Sarcolemmal Calcium Pump Research Articles

Specific Role of Neuronal Nitric-oxide Synthase when Tethered to the Plasma Membrane Calcium Pump in Regulating the β-Adrenergic Signal in the Myocardium

The cardiac neuronal nitric-oxide synthase (nNOS) has been described as a modulator of cardiac contractility. We have demonstrated previously that isoform 4b of the sarcolemmal calcium pump (PMCA4b) binds to nNOS in the heart and that this complex regulates beta-adrenergic signal transmission in vivo. Here, we investigated whether the nNOS-PMCA4b complex serves as a specific signaling modulator in the heart. PMCA4b transgenic mice (PMCA4b-TG) showed a significant reduction in nNOS and total NOS activities as well as in cGMP levels in the heart compared with their wild type (WT) littermates. In contrast, PMCA4b-TG hearts showed an elevation in cAMP levels compared with the WT. Adult cardiomyocytes isolated from PMCA4b-TG mice demonstrated a 3-fold increase in Ser(16) phospholamban (PLB) phosphorylation as well as Ser(22) and Ser(23) cardiac troponin I (cTnI) phosphorylation at base line compared with the WT. In addition, the relative induction of PLB phosphorylation and cTnI phosphorylation following isoproterenol treatment was severely reduced in PMCA4b-TG myocytes, explaining the blunted physiological response to the beta-adrenergic stimulation. In keeping with the data from the transgenic animals, neonatal rat cardiomyocytes overexpressing PMCA4b showed a significant reduction in nitric oxide and cGMP levels. This was accompanied by an increase in cAMP levels, which led to an increase in both PLB and cTnI phosphorylation at base line. Elevated cAMP levels were likely due to the modulation of cardiac phosphodiesterase, which determined the balance between cGMP and cAMP following PMCA4b overexpression. In conclusion, these results showed that the nNOS-PMCA4b complex regulates contractility via cAMP and phosphorylation of both PLB and cTnI.

Targeting the Sarcolemmal Calcium Pump: A Potential Novel Strategy for the Treatment of Cardiovascular Disease

Intracellular calcium dynamics play a very important role in mediating contraction and signalling in cardiomyocytes and vascular smooth muscle cells. A number of calcium transporters have been identified that orchestrate a complex process of excitation-contraction coupling and molecular signalling. Despite the variability of the calcium transporters expressed in cardiomyocytes, most calcium channel blockers used therapeutically target the L-type calcium channel and exhibit antihypertensive and/or vasodilating activities. Recently, another calcium pump which is located in the sarcolemma has been shown to mediate cardiac contractility and vascular tone. Interestingly, this sarcolemmal calcium pump (also known as Plasma Membrane Calcium/calmodulin dependent ATPase or PMCA) exerts its function not by altering global calcium concentration, but by mediating signal transduction pathways. This review will discuss recent advances that support the key roles of PMCA as signalling molecule and the potential to target this calcium pump as a novel approach for the treatment of cardiovascular disease.

The sarcolemmal calcium pump (PMCA4b) is involved in the development of pressure overload induced hypertrophy

The sarcolemmal calcium pump (PMCA4b) is involved in the development of pressure overload induced hypertrophy

Role of Na/Ca exchange and the plasma membrane Ca 2+ ‐ATPase in cell function

The Fifth International Conference on Na/Ca Exchange took place between 23 and 27 August 2006 in Brussels, Belgium, and was organized by A. Herchuelz. Image taken from the meeting poster showing hypothetical models for the Na/Ca exchanger (left) and the plasma membrane Ca2+‐ATPase (right), courtesy of M. Hilge. ![][1] Cytosolic Ca2+ regulates several cellular processes and its concentration is, in turn, finely regulated by various channels, pumps and exchangers. The Na/Ca exchanger (NCX) and the plasma membrane Ca2+‐ATPase (PMCA) pump are two concurrent mechanisms for Ca2+ extrusion from the cell (Carafoli, 1994; Blaustein & Lederer, 1999; Philipson & Nicoll, 2000; Strehler & Treiman, 2004; Prasad et al , 2004). PMCA and NCX were cloned 17 and 19 years ago, respectively; four mammalian isoforms have been identified for the former (PMCA1–4) and three have been identified for the latter (NCX1–3; Carafoli, 1994; Philipson & Nicoll, 2000). Na/Ca exchange (NCX) is a unique mechanism that allows Ca2+ extrusion from the cell against its gradient without energy consumption. Indeed, it is the entry of Na+ along its electrochemical gradient that provides the energy for Ca2+ extrusion. In addition, because NCX is electrogenic and voltage sensitive, it can reverse during cellular activation and contribute to Ca2+ entry into the cell (Blaustein & Lederer, 1999). Recently, the crucial role of NCX in cellular Ca2+ homeostasis has been definitively shown by the generation of mice with functional destruction of one Ncx gene. Homozygous Ncx1 ‐deficient mice are not viable, and die between embryonic days 9 and 10, owing to the lack of development of the cardiovascular system (Wakimoto et al , 2000); therefore, NCX is essential for life. PMCA belongs to the P‐type family of transport ATPases, which form a phosphorylated intermediate … [1]: /embed/graphic-1.gif

Neuronal Nitric Oxide Synthase Signaling in the Heart Is Regulated by the Sarcolemmal Calcium Pump 4b

Neuronal nitric oxide synthase (nNOS) has recently been shown to be a major regulator of cardiac contractility. In a cellular system, we have previously shown that nNOS is regulated by the isoform 4b of plasma membrane calcium/calmodulin-dependent ATPase (PMCA4b) through direct interaction mediated by a PDZ domain (PSD 95, Drosophilia Discs large protein and Zona occludens-1) on nNOS and a cognate ligand on PMCA4b. It remains unknown, however, whether this interaction has physiological relevance in the heart in vivo. We generated 2 strains of transgenic mice overexpressing either human PMCA4b or PMCA ct120 in the heart. PMCA ct120 is a highly active mutant form of the pump that does not interact with or modulate nNOS function. Calcium was extruded normally from PMCA4b-overexpressing cardiomyocytes, but in vivo, overexpression of PMCA4b reduced the beta-adrenergic contractile response. This attenuated response was not observed in ct120 transgenic mice. Treatment with a specific nNOS inhibitor (N omega-propyl-L-arginine) reduced the beta-adrenergic response in wild-type and ct120 transgenic mice to levels comparable to those of PMCA4b transgenic animals. No differences in lusitropic response were observed in either transgenic strain compared with wild-type littermates. These data demonstrate the physiological relevance of the interaction between PMCA4b and nNOS and suggests its signaling role in the heart.

The sarcolemmal calcium pump, alpha-1 syntrophin and neuronal nitric oxide synthase are part of a macromolecular protein complex

The sarcolemmal calcium pump, alpha-1 syntrophin and neuronal nitric oxide synthase are part of a macromolecular protein complex

The Sarcolemmal Calcium Pump Inhibits the Calcineurin/Nuclear Factor of Activated T-cell Pathway via Interaction with the Calcineurin A Catalytic Subunit

The calcineurin/nuclear factor of activated T-cell (NFAT) pathway represents a crucial transducer of cellular function. There is increasing evidence placing the sarcolemmal calcium pump, or plasma membrane calcium/calmodulin ATPase pump (PMCA), as a potential modulator of signal transduction pathways. We demonstrate a novel interaction between PMCA and the calcium/calmodulin-dependent phosphatase, calcineurin, in mammalian cells. The interaction domains were located to the catalytic domain of PMCA4b and the catalytic domain of the calcineurin A subunit. Endogenous calcineurin activity, assessed by measuring the transcriptional activity of its best characterized substrate, NFAT, was significantly inhibited by 60% in the presence of ectopic PMCA4b. This inhibition was notably reversed by the co-expression of the PMCA4b interaction domain, demonstrating the functional significance of this interaction. PMCA4b was, however, unable to confer its inhibitory effect in the presence of a calcium/calmodulin-independent constitutively active mutant calcineurin A suggesting a calcium/calmodulin-dependent mechanism. The modulatory function of PMCA4b is further supported by the observation that endogenous calcineurin moves from the cytoplasm to the plasma membrane when PMCA4b is overexpressed. We suggest recruitment by PMCA4b of calcineurin to a low calcium environment as a possible explanation for these findings. In summary, our results offer strong evidence for a novel functional interaction between PMCA and calcineurin, suggesting a role for PMCA as a negative modulator of calcineurin-mediated signaling pathways in mammalian cells. This study reinforces the emerging role of PMCA as a molecular organizer and regulator of signaling transduction pathways.

114 A novel role of the sarcolemmal calcium pump in the regulation of the calcineurin/NFAT hypertrophic pathway in the heart

114 A novel role of the sarcolemmal calcium pump in the regulation of the calcineurin/NFAT hypertrophic pathway in the heart

Regulation of Vascular Tone in Animals Overexpressing the Sarcolemmal Calcium Pump

The mechanisms governing vascular smooth muscle tone are incompletely understood. In particular, the role of the sarcolemmal calcium pump PMCA (plasma membrane calmodulin-dependent calcium ATPase), which extrudes Ca2+ from the cytosol, and its importance compared with the sodium/calcium exchanger remain speculative. To test whether the PMCA is a regulator of vascular tone, we generated transgenic mice overexpressing the human PMCA4b under control of the arterial smooth muscle-specific SM22alpha promoter. This resulted in an elevated systolic blood pressure compared with littermate controls. In PMCA-overexpressing mice, endothelium-dependent relaxation of norepinephrine-preconstricted aortic rings to acetylcholine did not differ from wild type controls (76 +/- 8% versus 79 +/- 8% of maximum relaxation; n = 12, n.s.). De-endothelialized aortas of transgenic mice exhibited stronger maximum contraction to KCl (100 mmol/liter) compared with controls (86 +/- 6% versus 68 +/- 7% of reference KCl contraction at the beginning of the experiment; p <0.05). Preincubation of de-endothelialized vessels with the nitric oxide synthase (NOS) inhibitor l-NAME (l-N(G)-nitroarginine methyl ester) (10-5 mol/liter) resulted in a stronger contraction to KCl (p <0.05 versus without l-NAME), thus unmasking vasodilatory effects of inherent NO production. Maximum contraction to KCl after preincubation with l-NAME did not differ between PMCA mice and controls. In analogy to the results in PMCA-overexpressing mice, contractions of de-endothelialized aortas of neuronal NOS-deficient mice to KCl were significantly increased compared with controls (151 +/- 5% versus 131 +/- 6% of reference KCl contraction; p <0.05). In conclusion, our data suggest a model in which the sarcolemmal Ca2+ pump down-regulates activity of the vascular smooth muscle Ca2+/calmodulin-dependent neuronal NOS by a functionally relevant interaction. Therefore, the PMCA represents a novel regulator of vascular tone.

Overexpression of sarcolemmal calcium pump attenuates induction of cardiac gene expression in response to ET-1.

The function of the plasma membrane calmodulin-dependent calcium ATPase (PMCA) in myocardium is unknown. PMCA is localized in caveolae, 50- to 100-nm membrane invaginations, which also contain receptors for endothelin-1 (ET-1) and various other ligands. PMCA has been suggested to play a role in regulation of caveolar signal transduction. We studied the effects of the hypertrophic agonist ET-1 and increased coronary perfusion pressure on cardiac synthesis of B-type natriuretic peptide (BNP) in transgenic rats overexpressing the human PMCA 4CI in isolated perfused heart preparation. ET-1 infusion for 2 h increased BNP mRNA levels twofold in left ventricles (LV) of nontransgenic rats, whereas no increase was noted in PMCA rat hearts. Similar responses were seen in adrenomedullin and c-fos mRNA levels, and in immunoreactive BNP secretion. Increased mechanical load produced by elevated perfusion pressure induced similar 1.5- to 1.6-fold increases in LV BNP mRNA in both nontransgenic and PMCA rat hearts. These results show that cardiac overexpression of PMCA attenuates ET-1-stimulated early induction of cardiac gene expression, suggesting that PMCA may modulate myocardial growth responses.

147 Reduced expression of the sarcolemmal calcium pump ??? a novel abnormality in hypertensive cardiac hypertrophy

147 Reduced expression of the sarcolemmal calcium pump ??? a novel abnormality in hypertensive cardiac hypertrophy

Competition and redistribution among calcium transport systems in rabbit cardiac myocytes.

Normally the sarcoplasmic reticular calcium pump and sarcolemmal Na/Ca exchange compete and are the primary mechanisms responsible for reducing [Ca]i during cardiac relaxation. The sarcolemmal calcium pump and mitochondrial calcium uptake are much slower, but may also participate in [Ca]i regulation. The aim of this study was to provide a clearer understanding of the interaction of these mechanisms. Myocyte shortening and [Ca]i transients (using indo-1 fluorescence) were measured in rabbit ventricular myocytes, with similar results. The t1/2 of twitch relaxation was 0.17(SEM 0.03) s. Contractures induced by 10 mM caffeine (caffeine contraction) relaxed more slowly [t1/2 = 0.54(0.07) s] due to prevention of sarcoplasmic reticular calcium uptake. When the Na/Ca exchange was also blocked by perfusion with 0Na,0Ca solution, the peak of the caffeine contraction was increased (by 44%) and relaxation was slowed about 16-fold [t1/2 = 8.8(0.8) s]. Blocking mitochondrial calcium uptake by including 1 microM FCCP + 1 microM oligomycin in the 0Na,0Ca solution slowed the relaxation of the caffeine contraction further [t1/2 = 19.7(3.2) s]. Inhibition of the sarcolemmal calcium pump by perfusion with 0Na, 10 mM Ca during caffeine contraction also increased the relaxation t1/2 to 27.5(6.9) s. Inhibition of all four calcium transport systems almost abolished relaxation. It is also shown that calcium which was taken up by the mitochondria during relaxation of the caffeine contraction in 0Na,0Ca gradually redistributed (with tau = 41 s) back to the sarcoplasmic reticulum after caffeine was removed. A second caffeine contraction could then be elicited.(ABSTRACT TRUNCATED AT 250 WORDS)

Stunning: A radical re-view

The recovery from trauma, whether ischemia or some other form of tissue injury, is never instantaneous; time is always required for repair and the return of normal metabolism and function. To what extent the delay in recovery of contractile activity (stunning) after a brief period of ischemia represents convalescence from ischemia-induced injury, as opposed to the expression of reperfusion-induced injury, is perhaps not as clear as the proponents of stunning would hope. Definitive evidence for a distinct reperfusion-induced pathology, which compromises the recovery of contractile function from the depressed state induced by ischemia, is elusive. If reperfusion-induced injury accounts for a significant proportion of stunning, then the molecular mechanisms responsible for initiating the event and those responsible for orchestrating the event at the level of the contractile protein are far from clear. Perturbations of calcium homeostasis are frequently cited as responsible for the depressed contractile state, however, some metabolic derangement must precede any pathologically induced ionic disturbance. In this connection, evidence indicates that free-radical-induced oxidant stress, during the early moments of reperfusion, may modify the activity of a number of thiol-regulated proteins that are directly, or indirectly, responsible for controlling the movement of calcium. Sarcolemmal sodium-calcium exchange and the calcium release channel of the sarcoplasmic reticulum may be activated, whereas the sarcolemmal calcium pump and sodium-potassium ATPase, together with the calcium pump of the sarcoplasmic reticulum, may be inhibited. Under the conditions prevailing during ischemia and reperfusion, this would be expected to promote an early intracellular calcium overload. It is difficult to reconcile such a change with the decreased inotropic state that characterizes stunning; however, it seems likely that the calcium overload is transient and that the stunned myocardium rapidly reestablishes normal levels of intracellular calcium. It is still difficult to explain adequately the reduced inotropic state; clearly, the mechanism of stunning is not quite as simple as its definition.

Taurine deficiency and doxorubicin: interaction with the cardiac sarcolemmal calcium pump

An anticancer drug, doxorubicin, and a naturally occurring β-amino acid, taurine, exert opposing actions on myocardial calcium content and lipid peroxidation. Thus, we tested the hypothesis that the two agents may interact to modify cardiac calcium metabolism and indices of lipid peroxidation. Cardiac taurine levels were reduced by half in rats given tap water containing a β-amino transport inhibitor, β-alanine. Taurine deficiency was associated with an increased susceptibility of the heart to doxorubicin-mediated calcium accumulation, a phenomenon commonly associated with doxorubicin cardiotoxicity. Taurine deficiency also predisposed the heart to enhanced formation of malondialdehyde caused by doxorubicin administration. While increases in malondialdehyde levels are often associated with lipid peroxidation, the failure of doxorubicin to cause changes in oxidized glutathione content makes peroxidative mechanisms a less likely explanation for the potentiation of doxorubicin-mediated myocardial calcium accumulation in taurine-deficient rats. A more likely possibility is the interaction between taurine deficiency and doxorubicin to inhibit the sarcolemmal calcium pump. The data also suggest that the interaction between doxorubicin and taurine deficiency does not involve alterations in the pharmacokinetics of doxorubicin or the cardiotoxic metabolite, doxorubicinol. It is concluded that reduction in sarcolemmal calcium pump activity by taurine deficiency may contribute to myocardial calcium accumulation in hearts whose calcium homeostasis has been compromised by doxorubicin.

The effect of sulfonylurea therapy on defective calcium movement associated with diabetic cardiomyopathy

Adult rats exposed to 70 mg/kg streptozocin developed characteristic symptoms of overt diabetes, such as muscle wasting and severely elevated blood glucose levels. Chronic treatment of these rats with the sulfonylurea glyburide for a period of 5 weeks did not affect either the weight of the animal or the degree of hyperglycemia. The drug also failed to influence myocardial glucose metabolism. Nevertheless, the decline in myocardial function associated with the diabetic cardiomyopathy was less in the glyburide-treated rats. At higher preload, myocardial work was significantly reduced in the untreated diabetic but was only moderately depressed in the glyburide-treated heart relative to the nondiabetic heart. The improvement in mechanical function was associated with partial recovery of sarcolemmal calcium pump activity. The drug did not alter the initial rate of Na+-Ca2+ exchange, but decreased the capacity of the transport system. The results indicate that glyburide benefits the diabetic heart by a mechanism independent of carbohydrate metabolism.

Regulation of calcium uptake in bovine aortic sarcoplasmic reticulum by cyclic AMP-dependent protein kinase

The effect of cAMP-dependent protein kinase on calcium uptake and protein phosphorylation in bovine aortic microsomes was examined. Acid gel electrophoresis demonstrated that the aortic microsomes contained a Ca2+-dependent, hydroxylamine-sensitive phosphoenzyme (Mr 110 kDa), characteristic of the calcium pump in sarcoplasmic reticulum, but showed no evidence of a sarcolemmal calcium pump. Calcium uptake by these aortic vesicles was markedly stimulated by oxalate, whereas calcium uptake by canine cardiac sarcolemmal vesicles was oxalate-independent. Both cAMP plus protein kinase (cAMP-PK) and catalytic subunit of protein kinase stimulated oxalate-supported calcium uptake by bovine aortic microsomes 23 +/- 3% (P less than 0.05) at 0.3 microM Ca2+, but had no effect at 6 to 10 microM Ca2+. Catalytic subunit of protein kinase and cAMP-PK phosphorylated an 11 kDa protein in bovine aortic microsomes which comigrated with canine cardiac phospholamban after boiling in sodium dodecylsulfate. The stoichiometry of the aortic 11 kDa phosphoprotein to 110 kDa phosphoenzyme was approximately 1:1. These data are consistent with the recent identification of phospholamban in various smooth muscles, and suggest that cAMP-mediated vascular relaxation may in part be attributable to stimulation of calcium uptake by the sarcoplasmic reticulum.

Cardiac sarcolemmal calcium pump activity is depressed after treatment with phosphatidylinositol-specific phospholipase C

Cardiac sarcolemmal calcium pump activity is depressed after treatment with phosphatidylinositol-specific phospholipase C

Direct regulatory effect of cholesterol on the calmodulin stimulated calcium pump of cardiac sarcolemma

A great controversy has been set for several years related to an indirect versus a direct effect of cholesterol upon the muscle calcium pump. Employing an enriched cardiac sarcolemma preparation and a (Ca2+, Mg2+)-ATPase fraction isolated from this preparation, this study demonstrates that cholesterol directly interacts with the sarcolemmal calcium pump importantly inhibiting its enzyme activity. It was discovered that this inhibition can be in part explained by a total sensitivity loss of the pump to calmodulin. These results can be considered of importance in the correlation of plasma membrane cholesterol levels with deficiencies in calcium transport and cardiac muscle cell damage.