Ca ATPase Pump Research Articles

Calcium Handling in Human Induced Pluripotent Stem Cell Derived Cardiomyocytes

BackgroundThe ability to establish human induced pluripotent stem cells (hiPSCs) by reprogramming of adult fibroblasts and to coax their differentiation into cardiomyocytes opens unique opportunities for cardiovascular regenerative and personalized medicine. In the current study, we investigated the Ca2+-handling properties of hiPSCs derived-cardiomyocytes (hiPSC-CMs).Methodology/Principal FindingsRT-PCR and immunocytochemistry experiments identified the expression of key Ca2+-handling proteins. Detailed laser confocal Ca2+ imaging demonstrated spontaneous whole-cell [Ca2+]i transients. These transients required Ca2+ influx via L-type Ca2+ channels, as demonstrated by their elimination in the absence of extracellular Ca2+ or by administration of the L-type Ca2+ channel blocker nifedipine. The presence of a functional ryanodine receptor (RyR)-mediated sarcoplasmic reticulum (SR) Ca2+ store, contributing to [Ca2+]i transients, was established by application of caffeine (triggering a rapid increase in cytosolic Ca2+) and ryanodine (decreasing [Ca2+]i). Similarly, the importance of Ca2+ reuptake into the SR via the SR Ca2+ ATPase (SERCA) pump was demonstrated by the inhibiting effect of its blocker (thapsigargin), which led to [Ca2+]i transients elimination. Finally, the presence of an IP3-releasable Ca2+ pool in hiPSC-CMs and its contribution to whole-cell [Ca2+]i transients was demonstrated by the inhibitory effects induced by the IP3-receptor blocker 2-Aminoethoxydiphenyl borate (2-APB) and the phosopholipase C inhibitor U73122.Conclusions/SignificanceOur study establishes the presence of a functional, SERCA-sequestering, RyR-mediated SR Ca2+ store in hiPSC-CMs. Furthermore, it demonstrates the dependency of whole-cell [Ca2+]i transients in hiPSC-CMs on both sarcolemmal Ca2+ entry via L-type Ca2+ channels and intracellular store Ca2+ release.

Overexpression of ornithine decarboxylase suppresses thapsigargin-induced apoptosis.

Ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis, has paradoxical roles in apoptosis. Our published papers show overexpression of ODC prevents the apoptosis induced by many cytotoxic drugs. Thapsigargin (TG) is an inhibitor of the sarcoplasmic/endoplasmic reticulum (ER) Ca(2+) ATPase (SERCA) pumps and causes ER stress-induced apoptosis. We used ODC overexpressing cell lines to examine whether overexpression of ODC inhibits TG-induced apoptosis. Our results indicated overexpression of ODC attenuated TG-induced apoptosis. Overexpression of ODC blocked procaspase-4 cleavage and phosphorylation of protein kinase-like ER-resident kinase (PERK), triggered by TG. It also attenuated the increase in CAAT/enhancer binding protein homologous protein (CHOP). Cells with overexpressed ODC had greater Bcl-2 expression. Overexpression of ODC preserved the expression of Bcl-2, inhibited the increase in Bak and stabilized mitochondrial membrane potential without the influences of TG. Cytochrome c release and down-stream caspase activation were blocked. That is, overexpression of ODC inhibits the mitochondria-mediated apoptotic pathway, induced by TG. Finally, overexpression of ODC maintains the protein and mRNA expression of SERCA. In conclusion, overexpression of ODC suppresses TG-induced apoptosis by blocking caspase-4 activation and PERK phosphorylation, attenuating CHOP expression and inhibiting the mitochondria-mediated apoptotic pathway.

Calcium balance and mechanotransduction in rat cochlear hair cells.

Auditory transduction occurs by opening of Ca(2+)-permeable mechanotransducer (MT) channels in hair cell stereociliary bundles. Ca(2+) clearance from bundles was followed in rat outer hair cells (OHCs) using fast imaging of fluorescent indicators. Bundle deflection caused a rapid rise in Ca(2+) that decayed after the stimulus, with a time constant of about 50 ms. The time constant was increased by blocking Ca(2+) uptake into the subcuticular plate mitochondria or by inhibiting the hair bundle plasma membrane Ca(2+) ATPase (PMCA) pump. Such manipulations raised intracellular Ca(2+) and desensitized the MT channels. Measurement of the electrogenic PMCA pump current, which saturated at 18 pA with increasing Ca(2+) loads, indicated a maximum Ca(2+) extrusion rate of 3.7 fmol x s(-1). The amplitude of the Ca(2+) transient decreased in proportion to the Ca(2+) concentration bathing the bundle and in artificial endolymph (160 mM K(+), 20 microM Ca(2+)), Ca(2+) carried 0.2% of the MT current. Nevertheless, MT currents in endolymph displayed fast adaptation with a submillisecond time constant. In endolymph, roughly 40% of the MT current was activated at rest when using 1 mM intracellular BAPTA compared with 12% with 1 mM EGTA, which enabled estimation of the in vivo Ca(2+) load as 3 pA at rest. The results were reproduced by a model of hair bundle Ca(2+) diffusion, showing that the measured PMCA pump density could handle Ca(2+) loads incurred from resting and maximal MT currents in endolymph. The model also indicated the endogenous mobile buffer was equivalent to 1 mM BAPTA.

A Model to Study Effect of Rapid Buffers and Na+ on Ca2+ Oscillations in Neuron Cell

$Ca^{2+}$ plays a vital role in muscle mechanics, cardiacelectrophysiology, secretion, hair cells, and adaptation inphotoreceptors. It is a vital second messenger used in signaltransduction. Calcium controls cell movement, cell differentiation,ciliary beating. Many cells exhibit oscillations in intracellular[Ca$^{2+}$] in response to agonist such as hormones andneurotransmitters. Many cells use oscillations in calciumconcentration to transmit messages (Sneyd J. et al, 2006, p.151-163). In this paper, an attempt has been made to develop a modelto study calcium oscillations in neuron cells. This modelincorporates the effect of variable Na$^{+}$ influx, sodium-calciumexchange (NCX) protein, Sarcolemmal Calcium ATPase (SL) pump,Sarco-Endoplasmic Reticulum CaATPase (SERCA) pump, sodium andcalcium channels, and $IP_{3}R$ channel. The proposed mathematicalmodel leads to a system of partial differential equations which hasbeen solved numerically using Forward Time Centered Space (FTCS)approach. The numerical results have been used to study therelationships among different types of parameters such as bufferconcentration, disassociation rate, calcium permeability, etc.

The anti-apoptotic protein HAX-1 is a regulator of cardiac function

The HS-1 associated protein X-1 (HAX-1) is a ubiquitously expressed protein that protects cardiomyocytes from programmed cell death. Here we identify HAX-1 as a regulator of contractility and calcium cycling in the heart. HAX-1 overexpression reduced sarcoplasmic reticulum Ca-ATPase (SERCA2) pump activity in isolated cardiomyocytes and in vivo, leading to depressed myocyte calcium kinetics and mechanics. Conversely, downregulation of HAX-1 enhanced calcium cycling and contractility. The inhibitory effects of HAX-1 were abolished upon phosphorylation of phospholamban, which plays a fundamental role in controlling basal contractility and constitutes a key downstream effector of the beta-adrenergic signaling cascade. Mechanistically, HAX-1 promoted formation of phospholamban monomers, the active/inhibitory units of the calcium pump. Indeed, ablation of PLN rescued HAX-1 inhibition of contractility in vivo. Thus, HAX-1 represents a regulatory mechanism in cardiac calcium cycling and its responses to sympathetic stimulation, implicating its importance in calcium homeostasis and cell survival.

What about the other two-thirds of the cardiac cycle?

Diastolic dysfunction is a relatively new clinical diagnosis being made in patients where the observed echocardiographic findings are deemed to be consistent with abnormalities of left ventricular (LV) filling. Left ventricular filling is dependent on the complex interaction of three processes: LV relaxation, LV compliance, and left atrial (LA) contraction. Left ventricular relaxation is an active process involving the uptake of Ca into the sarcoplasmic reticulum by the sarcoendoplasmic reticulum Ca ATPase pump and removal of Ca from the cell by way of the sodium-calcium exchanger. Sarcoendoplasmic reticulum Ca ATPase pump expression and activity is reduced in the cardiomyocyte (and diastolic function is altered) with the onset of senescence, hypertrophy, and heart failure. Left ventricular compliance is a measure of the relationship between LV volume and LV pressure development during LV filling (dV/dp). Left ventricular compliance is influenced by: 1) the presence of ventricular hypertrophy; 2) the functional state of the right ventricle (direct diastolic ventricular interaction), since both ventricles share the septum as a common wall; and 3) the properties of the pericardium. Abnormal LV compliance is a feature of longstanding poorly-controlled hypertension, right ventricular dysfunction, and pericardial disease. In the presence of abnormal LV relaxation and/or compliance, LA contraction assumes a key role in maintaining LV filling. Under physiological conditions in youthful individuals, atrial contraction contributes approximately 20% of the total filling volume of the LV; whereas, in patients with LV diastolic dysfunction, LA contraction may account for over 50% of LV volume at enddiastole. Absence of LA contraction, as occurs with the development of atrial fibrillation, is a harbinger of heart failure and is a marker of an increased risk of premature death. The occurrence of abnormal LV relaxation and compliance, as a consequence of hypertensive heart disease, is one of the most common determinants of LA enlargement, poor LA contractile function, and LA dysrhythmia development. It is difficult, if not impossible, to individually assess the components of LV filling in humans; however, relatively precise global estimates of diastolic function can be performed following cardiac catheterization. This approach is invasive, time consuming, and not without risk. In reality, both our knowledge of diastolic function in the broader clinical environment and our interest in the topic did not materialize until the introduction of pulse wave Doppler echocardiography. Pulse wave Doppler echocardiography allows the timing and amplitude of the blood flow and tissue velocity profiles within the heart to be ascertained and graphically displayed. Extensive research has determined which blood flow/tissue velocity profile patterns are considered normal and which appear to be characteristic of diastolic dysfunction. The mitral inflow velocity profile, one of the earliest patterns studied, offers a relatively simple example of the deductive reasoning underlying the evolution of Doppler derived diastolic function assessment. Under ‘‘normal’’ circumstances, flow velocity across the mitral valve is biphasic, i.e., greater during early diastole (E wave) than during subsequent atrial contraction (A wave). In the presence of mild diastolic dysfunction associated with impaired ventricular relaxation, the early diastolic passive flow velocity is reduced while the flow velocity associated with atrial contraction is enhanced (reversed E/A ratio). It is clear, however, that many factors B. A. Finegan, MB (&) Department of Anesthesiology and Pain Medicine, University of Alberta, 8-120 Clinical Sciences Building, Edmonton, AB T6G 2G3, Canada e-mail: chassist@ualberta.ca

Role of 5-Hydroxytryptamine2CReceptors in Ca2+-Dependent Ethanol Potentiation of GABA Release onto Ventral Tegmental Area Dopamine Neurons

Activation of ventral tegmental area (VTA)-dopaminergic (DA) neurons by ethanol has been implicated in the rewarding and reinforcing actions of ethanol. GABAergic transmission is thought to play an important role in regulating the activity of DA neurons. We have reported previously that ethanol enhances GABA release onto VTA-DA neurons in a brain slice preparation. Because intraterminal Ca(2+) levels regulate neurotransmitter release, we investigated the roles of Ca(2+)-dependent mechanisms in ethanol-induced enhancement of GABA release. Acute ethanol enhanced miniature inhibitory postsynaptic current (mIPSC) frequency in the presence of the nonspecific voltage-gated Ca(2+) channel inhibitor, cadmium chloride, even though basal mIPSC frequency was reduced by cadmium. Conversely, the inositol-1,4,5-triphosphate receptor inhibitor, 2-aminoethoxydiphenylborane, and the sarco/endoplasmic reticulum Ca(2+) ATPase pump inhibitor, cyclopiazonic acid, eliminated the ethanol enhancement of mIPSC frequency. Recent studies suggest that the G protein-coupled receptor, 5-hydroxytryptamine (5-HT)(2C), may modulate GABA release in the VTA. Thus, we also investigated the role of 5-HT(2C) receptors in ethanol enhancement of GABAergic transmission. Application of 5-HT and the 5-HT(2C) receptor agonist, Ro-60-0175 [(alphaS)-6-chloro-5-fluoro-alpha-methyl-1H-indole-1-ethanamine fumarate], alone enhanced mIPSC frequency of which the latter was abolished by the 5-HT(2C) receptor antagonist, SB200646 [N-(1-methyl-5-indoyl)-N-(3-pyridyl)urea hydrochloride], and substantially diminished by cyclopiazonic acid. Furthermore, SB200646 abolished the ethanol-induced increase in mIPSC frequency and had no effect on basal mIPSC frequency. These observations suggest that an increase in Ca(2+) release from intracellular stores via 5-HT(2C) receptor activation is involved in the ethanol-induced enhancement of GABA release onto VTA-DA neurons.

Effect of uncoupling endothelial nitric oxide synthase on calcium homeostasis in aged porcine endothelial cells

The requirement of endothelial NO synthase (NOS3) calcium to produce NO is well described, although the effect of NO on intracellular calcium levels [Ca(2+)](i) is still confusing. Therefore, NO and [Ca(2+)](i) cross-talk were studied in parallel in endothelial cells possessing a functional or a dysfunctional NO pathway. Dysfunctional porcine endothelial cells were obtained either in vitro by successive passages or in vivo from regenerated endothelium 1 month after coronary angioplasty. Activity of NOS3 was characterized by conversion of arginine to citrulline, BH(4) intracellular availability, cGMP, and superoxide anion production. Imaging of the Ca(2+) indicator FURA 2-AM was recorded and sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) pump activity was analysed by (45)Ca(2+) uptake into cells. In endothelial cells with a functional NO pathway, NOS3 inhibition increased [Ca(2+)](i) and, conversely, an NO donor decreased it. In aged cells with an uncoupled NOS3 as shown by the reduced BH(4) level, the increase in superoxide anion and the lower production of cGMP and the decrease in NO bioavailability were linearly correlated with the increase in basal [Ca(2+)](i). Moreover, when stimulated by bradykinin, the calcium response was reduced while its decay was slowed down. These effects on the calcium signalling were abolished in calcium-free buffer and were similarly induced by SERCA inhibitors. In aged cells, NO improved the reduced SERCA activity and tended to normalize the agonist calcium response. In control endothelial cells, NO exerts a negative feedback on cytosolic Ca(2+) homeostasis. In aged cells, uncoupled NOS3 produced NO that was insufficient to control the [Ca(2+)](i). Consequently, under resting conditions, SERCA activity decreased and [Ca(2+)](i) increased. These alterations were reversible as exogenous NO, in a cGMP-independent way, refilled intracellular calcium stores, reduced calcium influx, and improved the agonist-evoked calcium response. Therefore, prevention of the decrease in NO in dysfunctional endothelium would normalize the calcium-dependent functions.

Intermittent hypoxia protects cardiomyocytes against ischemia-reperfusion injury-induced alterations in Ca2+ homeostasis and contraction via the sarcoplasmic reticulum and Na+/Ca2+ exchange mechanisms

We have previously demonstrated that intermittent high-altitude (IHA) hypoxia significantly attenuates ischemia-reperfusion (I/R) injury-induced excessive increase in resting intracellular Ca(2+) concentrations ([Ca(2+)](i)). Because the sarcoplasmic reticulum (SR) and Na(+)/Ca(2+) exchanger (NCX) play crucial roles in regulating [Ca(2+)](i) and both are dysfunctional during I/R, we tested the hypothesis that IHA hypoxia may prevent I/R-induced Ca(2+) overload by maintaining Ca(2+) homeostasis via SR and NCX mechanisms. We thus determined the dynamics of Ca(2+) transients and cell shortening during preischemia and I/R injury in ventricular cardiomyocytes from normoxic and IHA hypoxic rats. IHA hypoxia did not affect the preischemic dynamics of Ca(2+) transients and cell shortening, but it significantly suppressed the I/R-induced increase in resting [Ca(2+)](i) levels and attenuated the depression of the Ca(2+) transients and cell shortening during reperfusion. Moreover, IHA hypoxia significantly attenuated I/R-induced depression of the protein contents of SR Ca(2+) release channels and/or ryanodine receptors (RyRs) and SR Ca(2+) pump ATPase (SERCA2) and SR Ca(2+) release and uptake. In addition, a delayed decay rate time constant of Ca(2+) transients and cell shortening of Ca(2+) transients observed during ischemia was accompanied by markedly inhibited NCX currents, which were prevented by IHA hypoxia. These findings indicate that IHA hypoxia may preserve Ca(2+) homeostasis and contraction by preserving RyRs and SERCA2 proteins as well as NCX activity during I/R.

Sarcoplasmic reticulum Ca-ATPase–phospholamban interactions and dilated cardiomyopathy

Dilated cardiomyopathy is a disease of the heart muscle resulting from a diverse array of conditions that damages the heart and impairs myocardial function. Heart failure occurs when the heart is unable to pump blood at a rate which can accommodate the heart muscle’s metabolic requirements. Several signaling pathways have been shown to be involved in the induction of cardiac disease and heart failure. Many of these pathways are linked to cardiac sarcoplasmic reticulum (SR) Ca cycling directly or indirectly. A large body of evidence points to the central role of abnormal Ca handling by SR proteins, Ca-ATPase pump (SERCA2a) and phospholamban (PLN), in pathophysiological heart conditions, compromising the contractile state of the cardiomyocytes. This review summarizes studies which highlight the key role of these two SR proteins in the regulation of cardiac function, the significance of SERCA2a–PLN interactions using transgenic approaches, and the recent discoveries of human PLN mutations leading to disease states. Finally, we will discuss extrapolation of experimental paradigms generated in animal models to the human condition.

Mechanism of Ca2+-mediated Regulation of NDR Protein Kinase through Autophosphorylation and Phosphorylation by an Upstream Kinase

NDR1 (nuclear Dbf2-related) is a serine/threonine protein kinase belonging to subfamily of kinases implicated in the regulation of cell division and morphology. Previously, we demonstrated that the activity of NDR1 is controlled by phosphorylation of two regulatory residues, Ser-281 and Thr-444. Moreover, we found that NDR1 becomes activated through a direct interaction with EF-hand Ca(2+)-binding proteins of the S100 family. In this work, we characterize this regulatory mechanism in detail. We found that NDR1 autophosphorylates in vitro predominantly on Ser-281 and to a lesser extent on Thr-74 and Thr-444. All of these residues proved to be crucial also for NDR1 activity in vivo; however, in contrast to Ser-281 and Thr-444, Thr-74 seems to be involved only in binding to S100B rather than directly regulating NDR1 activity per se. When we added Ca(2+)/S100B, we observed an increased autophosphorylation on Ser-281 and Thr-444, resulting in stimulation of NDR1 activity in vitro. Using phosphospecific antibodies, we found that Ser-281 also becomes autophosphorylated in vivo, whereas Thr-444 is targeted predominantly by an as yet unidentified upstream kinase. Significantly, the Ca(2+)-chelating agent BAPTA-AM suppressed the activity and phosphorylation of NDR1 on both Ser-281 and Thr-444, and specifically, these effects were reversed when we added the sarcoplasmic-endoplasmic reticulum Ca(2+) ATPase pump inhibitor thapsigargin.

Asymmetrical, agonist-induced fluctuations in local extracellular [Ca(2+)] in intact polarized epithelia.

We recently proposed that extracellular Ca(2+) ions participate in a novel form of intercellular communication involving the extracellular Ca(2+)-sensing receptor (CaR). Here, using Ca(2+)-selective microelectrodes, we directly measured the profile of agonist-induced [Ca(2+)]ext changes in restricted domains near the basolateral or luminal membranes of polarized gastric acid-secreting cells. The Ca(2+)-mobilizing agonist carbachol elicited a transient, La(3+)-sensitive decrease in basolateral [Ca(2+)] (average approximately 250 microM, but as large as 530 microM). Conversely, carbachol evoked an HgCl2-sensitive increase in [Ca(2+)] (average approximately 400 microM, but as large as 520 microM) in the lumen of single gastric glands. Both responses were significantly reduced by pre-treatment with sarco-endoplasmic reticulum Ca(2+) ATPase (SERCA) pump inhibitors or with the intracellular Ca(2+) chelator BAPTA-AM. Immunofluorescence experiments demonstrated an asymmetric localization of plasma membrane Ca(2+) ATPase (PMCA), which appeared to be partially co-localized with CaR and the gastric H(+)/K(+)-ATPase in the apical membrane of the acid-secreting cells. Our data indicate that agonist stimulation results in local fluctuations in [Ca(2+)]ext that would be sufficient to modulate the activity of the CaR on neighboring cells.

Sarcoplasmic reticulum Ca(2+)/Calmodulin-dependent protein kinase is altered in heart failure.

Although Ca(2+)/calmodulin-dependent protein kinase-II (CaMK) is known to phosphorylate different Ca(2+) cycling proteins in the cardiac sarcoplasmic reticulum (SR) and regulate its function, the status of CaMK in heart failure has not been investigated previously. In this study, we examined the hypothesis that changes in the CaMK-mediated phosphorylation of the SR Ca(2+) cycling proteins are associated with heart failure. For this purpose, heart failure in rats was induced by occluding the coronary artery for 8 weeks, and animals with >30% infarct of the left ventricle wall plus septum mass were used. Noninfarcted left ventricle was used for biochemical assessment; sham-operated animals served as control. A significant depression in SR Ca(2+) uptake and release activities was associated with a decrease in SR CaMK phosphorylation of the SR proteins, ryanodine receptor (RyR), Ca(2+) pump ATPase (SR/endoplasmic reticulum Ca(2+) ATPase [SERCA2a]), and phospholamban (PLB) in the failing heart. The SR protein contents for RyR, SERCA2a, and PLB were decreased in the failing hearts. Although the SR Ca(2+)/calmodulin-dependent CaMK activity, CaMK content, and CaMK autophosphorylation were depressed, the SR phosphatase activity was enhanced in the failing heart. On the other hand, the cAMP-dependent protein kinase-mediated phosphorylation of RyR and PLB was not affected in the failing heart. On the basis of these results, we conclude that alterations in SR CaMK-mediated phosphorylation may be partly responsible for impaired SR function in heart failure.

Mechanisms of calcium transport in human endothelial cells subjected to hyperthermia

We have probed the mechanisms by which severe heat alters cytosolic calcium ion concentrations (Cai) in individual cultured human endothelial cells (ATCC ♯1998). Cells adhering to glass coverslips were heated to as high as 50°C and Cai determined by means of a fluorescence laser imaging system using the calcium-sensitive dye, indo-1, in the presence of thapsigargin, and in Na-free and Ca-free media. Baseline Cai varied between 175–225 nM. When cells were heated to 50°C in a complete Ca-containing medium, there was first a transient fall in Cai, then a rapid rise of 50–100 nM in Cai, followed by a slower, secondary rise of 50–75 nM. Depleting the intracellular calcium stores with thapsigargin blocked both the transient fall and the secondary rise in Cai. Placement of the cells into a Ca-free medium blocked both the transient fall and the initial rapid rise, while use of a Na-free buffer prevented the initial rapid rise only. These data suggest that in human endothelial cells, extreme heat accelerates the CaATPase pumps of the intracellular Ca stores causing the transient fall in Cai which is soon followed by activation of the reverse mode of the Na/Ca exchanger to cause the initial rapid rise in Cai. The Ca-release channels of the intracellular stores become activated by heat to cause the secondary, slow rise in Cai. This preliminary work indicates that the application of heat to cultured cells can be a useful probe to examine the kinetics and unmask mechanisms of intracellular Ca fluctuations.

Functional effects of uridine triphosphate on human skinned skeletal muscle fibers

Chemically skinned human skeletal muscle fibers were used to study the effects of uridine triphosphate (UTP) on the tension-pCa relationship and on Ca2+ uptake and release by the sarcoplasmic reticulum (SR). Total replacement (2.5 mM) of adenosine triphosphate (ATP) with UTP (i) displaced the tension-pCa relationship to the left along the abcissae and increased maximum Ca(2+)-activated tension, both effects being larger in slow- than in fast-type fibers; (ii) markedly reduced Ca2+ uptake by the SR (evaluated by the caffeine-evoked tension) in both fiber types; (iii) had no effect on the rate of depletion of caffeine-sensitive Ca2+ stores during soaking in relaxing solutions; (iv) induced tension in slow- but not in fast-type fibers. The effects on the SR functional properties are consistent with the notion that UTP is a poor substitute for ATP as a substrate for the Ca ATPase pump and as an agonist of the ryanodine-sensitive Ca(2+)-release channel. The UTP-induced tension in human slow-type fibers is attributed to effect(s) of the nucleotide on the tension-pCa relationship of the contractile machinery. The present data reveal important differences between the effects of UTP on human versus rat muscle fibers.

A minimal model for calcium signal generated by tyrosine kinase and G protein linked receptors; a stochastic computer simulation with CALSIM

A software was designed to simulate the calcium signal following hormone or growth factor stimulation in epithelial cells. The software written in C runs on a PC under Windows environment. It is based on a Markov process where the dynamic of the system is characterised by phenomenological transition probabilities. Moreover a minimal model is proposed to analyse the role of plasma channels and IP3 receptors, together with the opposite action of the CaATPase pumps, in the cytosolic and endoplasmic reticulum (ER) calcium signal control. The simulation is applied on the calcium response following stimulation by carbacol (protein G coupled receptors) or epidermal growth factor (tyrosine kinase type receptors) in A431 epithelial cells. The experimental calcium signals can be grouped in three classes; a spike and a return to the basal level (signal A), a spike and a decrease to a plateau level (signal B) or a slow increase to a plateau (signal C). Epidermal growth factor induces signal A and B while carbacol gives signal B and C. When a `pseudo' steady state is reached oscillations occur. Computer simulations show that signal A can result from the activation of IP3 receptors while signal C would result from the activation of the plasma channels; signal B appears as the additive contribution of both channels, while oscillations are compatible with a calcium induced calcium release mechanism. Simulations suggest that the calcium dynamic in the ER is a mirror of cytosolic calcium but that a simple way to produce similar calcium elevation in these two compartments is to activate plasma channels. Implications of such a mechanism is discussed.

SR Ca 2+-ATPase/phospholamban incardiomyocyte function

Ca ATPase regulates intracellular Ca levels by pumping Ca into sarcoplasmic and endoplasmic reticulum (SER). Phospholamban was first identified as a phosphoprotein in cardiac myocytes. Functional properties of phospholamban by steady-state and presteady-state kinetic studies of Ca pump ATPase suggest that phospholamban functions as an inhibitory co-factor for cardiac Ca ATPase (SERCA 2). Protein kinase A-catalyzed phosphorylation of phospholamban results in the dissociation of phospholamban from the Ca ATPase, thus augmenting the ATPase activity. Phospholamban is found as a homo-pentamer, formed from subunits of 6080 Da in size. PKA-catalyzed and CAM kinase- catalyzed phosphorylation residues (Ser 16 and Thr 17) are located in the N-terminal cytoplasmic domain, whereas the C-terminal 22 residues are extremely hydrophobic and are considered to be embedded in the SR membrane. At least three kinds of Ca ATPase have been found. SERCA 1 is expressed in fast-twitch skeletal muscle, while the SERCA 2 gene encodes two alternatively spliced products, SERCA 2a and 2b. SERCA 2a is expressed in cardiac and slow-twitch skeletal muscles; SERCA 2b in smooth muscle and non-muscle tissues. SERCA 3 is expressed in a broad variety of muscle and non-muscle tissues. In vitro expression systems revealed that the functional properties of Ca transport of SERCA 2 are identical to SERCA 1, but not SERCA 3. In particular, the Ca affinity for Ca transport of SERCA 1 or 2 is lowered by co-expression with phospholamban, whereas that of SERCA3 is not. Identification of the interaction sites of phospholamban and SERCA 2 helps definingthe molecular mode of interaction between the two proteins. Photoactivated crosslinking studies indicated that potential binding residues are located just downstream of the active ATPase site (Asp 351) of SERCA 2, but SERCA 3 is devoid of this sequence. If a chimeric Ca ATPase (CH2) is made from SERCA 2 and 3, in which the SERCA 3 region corresponding to the phospholamban-binding sequence of SERCA 2 is introduced into the remainder of the SERCA 2 molecule, then the interaction with phospholamban is lost. These results suggest that this region of SERCA 2 contains amino acids which are involved in the interaction with phospholamban. By site-directed mutagenesis of amino acids of this region, we were able to show that 6 residues, Lys-Asp-Asp-Lys-Pro-Va1402, of SERCA 2 are functionally important for the interaction. When the chimera CH2 was mutated back to SERCA 2 type, mutated CH2 containing these 6 residues of SERCA 2 restored the interaction with phospholamban. Altogether, these 6 residues of SERCA 2 represent the interaction sites for phospholamban. Mutagenesis studies of phospholamban also demonstrated that the hydrophilic, cytoplasmic region of phospholamban contains a potential binding site for SERCA 2. We therefore conclude that the functional interaction between the two proteins occurs in the cytoplasmic region.

Effects of hypocaloric feeding and refeeding on myocardial Ca and ATP cycling in the rat.

Hypocaloric feeding (HCF) depresses heart function causing cardiac atrophy, bradycardia, and decreased cardiac output. We tested the hypothesis that HCF results in decreased myocardial Ca- and ATP cycling. We reduced protein-calorie intake of adult rats by 20% for 7 days and then allowed them to recover for 3 days. Changes in ionized Ca concentration (nM/s) of 2.5% myocardial homogenates that were attributable to the Ca-ATPase pump and Ca-release channel (CRC), respectively, of the sarcoplasmic reticulum (SR) were depressed 41 and 85% by HCF from 61.6 +/- 9.4 and 24.7 +/- 3.3, to 36.1 +/- 2.8 and 3.6 +/- 2.9. Activity of the Ca-pump was restored after 3 days of refeeding, whereas the CRC remained 23% depressed (all p < 0.05). Additionally, the CRC activity was inhibited to a 3-fold greater extent than controls by HCF, but was disinhibited within one day of refeeding. The greater effect on CRC than Ca-pump activity resulted in net Ca-uptake being unaffected by HCF. In addition to depression of Ca-cycling, ATP sythetase and total ATPase activities (IU/g), respectively, were depressed 20 and 15% by HCF from 174 +/- 19 and 51.3 +/- 3.8 to 140 +/- 15 and 43.7 +/- 4.7, but were restored to control values within one day of refeeding. We conclude that HCF produces a compensatory, reversible, and asymmetric downregulation and inhibition of Ca-cycling, with the CRC being preferentially affected.

Sarcoplasmic reticulum Ca-release channel and ATP-synthesis activities are early myocardial markers of heart failure produced by rapid ventricular pacing in dogs.

The contraction-relaxation cycle of the heart is dependent on a cycle of ATP production and utilization and a cycle of Ca uptake and Ca release by the sarcoplasmic reticulum (SR). Heart failure (HF) is associated with abnormalities of myocardial Ca and ATP cycling, but the time course of their development is unknown. This study tested the hypothesis that, compared with ATP-utilizing and Ca-uptake activities, decreases in ATP-synthesis and Ca-release activities occurred earlier in the development of HF and persisted longer during recovery from HF. HF was induced by right ventricular pacing of dogs at 250 beats/min. Dogs were studied after 1 week of pacing (n = 8, early HF), at HF (n = 11, severe HF), and 4 weeks after cessation of pacing (n = 9) and were compared with dogs not subjected to pacing. At early HF, there were decreased activities (p < 0.05) of the SR Ca-release channel (rate constant from 199 +/- 36 x 10(-4) to 90 +/- 16 x 10(-4) s-1), mitochondrial ATP synthesis (from 11.2 +/- 2.4 to 7.0 +/- 2.2 international units (IU)/g), and creatine kinase (CK) from 2028 +/- 266 to 1811 +/- 79 IU/g). The decreased Ca-channel activity was due to a 32% decrease in maximal activity (rate constant from 249 +/- 50 x 10(-4) to 170 +/- 29 x 10(-4) s-1) and to a 2-fold increase (from 19.1 +/- 12.4 to 42.0 +/- 14.2%) in inhibition of maximal channel activity (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Different patterns of calcium signaling triggered through two components of the B lymphocyte antigen receptor.

The engagement of the B cell antigen receptor is the first step of antigenic stimulation of B lymphocytes. This step is followed by a series of biochemical events, including the activation of protein-tyrosine kinases, phosphoinositide turnover, and multiple patterns of calcium mobilization, which lead to the regulation of gene transcription and cellular responses. The B cell antigen receptor complex is composed of membrane immunoglobulins (as antigen recognition subunits) and associated chains (Ig-alpha and Ig-beta) that couple the receptor to cytoplasmic protein kinases. To investigate independently the relative signaling capacity of Ig-alpha and Ig-beta, chimeric proteins containing their cytoplasmic domains were expressed in a B cell line. We found that Ig-alpha and Ig-beta activate two distinct intracellular signaling pathways. The engagement of Ig-alpha chimeras induces a complete release of calcium from intracellular stores, followed by transmembrane calcium influx and late cell activation signals, detected by lymphokine secretion. In contrast, Ig-beta chimeras do not induce lymphokine secretion or calcium influx, but induce short oscillatory release of calcium, dependent on the activity of the Ca-ATPase pump of the endoplasmic reticulum. These results provide a structural basis for the diversity of B cell responses.