败血症大鼠肝细胞核Ca^2＋转运功能的改变

本实验观察败血症时肝细胞核钙转运的变化。早期败血症（结扎盲肠及穿刺后,９ｈ）大鼠肝细胞和肝细胞核钙含量分别增加２０％和３６％（Ｐ〈０．０５）。败血症大鼠肝细胞核Ｃａ＾２＋－ＡＴＰａｓｅ活性增加９４％（Ｐ〈０．０１）,核＾４５Ｃａ＾２＋转运显著增强（增加３２％,Ｐ〈０．０１）。核＾４５Ｃａ＾２＋转运与Ｃａ＾２＋－ＡＴＰａｓｅ活性呈明显正相关（ｒ＝０．９１４,Ｐ〈０．０１）。加入钙调素显著刺激而加入钙     

不同频率电刺激膈神经导致膈肌肌浆网Ca~(2 )-ATPase和Ca~(2 )释放-摄取动力学改变

研究不同频率慢性电刺激（ＣＥＳ）后兔膈肌肌浆网（ＳＲ）Ｃａ２＋－ＡＴＰａｓｅ活性以及ＳＲ Ｃａ２＋摄取－释放动力学对不同频率ＣＥＳ的适应性变化。建立不同频率ＣＥＳ组;用定磷法测定ＳＲ Ｃａ２＋－ＡＴＰａｓｅ活性;用Ｆｕｒａ－２荧光法测定ＳＲ　Ｃａ２＋摄取－释放动力学。与对照组比较,慢性低频电刺激１０ Ｈｚ和２０Ｈｚ组的ＳＲ　Ｃａ２＋－ＡＴＰａｓｅ活性明显降低（Ｐ＜０．０１）,Ｃａ２＋释放－摄取动力学也显著降低（Ｐ＜０．０１）;慢性高频电刺激５０ Ｈｚ和１００Ｈｚ组的ＳＲ Ｃａ２＋－ＡＴＰａｓｅ活性则显著升高（Ｐ＜０．０１）,Ｃａ２＋释放－摄取动力学亦明显升高（Ｐ＜０．０１）。实验提示,ＣＥＳ后不同频率ＣＥＳ导致膈肌ＳＲＣａ２＋－ＡＴＰａｓｅ、Ｃａ２＋摄取－释放动力学产生不同的适应性变化;对不同功能状态的膈肌应用不同频谱的慢性电刺激可能具有重要的临床意义。     

氧化脂质及对大鼠心肌肌质网Ca^2＋—ATPase磷酸化微…

用ＴＡＢ法测定了三尖杉酯碱的膜脂氧化效应;用纳秒荧光偏振技术研究了氧化膜脂对ＤＰＨ标记大鼠心肌肌质网膜脂、ＡＮＭ标记心肌肌质网Ｃａ＾２＋－ＡＴＰａ功能及磷酸化微区运动状态的影响。随膜脂中氧化磷脂的增加,肌质网膜脂双层的微粘度增加,磷脂分子摆动角减小;ＤＰＨ的荧光强度减弱,荧光寿命缩短。Ｃａ＾２＋－ＡＴＰａｓｅ的ＡＴＰ水解活性降低。ＡＮＭ标记Ｃａ＾２＋－ＡＴＰａｓｅ磷酸化微区的ｒ（ｔ）曲线半衰期减至     

花生幼苗下胚轴质膜Ca2+-ATP酶及其对低温胁迫的反应

经６％－１２％ＤｅｘｔｒａｎＴ７０密度梯度离心,获得了纯度较高的７ｄ龄花生幼苗下胚轴质膜制剂,质膜Ｃａ＾２＋－ＡＴＰａｓｅ在反应系统不存在Ｍｇ＾２＋时,可正常表现水解ＡＴＰ的活性,但此活性明显低于Ｍｇ＾２＋激活的ＡＴＰａｓｅ,Ｃａ＾２＋－ＡＴＰａｓｅ不受Ｎａ３ＶＯ４抑制,不被Ｋ＾＋激活,而被Ｃｌ＾－抑制,Ｃａ＾２＋－ＡＴＰａｓｅ的最适,ｐＨ不同于Ｍｇ＾２＋激活的ＡＴＰａｓｅ,低温胁迫显著提高质膜Ｃ     

水分胁迫下钙螯合剂与CPZ抑制剂对棉花根和下胚轴两种ATPase…

水分胁迫下棉花根和下胚轴质膜Ｈ＾＋－ＡＴＰａｓｅ和Ｃａ＾２＋－ＡＴＰａｓｅ活力,表现Ｋｍ值以及Ｖｍａｘ降低。－０．３ＭＰａ和－１．１ＭＰａ胁迫下质膜ＡＴ－Ｐａｓｅ活力随时间延长分别呈“Ｖ”字形变化和下降趋势。钙螯合剂、ＣａＭ抑制剂对棉花根和下胚轴质膜ＡＴＰａｓｅ活性有明显的抑制效应,抑制程度为－１．１ＭＰａ大于－０．３ＭＰａ大于对照。     

牛磺酸对损伤的心肌肌膜ATPase活性影响的研究

本研究以异丙肾上腺素（Ｉｓｏｐｒｏｔｅｒｎｏｌ,Ｉｓｏ．）诱导的大鼠心肌损伤为模型,观察牛磺酸（Ｔａｕｒｉｎｅ,Ｔａｕ）对损伤心肌肌膜上Ｎ＿ａ￣＋—Ｋ＿－￣＋ＡＴＰａｓｅ、Ｃ＿ａ￣（２＋）－ＡＴＰａｓｅ、Ｍ＿ｇ￣（２＋）ＡＴＰＡＳＥ活性的影响。按Ｄａｈａｌｌａ方法分离及鉴定心肌肌膜同时分别测定三种ＡＴＰａｓｅ活性,结果：Ｉｓｏ组,与Ｔａｕ＋Ｉｓｏ组的三种ＡＴＰａｓｅ活性明显低于对照组与Ｔａｕ组,且同时Ｔａｕ＋Ｉｓｏ组明显高于Ｉｓｏ组但对照组与Ｔａｕ组之间差别无显著性。结果提示：牛磺酸能提高异丙肾上腺素诱导心肌损伤的心肌膜ＡＴＰａｓｅ活性,可能通过其保护心肌肌膜的结构及功能而实现的。     

稀土离子对CaM及Ca2+-Mg2+-ATPase活力及CD研究

研究了稀土离子（Ｌｎ３＋）对钙调蛋白（ＣａＭ）调控的Ｃａ２＋－Ｍｇ２＋－ＡＴＰａｓｅ的活力影响。结果表明,在ＣａＭ和Ｃａ２＋－Ｍｇ２＋－ＡＴＰａｓｅ的体系中,一些Ｌｎ３＋（Ｌａ３＋、Ｇｄ３＋）对由ＣａＭ调节的Ｃａ２＋－Ｍｇ２＋－ＡＴＰａｓｅ的活力影响呈现双相效应,即Ｌｎ３＋在低浓度时,能提高激活Ｃａ２＋－Ｍｇ２＋－ＡＴＰａｓｅ的水解活力;在高浓度时,则抑制ＣａＭ调节Ｃａ２＋－Ｍｇ２＋－ＡＴＰａｓｅ活力的能力;少数Ｌｎ３＋（Ｓｍ３＋）仅表现出抑制效应。在无ＣａＭ的Ｃａ２＋－Ｍｇ２＋－ＡＴＰａｓｅ体系中,高浓度的Ｌｎ３＋抑制Ｃａ２＋－Ｍｇ２＋－ＡＴＰａｓｅ的基础活力。结合圆二色（ＣＤ）谱信息对Ｌｎ３＋和ＣａＭ相互作用的分子机制进行了初步的探讨。     

氧化脂质及对大鼠心肌肌质网Ca~（2＋）－ATPase磷酸化微区运动状态的影响

用ＴＢＡ法测定了三尖杉酯碱的膜脂氧化效应;用纳秒荧光偏振技术研究了氧化膜脂对ＤＰＨ标记大鼠心肌肌质网膜脂、ＡＮＭ标记心肌肌质网Ｃａ２＋－ＡＴＰａ功能及磷酸化微区运动状态的影响。随膜脂中氧化磷脂的增加,肌质网膜脂双层的微粘度增加,磷脂分子摆动角减小：ＤＰＨ的荧光强度减弱,荧光寿命缩短。Ｃａ２＋－ＡＴＰａｓｅ的ＡＴＰ水解活性降低。ＡＮＭ标记Ｃａ２＋－ＡＴＰａｓｅ磷酸化微区的ｒ（ｔ）曲线半衰期减至６８±４ｎｓｅｃ。结果提示,膜脂中氧化磷脂的含量影响膜脂双层的流动性及Ｃａ２＋－ＡＴＰａｓｅ的ＡＴＰ水解活性和磷酸化微区的微细结构。     

低氧、血管紧张素Ⅱ对肺内小动脉平滑肌细胞膜Ca2 ┐ATPase活力的影响

本课题观察了低氧及血管紧张素Ⅱ（ａｎｇｉｏｔｅｎｓｉｎⅡ,ＡｎｇⅡ）对分离培养家兔肺内小动脉平滑肌细胞（ＰＡＳＭ－Ｃｓ）膜Ｃａ２＋－ＡＴＰａｓｅ活力的影响,同时用钙通道阻断剂维拉帕米（ｖｅｒａｐａｍｉｌ,ＶＰ）进行干预,进一步了解细胞内钙与Ｃａ２＋－ＡＴＰａｓｅ活力的关系。结果表明：ＰＡＳＭＣｓ膜Ｃａ２＋－ＡＴＰａｓｅ活力对低氧具有短暂的耐受性,随低氧时间延长,Ｃａ２＋－ＡＴＰａｓｅ活力呈时间依赖性抑制;低氧、ＡＮＧⅡ均能抑制Ｃａ２＋－ＡＴＰａｓｅ活力（Ｐ＜０．０１）低氧＋ＡⅡ对Ｃａ２＋－ＡＴＰａｓｅ活力的抑制具叠加效应（Ｐ＜０．０５）;ＶＰ可逆转低氧、ＡｎｇⅡ、低氧＋ＡｎｇⅡ对Ｃａ２＋－ＡＴＰａｓｅ活力的抑制（Ｐ＜０．０１）。结果提示：低氧,ＡＮＧⅡ可通过抑制肺血管平滑肌细胞膜Ｃａ２＋－ＡＴＰａｓｅ活力而可能削弱肺血管平滑肌舒张功能也可能是低氧性肺动脉高压（ＨＰＨ）形成的原因之一。     

败血症大鼠心肌钙通道分布的变化

本研究用结扎盲肠及穿刺（ＣＬＰ）引起败血症。结果证明：大鼠心肌钙通道在早期败血症（ＥＳ,ＣＬＰ后９ｈ）时由心肌轻型囊泡向心肌肌膜转运增多;在晚期败血症（ＬＳ,ＣＬＰ后１８ｈ）时由心肌肌膜向心肌轻型囊泡转运增多。败血症时大鼠心肌肌膜和心肌轻型囊泡钙通道的再分布与ｃＡＭＰ依赖性蛋白激酶（ＰＫＡ）,Ｃａ（２＋）／钙调素依赖性蛋白激酶（ＰＫＭ）和蛋白激酶Ｃ（ＰＫＣ）磷酸化作用无关。败血症时大鼠心肌肌膜和心肌轻型囊泡上肾上腺能β－受体、Ｍ－胆碱受体和Ｎａ＋／Ｋ＋ＡＴＰａｓｅ的变化规律和钙通道的一样,它们可能是败血症时的一种非特异性变化。     

Mechanism of the stimulation of calcium ion dependent adenosine triphosphatase of cardiac sarcoplasmic reticulum by adenosine 3',5'-monophosphate dependent protein kinase

Canine cardiac sarcoplasmic reticulum (SR) is known to be phosphorylated by adenosine 3',5'-monophosphate (cAMP) dependent protein kinase on a 22 000-dalton protein. Phosphorylation enhances the initial rate of Ca2+ uptake and Ca2+-ATPase activity. To determine the molecular mechanism by which phosphorylation regulates the calcium pump in SR, we examined the effect of cAMP-dependent protein kinase on the individual steps of the Ca2+-ATPase reaction sequence. Cardiac sarcoplasmic reticulum was preincubated with cAMP and cAMP-dependent protein kinse in the presence (phosphorylated SR) and absence (control) of adenosine 5'-triphosphate (ATP). Control and phosphorylated SR were subsequently assayed for formation (4-200 ms) and decomposition (0-73 ms) of the acid-stable phosphorylated enzyme (E approximately P) of Ca2+-ATPase in media containing 100 microM [ATP] and various free [Ca2+]. cAMP-dependent phosphorylation of SR resulted in pronounced stimulation of initial rates and levels of E approximately P formed at low free [Ca2+] (less than or equal to 7 microM), but the effect was less at high free Ca2+ (greater than or equal to 10 microM). This stimulation was associated with a decrease in the dissociation constant for Ca2+ binding and a possible increase in Ca2+ sites. The observed rate constant for E approximately P formation of calcium-preincubated SR was not significantly altered by phosphorylation. Phosphorylation also increased the initial rate of E approximately P decomposition. These findings indicate that phosphorylation of cardiac SR by cAMP-dependent protein kinase regulates several steps in the Ca2+-ATPase reaction sequence which result in an overall stimulation of the calcium pump observed at steady state.     

Reversible inhibition of the calcium-pumping ATPase in native cardiac sarcoplasmic reticulum by a calmodulin-binding peptide. Evidence for calmodulin-dependent regulation of the V(max) of calcium transport

Calmodulin (CaM) and Ca(2+)/CaM-dependent protein kinase II (CaM kinase) are tightly associated with cardiac sarcoplasmic reticulum (SR) and are implicated in the regulation of transmembrane Ca(2+) cycling. In order to assess the importance of membrane-associated CaM in modulating the Ca(2+) pump (Ca(2+)-ATPase) function of SR, the present study investigated the effects of a synthetic, high affinity CaM-binding peptide (CaM BP; amino acid sequence, LKWKKLLKLLKKLLKLG) on the ATP-energized Ca(2+) uptake, Ca(2+)-stimulated ATP hydrolysis, and CaM kinase-mediated protein phosphorylation in rabbit cardiac SR vesicles. The results revealed a strong concentration-dependent inhibitory action of CaM BP on Ca(2+) uptake and Ca(2+)-ATPase activities of SR (50% inhibition at approximately 2-3 microM CaM BP). The inhibition, which followed the association of CaM BP with its SR target(s), was of rapid onset (manifested within 30 s) and was accompanied by a decrease in V(max) of Ca(2+) uptake, unaltered K(0.5) for Ca(2+) activation of Ca(2+) transport, and a 10-fold decrease in the apparent affinity of the Ca(2+)-ATPase for its substrate, ATP. Thus, the mechanism of inhibition involved alterations at the catalytic site but not the Ca(2+)-binding sites of the Ca(2+)-ATPase. Endogenous CaM kinase-mediated phosphorylation of Ca(2+)-ATPase, phospholamban, and ryanodine receptor-Ca(2+) release channel was also strongly inhibited by CaM BP. The inhibitory action of CaM BP on SR Ca(2+) pump function and protein phosphorylation was fully reversed by exogenous CaM (1-3 microM). A peptide inhibitor of CaM kinase markedly attenuated the ability of CaM to reverse CaM BP-mediated inhibition of Ca(2+) transport. These findings suggest a critical role for membrane-bound CaM in controlling the velocity of Ca(2+) pumping in native cardiac SR. Consistent with its ability to inhibit SR Ca(2+) pump function, CaM BP (1-2.5 microM) caused marked depression of contractility and diastolic dysfunction in isolated perfused, spontaneously beating rabbit heart preparations. Full or partial recovery of contractile function occurred gradually following withdrawal of CaM BP from the perfusate, presumably due to slow dissociation of CaM BP from its target sites promoted by endogenous cytosolic CaM.     

Gingerol, a novel cardiotonic agent, activates the Ca2+-pumping ATPase in skeletal and cardiac sarcoplasmic reticulum

Gingerol, isolated as a potent cardiotonic agent from the rhizome of ginger, stimulated the Ca2+-pumping activity of fragmented sarcoplasmic reticulum (SR) prepared from rabbit skeletal and dog cardiac muscles. The extravesicular Ca2+ concentrations of the heavy fraction of the fragmented SR (HSR) were measured directly with a Ca2+ electrode to examine the effect of gingerol on the SR. Gingerol (3-30 microM) accelerated the Ca2+-pumping rate of skeletal and cardiac SR in a concentration-dependent manner. The rate of 45Ca2+ uptake of HSR was also increased markedly by 30 microM gingerol without affecting the 45Ca2+ efflux from HSR. Furthermore, gingerol activated Ca2+-ATPase activities of skeletal and cardiac SR (EC50, 4 microM). The activation of SR Ca2+-ATPase activity by gingerol (30 microM) was completely reversed by 100-fold dilution with the fresh saline solution. Kinetic analysis of activating effects of gingerol suggests that the activation of SR Ca2+-ATPase is uncompetitive and competitive with respect to Mg . ATP at concentrations of 0.2-0.5 mM and above 1 mM, respectively. Kinetic analysis also suggests that the activation by gingerol is mixed-type with respect to free Ca2+ and this enzyme is activated probably due to the acceleration of enzyme-substrate complex breakdown. Gingerol had no significant effect on sarcolemmal Ca2+-ATPase, myosin Ca2+-ATPase, actin-activated myosin ATPase and cAMP-phosphodiesterase activities, indicating that the effect of gingerol is rather specific to SR Ca2+-ATPase activity. Gingerol may provide a valuable chemical tool for studies aimed at clarifying the regulatory mechanisms of SR Ca2+-pumping systems and the causal relationship between the Ca2+-pumping activity of SR and muscle contractility.     

Diabetic alterations in cardiac sarcoplasmic reticulum Ca2+-ATPase and phospholamban protein expression.

Diabetic cardiomyopathy has been suggested to be caused by abnormal intracellular Ca2+ homeostasis in the myocardium, which is partly due to a defect in calcium transport by the cardiac sarcoplasmic reticulum (SR). In the present study, the underlying mechanism for this functional derangement was investigated with respect to SR Ca2+-ATPase and phospholamban (the inhibitor of SR Ca2+-ATPase). The maximal Ca2+ uptake and the affinity of Ca2+-ATPase for Ca2+ were decreased, and exogenous phosphorylation level of phospholamban was higher in streptozotocin-induced diabetic rat SR. Levels of both mRNA and protein of phospholamban were significantly increased in the diabetic hearts, whereas those of SR Ca2+-ATPase were significantly decreased. Consequently, the relative phospholamban/Ca2+-ATPase ratio was 1.88 in the diabetic hearts, and these changes were correlated with changes in the rates of SR Ca2+ uptake. However, phosphatase pretreatment of phospholamban for dephosphorylation of the sites phosphorylated in vivo did not change the levels of subsequent phospholamban phosphorylation in either control or diabetic rat hearts. The above data indicated that the increased phospholamban phosphorylation was not due to autonomic dysfunction but possibly due to increased phospholamban expression. These findings suggest that reduction of the SR Ca2+-ATPase level would contribute to decreased rates of SR Ca2+ uptake and that this function is further impaired by the enhanced inhibition by phospholamban due to its increased expression in the diabetic heart.     

Ca2+/calmodulin-dependent phosphorylation of the Ca2+-ATPase, uncoupled from phospholamban, stimulates Ca2+-pumping in native cardiac sarcoplasmic reticulum

Recent studies have demonstrated phosphorylation of the cardiac and slow-twitch muscle isoform (SERCA2a) of the sarcoplasmic reticulum (SR) Ca2+-ATPase (at Ser38) by a membrane-associated Ca2+/calmodulin-dependent protein kinase (CaM kinase). Analysis of the functional consequence of Ca2+-ATPase phosphorylation in the native SR membranes, however, is complicated by the concurrent phosphorylation of the SR proteins phospholamban (PLN) which stimulates Ca2+ sequestration by the Ca2+-ATPase, and the ryanodine receptor-Ca2+ release channel (RYR-CRC) which likely augments Ca2+ release from the SR. In the present study, we achieved selective phosphorylation of the Ca2+-ATPase by endogenous CaM kinase in isolated rabbit cardiac SR vesicles utilizing a PLN monoclonal antibody (PLN AB) which inhibits PLN phosphorylation, and the RYR-CRC blocking drug, ruthenium red, which inhibits phosphorylation of RYR-CRC. Analysis of the Ca2+ concentration-dependence of ATP-energized Ca2+ uptake by SR showed that endogenous CaM kinase mediated phosphorylation of the Ca2+-ATPase, in the absence of PLN and/or RYR-CRC phosphorylation, results in a significant increase (approximately 50-70%) in the Vmax of Ca2+ sequestration without any change in the k0.5 for Ca2+ activation of the Ca2+ transport rate. On the other hand, treatment of SR with PLN AB (which mimics the effect of PLN phosphorylation by uncoupling Ca2+-ATPase from PLN) resulted in approximately 2-fold decrease in k0.5 for Ca2+ without any change in Vmax of Ca2+ sequestration. These findings suggest that, besides PLN phosphorylation, direct phosphorylation of the Ca2+-ATPase by SR-associated CaM kinase serves to enhance the speed of cardiac muscle relaxation.     

Effects of exercise of varying duration on sarcoplasmic reticulum function

Sarcoplasmic reticulum (SR) Ca2+ uptake and Ca2+-Mg2+-ATPase activity were examined in muscle homogenates and the purified SR fraction of the superficial and deep fibers of the gastrocnemius and vastus muscles of the rat after treadmill runs of 20 or 45 min or to exhaustion (avg time to exhaustion 140 min). Vesicle intactness and cross-contamination of isolated SR were estimated using a calcium ionophore and mitochondrial and sarcolemmal marker enzymes, respectively. Present findings confirm previously reported fiber-type specific depression in the initial rate and maximum capacity of Ca2+ uptake and altered ATPase activity after exercise. Depression of the Ca2+-stimulated ATPase activity of the enzyme was evident after greater than or equal to 20 min of exercise in SR isolated from the deep fibers of these muscles. The lowered ATPase activity was followed by a depression in the initial rate of Ca2+ uptake in both muscle homogenates and isolated SR fractions after greater than or equal to 45 min of exercise. Maximum Ca2+ uptake capacity was lower in isolated SR only after exhaustive exercise. Ca2+ uptake and Ca2+-sensitive ATPase activity were not affected at any duration of exercise in SR isolated from superficial fibers of these muscles; however, the Mg2+-dependent ATPase activity was increased after 45 min and exhaustive exercise bouts. The alterations in SR function could not be attributed to disrupted vesicles or differential contamination in the SR from exercise groups and were reinforced by similar changes in Ca2+ uptake in crude muscle homogenates.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of cAMP-dependent protein kinase phosphorylation on the external Ca2+ binding sites of cardiac sarcoplasmic reticulum

Canine cardiac sarcoplasmic reticulum (SR) is known to be phosphorylated by adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase on a 22,000-dalton protein, Phosphorylation is associated with an increase in both the initial rate of Ca2+ uptake and the Ca(2+)-ATPase activity which is partially due to an increase in the affinity of the Ca(2+)-Mg(2+)-ATPase (E) of sarcoplasmic reticulum for calcium. In this study, the effect of cAMP-dependent protein kinase phosphorylation on the binding of calcium to the SR and on the dissociation of calcium from the SR was examined. The rate of dissociation of the E x Ca2 was measured directly and was not found to be significantly altered by cAMP-dependent protein kinase phosphorylation. Since the affinity of the enzyme for Ca2+ is equal to the ratio of the on and off rates of calcium, these results demonstrate that the observed change in affinity must be due to an increase in the rate of calcium binding to the Ca(2+)-Mg(2+)-ATPase of SR. In addition, an increase in the degree of positive cooperativity between the two calcium binding sites was associated with protein kinase phosphorylation.     

Functional reconstitution of the cardiac sarcoplasmic reticulum Ca2(+)-ATPase with phospholamban in phospholipid vesicles

The Ca2(+)-ATPase in cardiac sarcoplasmic reticulum (SR) is under regulation by phospholamban, an oligomeric proteolipid. To determine the molecular mechanism by which phospholamban regulates the Ca2(+)-ATPase, a reconstitution system was developed, using a freeze-thaw sonication procedure. The best rates of Ca2+ uptake (700 nmol/min/mg reconstituted vesicles compared with 800 nmol/min/mg SR vesicles) were observed when cholate and phosphatidylcholine were used at a ratio of cholate/phosphatidylcholine/Ca2(+)-ATPase of 2:80:1. The EC50 values for Ca2+ were 0.05 microM for both Ca2+ uptake and Ca2(+)-ATPase activity in the reconstituted vesicles compared with 0.63 microM Ca2+ in native SR vesicles. Inclusion of phospholamban in the reconstituted vesicles was associated with a significant inhibition of the initial rates of Ca2+ uptake at pCa 6.0. However, phosphorylation of phospholamban by the catalytic subunit of the cAMP-dependent protein kinase reversed the inhibitory effect on the Ca2+ pump. Similar findings were observed when a peptide, corresponding to amino acids 1-25 of phospholamban, was used. These findings indicate that phospholamban is an inhibitor of the Ca2(+)-ATPase in cardiac SR and phosphorylation of phospholamban relieves this inhibition. The mechanism by which phospholamban inhibits the Ca2+ pump is unknown, but our findings with the synthetic peptide suggest that a direct interaction between the Ca2(+)-ATPase and the hydrophilic portion of phospholamban may be one of the mechanisms for regulation.     

Regulation of the skeletal sarcoplasmic reticulum Ca2+ pump by phospholamban in reconstituted phospholipid vesicles

Phospholamban is the regulator of the Ca(2+)-ATPase in cardiac sarcoplasmic reticulum (SR). The mechanism of regulation appears to involve inhibition by dephosphorylated phospholamban, and phosphorylation may relieve this inhibition. Fast-twitch skeletal muscle SR does not contain phospholamban, and it is not known whether the Ca(2+)-ATPase isoform from this muscle may be also subject to regulation by phospholamban in a similar manner as the cardiac isoform. To determine this we reconstituted the skeletal isoform of the SR Ca(2+)-ATPase with phospholamban in phosphatidylcholine proteoliposomes. Inclusion of phospholamban was associated with significant inhibition of the initial rates of Ca2+ uptake at pCa 6.0, and phosphorylation of phospholamban by the catalytic subunit of cAMP-dependent protein kinase reversed the inhibitory effects on the Ca2+ pump. Similar effects of phospholamban were also observed using phosphatidylcholine:phosphatidylserine proteoliposomes, in which the Ca2+ pump was activated by the negatively charged phospholipids (24). Regulation of the Ca(2+)-ATPase appeared to involve binding with the hydrophilic portion of phospholamban, as evidenced by cross-linking experiments, using a synthetic peptide that corresponded to amino acids 1-25 of phospholamban. These findings suggest that the fast-twitch isoform of the SR Ca(2+)-ATPase may be also regulated by phospholamban, although this regulator is not expressed in fast-twitch skeletal muscles.     

Reversible inhibition of sarcoplasmic reticulum Ca-ATPase by altered neuromuscular activity in rabbit fast-twitch muscle

A 50% decrease in both the initial rate and the total capacity of Ca2+ uptake by the sarcoplasmic reticulum (SR) occurred 2 days after the onset of chronic (10 Hz) nerve stimulation in rabbit fast-twitch muscle. Prolonged stimulation (up to 28 days) did not lead to further decreases. This reduction, which was detected in muscle homogenates using a Ca2+-sensitive electrode, was reversible after 6 days cessation of stimulation and was not accompanied by changes in the immunochemically (ELISA) determined tissue level or isozyme characteristics of the SR Ca2+-ATPase protein. However, as measured in isolated SR, it correlated with a reduced specific activity of the Ca2+-ATPase. Kinetic analyses demonstrated that affinities of the SR Ca2+-ATPase towards Ca2+ and ATP were unaltered. Positive cooperativity for Ca2+ binding (h = 1.5) was maintained. However, a 50% decrease in Ca2+-dependent phosphoprotein formation indicated the presence of inactive forms of Ca2+-ATPase in stimulated muscle. The reduced phosphorylation of the enzyme was accompanied by an approximately 50% lowered binding of fluorescein isothiocyanate, a competitor at the ATP-binding site. In view of the unaltered affinity for ATP, this finding suggests that active Ca2+-ATPase molecules coexist in stimulated muscle with inactive enzyme molecules, the latter displaying altered properties at the nucleotide-binding site.