部分通过抑制MLCK活性而达到稳定血管屏障、改善肺水肿和炎症.     

Preliminary research on myosin light chain kinase in rabbit liver

AIM:To study preliminarily the properties of myosin lightchain kinase(MLCK)in rabbit liver.METHODS:The expression of MLCK was detected byreverse transcdption-polymerase chain reaction(RT-PCR);the MLCK was obtained from rabbit liver,and its activitywas analyzed by γ-~(32) p incorporation technique to detect thephosphorylation of myosin light chain.RESULTS:MLCK was expressed in rabbit liver,and theactivity of the enzyme was similar to rabbit smooth muscleMLCK,and calmodulin-dependent.When the concentrationwas 0.65 mg·L~(-1),the activity was at the highest level.CONCLUSION:MLCK expressed in rabbit liver may catalyzethe phosphorylation of myosin light chain,which may playimportant rolos in the regulation of hepatic cell functions.     

Dendroaspis natriuretic peptide relaxes gastric antral circular smooth muscle of guinea-pig through the cGMP/cGMP-dependent protein kinase pathway

AIM： To systematically investigate if cGMP/cGMPdependent protein kinase G （PKG） signaling pathway may participate in dendroaspis natriuretic peptide （DNP）-induced relaxation of gastric circular smooth muscle.
METHODS： The content of cGMP in guinea pig gastric antral smooth muscle tissue and perfusion solution were measured using radioimmunoassay; spontaneous contraction of gastric antral circular muscles recorded using a 4-channel physiograph; and Ca^2＋-activated K^＋ currents （IK（Ca）） and spontaneous transient outward currents （STOCs） in isolated gastric antral myocytes were recorded using the whole-cell patch clamp technique.
RESULTS： DNP markedly enhanced cGMP levels in gastric antral smooth muscle tissue and in the perfusion medium. DNP induced relaxation in gastric antral circular smooth muscle, which was inhibited by KT5823, a cGMP-dependent PKG inhibitor. DNP increased IK（Ca）. This effect was almost completely blocked by KT5823, and partially blocked by LY83583, an inhibitor of guanylate cyclase to change the production of cGMP. DNP also increased STOCs. The effect of DNP on STOCs was abolished in the presence of KT5823, but not affected by KT-5720, a PKA-specific inhibitor.
CONCLUSION： DNP activates IK（ca） and relaxes guinea-pig gastric antral circular smooth muscle via the cGMP/PKG-dependent singling axis instead of cAMP/ PKA pathway.     

自发性高血压鼠心脏组织肌球蛋白轻链磷酸酶水平的研究

目的:比较自发性高血压大鼠(SHR)和正常血压大鼠(WKY)心脏组织的心肌肌球蛋白磷酸酶(MLCP)的130000、38000、21000亚单位含量的差异,以探讨MLCP与SHR心脏舒缩机制的关系。方法:在4℃的环境下,取SHR与WKY大鼠各10只断头处死,迅速取其心脏组织,称重速冻后将其磨成匀浆,置于缓冲液中并通过离心提取蛋白质,校正2组总蛋白的浓度后,分别应用SDS-PAGE、Western印迹杂交和化学发光的方法测定SHR与WKY心脏组织MLCP的130000、38000、21000亚单位的含量,对显影结果进一步进行吸光度扫描分析。结果:SHR和WKY大鼠心脏组织的MLCP的亚单位的含量不同,SHR的MLCP的130000、38000、21000亚单位含量均高于WKY大鼠(P<0.01)。结论:SHR与WKY大鼠心脏组织MLCP的130000、38000、21000亚单位含量有着明显的差异,提示心脏组织的MLCP结构或功能的异常可能与SHR高血压的病理机制有关。     

自发性高血压大鼠血管壁肌球蛋白轻链磷酸酶含量的研究

目的：肌球蛋白轻链磷酸酶（MLCP）水平与血管的收缩与舒张功能有着重要的关系。本研究探讨MLCP与自发性高血压大鼠（SHR）高血压发病机制之间的关系。方法：在摄氏4度的环境下，取SHR与正常血压WKY大鼠各10只断头处死，迅速取其大动脉，称重速冻后将血管磨成匀浆，置于缓冲液中，并通过离心提取蛋白质，校正两组总蛋白的浓度后，分别应用（SDS-PAGE）、Western印迹杂交和化学发光方法测定SHR与WKY大鼠动脉血管壁MLCP的130kD、38kD、21kD三个亚单位的含量，对显影结果进一步进行吸光度扫描分析。并进行两组间的比较。结果：SHR的MLCP130kD亚单位的含量明显高于WKY大鼠（P〈0．01），而38kD和21kD亚单位含量则明显低于WKY大鼠（P〈0．01）。结论：SHR和WKY大鼠血管壁MLCP的130kD、38kD、21kD三个亚单位含量有着明显的差异，提示MLCP结构或功能的异常可能与SHR高血压的发病有关。     

Distinct temporal-spatial roles for rho kinase and myosin light chain kinase in epithelial purse-string wound closure

BACKGROUND & AIMS: Small epithelial wounds heal by purse-string contraction of an actomyosin ring that is regulated by myosin light chain (MLC) kinase (MLCK) and rho kinase (ROCK). These studies aimed to define the roles of these kinases in purse-string wound closure. METHODS: Oligocellular and single-cell wounds were created in intestinal epithelial monolayers. Fluorescence imaging and electrophysiologic data were collected during wound closure. Human biopsies were studied immunohistochemically. RESULTS: Live-cell imaging of enhanced green fluorescent protein-beta-actin defined rapid actin ring assembly within 2 minutes after wounding. This progressed to a circumferential ring within 8 minutes that subsequently contracted and closed the wound. We therefore divided this process into 2 phases: ring assembly and wound contraction. Activated rho and ROCK localized to the wound edge during ring assembly. Consistent with a primary role in the assembly phase, ROCK inhibition prevented actin ring assembly and wound closure. ROCK inhibition after ring assembly was complete had no effect. Recruitment and activation of MLCK occurred after ring assembly was complete and coincided with ring contraction. MLCK inhibition slowed and then stopped contraction but did not prevent ring assembly. MLCK inhibition also delayed barrier function recovery. Studies of human colonic biopsy specimens suggest that purse-string wound closure also occurs in vivo, because MLC phosphorylation was enhanced surrounding oligocellular wounds. CONCLUSIONS: These results suggest complementary roles for these kinases in purse-string closure of experimental and in vivo oligocellular epithelial wounds; rho and ROCK are critical for actin ring assembly, while the activity of MLCK drives contraction.     

Regulation of force in vascular smooth muscle

Vascular smooth muscle contraction plays a defining role in the regulation and maintenance of blood pressure, and its deregulation is associated with many clinical syndromes including hypertension, coronary vasospasm and congestive heart failure. Over the past 20 years, there has been a growing understanding of the regulation of 20 kDa myosin light chain phosphorylation by myosin light chain kinase and myosin light chain phosphatase, the role of splice-variant isoforms of both the myosin heavy chain and the essential myosin light chain, as well as the signaling pathways involved in smooth muscle contraction under normal and pathophysiological conditions. This review will attempt to recapitulate the data in the field, primarily focusing on the contractile response of smooth muscle, and the molecular determinants responsible for the regulation of vascular tone.     

Functional reconstitution of vascular smooth muscle cells with cGMP-dependent protein kinase I isoforms

The cGMP-dependent protein kinase type I (cGKI) is a major mediator of NO/cGMP-induced vasorelaxation. Smooth muscle expresses two isoforms of cGKI, cGKIalpha and cGKIbeta, but the specific role of each isoform in vascular smooth muscle cells (VSMCs) is poorly understood. We have used a genetic deletion/rescue strategy to analyze the functional significance of cGKI isoforms in the regulation of the cytosolic Ca(2+) concentration by NO/cGMP in VSMCs. Cultured mouse aortic VSMCs endogenously expressed both cGKIalpha and cGKIbeta. The NO donor diethylamine NONOate (DEA-NO) and the membrane-permeable cGMP analogue 8-bromo-cGMP inhibited noradrenaline-induced Ca(2+) transients in wild-type VSMCs but not in VSMCs genetically deficient for both cGKIalpha and cGKIbeta. The defective Ca(2+) regulation in cGKI-knockout cells could be rescued by transfection of a fusion construct consisting of cGKIalpha and enhanced green fluorescent protein (EGFP) but not by a cGKIbeta-EGFP construct. Fluorescence imaging indicated that the cGKIalpha-EGFP fusion protein was concentrated in the perinuclear/endoplasmic reticulum region of live VSMCs, whereas the cGKIbeta-EGFP protein was more homogeneously distributed in the cytoplasm. These results suggest that one component of NO/cGMP-induced smooth muscle relaxation is the activation of the cGKIalpha isoform, which decreases the noradrenaline-stimulated cytosolic Ca(2+) level.     

Myosin light-chain kinase of smooth muscle stimulates myosin ATPase activity without phosphorylating myosin light chain

Myosin light-chain kinase (MLCK) of smooth muscle is multifunctional, being composed of N-terminal actin-binding domain, central kinase domain, and C-terminal myosin-binding domain. The kinase domain is the best characterized; this domain activates the interaction of smooth-muscle myosin with actin by phosphorylating the myosin light chain. We have recently shown that the Met-1-Pro-41 sequence of MLCK binds to actin to inhibit this interaction. However, it is not known whether the myosin-binding domain modifies the actin-myosin interaction. We designed MLCK.cDNA to overexpress the Asp-777-Glu-972 sequence in Escherichia coli. The purified Asp-777-Glu-972 fragment, although devoid of the kinase activity, exerted a stimulatory effect on the ATPase activity of dephosphorylated myosin (Vmax = 7.36 +/- 0.44-fold, Km = 1.06 +/- 0. 20 microM, n = 4). When the N-terminal 39 residues of the fragment were deleted from the fragment, the resultant fragment, Met-816-Glu-972, lost the stimulatory activity. We synthesized the Ala-777-Ser-815 peptide that was deleted from the fragment and confirmed its stimulatory effect of the peptide (Vmax = 3.03 +/- 0. 22-fold, Km = 6.93 +/- 1.61 microM, n = 3). When this peptide was further divided into Asp-777-Met-795 and Ala-796-Ser-815 peptides, the stimulatory activity was found in the latter. We confirmed that the myosin phosphorylation did not occur during the experiments with the above fragments and peptides. Therefore, we suggest that phosphorylation is not obligatory for smooth-muscle myosin not to be active.     

Regulatory light chain mutations associated with cardiomyopathy affect myosin mechanics and kinetics

The myosin regulatory light chain (RLC) wraps around the alpha-helical neck region of myosin. This neck region has been proposed to act as a lever arm, amplifying small conformational changes in the myosin head to generate motion. The RLC serves an important structural role, supporting the myosin neck region and a modulatory role, tuning the kinetics of the actin myosin interaction. Given the importance of the RLC, it is not surprising that mutations of the RLC can lead to familial hypertrophic cardiomyopathy (FHC), the leading cause of sudden cardiac death in people under 30. Population studies identified two FHC mutations located near the cationic binding site of the RLC, R58Q and N47K. Although these mutations are close in sequence, they differ in clinical presentation and prognosis, with R58Q showing a more severe phenotype. We examined the molecular based changes in myosin that are responsible for the disease phenotype by purifying myosin from transgenic mouse hearts expressing mutant myosins and examining actin filament sliding using the in vitro motility assay. We found that both R58Q and N47K show reductions in force compared to the wild type that could result in compensatory hypertrophy. Furthermore, we observed a higher ATPase rate and an increased activation at submaximal calcium levels for the R58Q myosin that could lead to decreased efficiency and incomplete cardiac relaxation, potentially explaining the more severe phenotype for the R58Q mutation.     

MLCK-A, an unconventional myosin light chain kinase from Dictyostelium, is activated by a cGMP-dependent pathway

Dictyostelium myosin II is activated by phosphorylation of its regulatory light chain by myosin light chain kinase A (MLCK-A), an unconventional MLCK that is not regulated by Ca²⁺/calmodulin. MLCK-A is activated by autophosphorylation of threonine-289 outside of the catalytic domain and by phosphorylation of threonine-166 in the activation loop by an unidentified kinase, but the signals controlling these phosphorylations are unknown. Treatment of cells with Con A results in quantitative phosphorylation of the regulatory light chain by MLCK-A, providing an opportunity to study MLCK-A’s activation mechanism. MLCK-A does not alter its cellular location upon treatment of cells with Con A, nor does it localize to the myosin-rich caps that form after treatment. However, MLCK-A activity rapidly increases 2- to 13-fold when Dictyostelium cells are exposed to Con A. This activation can occur in the absence of MLCK-A autophosphorylation. cGMP is a promising candidate for an intracellular messenger mediating Con A-triggered MLCK-A activation, as addition of cGMP to fresh Dictyostelium lysates increases MLCK-A activity 3- to 12-fold. The specific activity of MLCK-A in cGMP-treated lysates is 210-fold higher than that of recombinant MLCK-A, which is fully autophosphorylated, but lacks threonine-166 phosphorylation. Purified MLCK-A is not directly activated by cGMP, indicating that additional cellular factors, perhaps a kinase that phosphorylates threonine-166, are involved.     

Purification of myosin light chain kinase from bovine cardiac muscle.

Myosin light chain kinase was purified > 100,000-fold to apparent homogeneity with a yield of 10% from bovine cardiac muscle. Sodium dodecyl sulfate gels of the purified kinase showed one stained band corresponding to a Mr of 94,000. The enzyme was activated > 10-fold in the presence of Ca2+ (apparent Ka = 0.6-1.2 microM) and calmodulin (apparent Ka = 3-5 nM). The purified enzyme had a specific activity of 20-30 mumol of phosphate transferred per min per mg from ATP to cardiac myosin light chain 2. One mole of phosphate was incorporated per 94,000 g of the kinase in the presence of Ca2+ and calmodulin or of cyclic AMP-dependent protein kinase or of both additions. In addition to myosin light chain kinase, a calmodulin-binding protein of unknown function was purified from bovine cardiac muscle. This protein had a Mr of 85,000, was composed of two dissimilar subunits (Mr of 61,000 and 15,000), and competed with myosin light chain kinase for calmodulin. The protein appears to be closely related to the calmodulin-binding protein I purified from brain.     

Effects of antibodies to myosin light chain kinase on contractility and myosin phosphorylation in chemically permeabilized smooth muscle

We have used an immunological approach to investigate the role of myosin light chain phosphorylation (MLC-Pi) in the control of contractility in smooth muscle. Our aim was to specifically inhibit myosin light chain kinase (MLCK) in the presence of physiologically activating levels of Ca2+ so that other putative Ca2(+)-dependent regulatory systems could be unmasked. Fab fragments were prepared by papain digestion of immunoglobulin G (IgG) molecules obtained from goats immunized with turkey gizzard MLCK. Anti-MLCK Fab was then purified by chromatography on an MLCK-Sepharose 4B column. These affinity-purified Fab fragments inhibit the activity of MLCK purified from turkey gizzard smooth muscle and interact monospecifically with MLCK in various mammalian smooth muscles as demonstrated by a Western blot analysis. The effect of these Fab fragments on the contractile properties was tested in guinea pig taenia coli made permeable (skinned) using Triton X-100. Skinned fibers, approximately 100 microns in diameter and 4 mm long, were mounted for isometric measurements and immersed in calcium-EGTA buffers. Fibers preincubated with anti-MLCK Fab in relaxing solution (Ca2+ less than 1 nM) for 75 minutes developed about 25% of the isometric force of a parallel control contraction when transferred to contracting solution (Ca2+ = 0.5 microM). When added to contracting solution at the peak of a contracture, anti-MLCK Fab elicited a relaxation that was complete in about 120 minutes despite the presence of Ca2+. No significant effect on isometric force was observed when fibers were incubated with another affinity-purified mouse Fab raised against the Fc region of human IgG (control Fab).(ABSTRACT TRUNCATED AT 250 WORDS)