H2O2-induced Ca2 Research Articles

Melatonin modulates apoptosis and TRPM2 channels in transfected cells activated by oxidative stress

Transient receptor potential melastatin-like 2 (TRPM2) is a non-selective Ca2+ permeable cation channel and is known to be activated by H2O2, one of the most important indicators of intracellular oxidative stress. A neurohormone melatonin may have a modulator role on TRPM2 channels activated by oxidative stress because it is a strong antioxidant. In this study we investigated the effects of melatonin on apoptosis, whole cell currents and Ca2+ influx arising from TRPM2 channels activated by H2O2.In whole-cell patch clamp experiments, TRPM2 channels in transfected Chinese hamster ovary (CHO) cells were activated by H2O2. However, the currents were inhibited either by intracellular or by extracellular melatonin. When intracellular melatonin was introduced by pipette, TRPM2 channel currents were not activated by H2O2 although H2O2-induced Ca2+ gating and release were not blocked 2-aminoethyldiphenyl borate (2-APB). Cytosolic Ca2+ release was measured by Fura-2 and was higher in H2O2 groups than in control. Melatonin also inhibited apoptosis in the transfected cells.In conclusion, we observed modulator roles of intracellular and extracellular melatonin on Ca2+ influx and apoptosis through a TRPM2 channel in transfected CHO cells.

Neuroprotective effects of phlorotannins isolated from a brown alga, Ecklonia cava, against H2O2-induced oxidative stress in murine hippocampal HT22 cells

Exposure of neurons to hydrogen peroxide (H2O2) results in oxidative stress and the activation of a cascade of intracellular toxic events resulting in oxidation, lipid peroxidation, and Ca2+ elevation, ultimately resulting in cell death. In this study, we attempted to characterize the neuroprotective effects of phlorotannins isolated from Ecklonia cava, including phloroglucinol, eckol, triphloroethol A, eckstolonol, and dieckol, against H2O2-induced cell damage in murine hippocampus neuronal (HT22) cells. We measured the reactive oxygen species (ROS) and lipid peroxidation levels and evaluated the resultant cell death and alterations in Ca2+-concentrations. All phlorotannins were to scavenge intracellular ROS and repress ROS accumulation, thus preventing lipid peroxidation. Consquently, all phlorotannins reduced H2O2-induced cell death in HT22 cells. Moreover, phlorotannins inhibited H2O2-induced Ca2+ release. This study provides a new useful strategy for preventing neuronal H2O2-induced oxidative stress.

Physiological Role of the Oxidative Stress-Sensitive TRPM2 Ca2+ Channel in Immunocytes

It is known that a large amount of reactive oxygen species (ROS) exist at inflamed sites. ROS induce chemokines responsible for the recruitment of inflammatory cells at inflamed sites. Here, we demonstrate that the plasma membrane Ca2+-permeable channel TRPM2 controls ROS-induced chemokine production in monocytes/macrophages. In monocytes from Trpm2-defecient mice, H2O2-induced Ca2+ influx and production of the macrophage inflammatory protein-2 (CXCL2), which exhibit potent neutrophil chemotactic activity, were impaired. In the inflammation model dextran sulfate sodium-induced colitis, CXCL2 expression was attenuated by Trpm2 disruption. Interestingly, the number of recruited neutrophils was significantly reduced in DSS-treated TRPM2 KO mice, whereas that of DSS-induced macrophages after infiltration into inflamed sites, was indistinguishable in WT and TRPM2 KO mice. Importantly, TRPM2 deficiency failed to impair important aspects of CXCL2-evoked neutrophil chemotaxis, including Ca2+ response, in vitro migration, and in vivo infiltration after DSS administration. Thus, TRPM2-mediated Ca2+ influx induces chemokine production in monocytes that aggravates inflammatory neutrophil infiltration. We propose functional inhibition of TRPM2 channels as a new therapeutic strategy for treating inflammatory diseases.

Effects of γ-Ketoaldehydes on Ca2+ Cuttrent Induced SR Ca2+ Release in Ventricular Myocytes

• Oxidation increases RyR2 channel activity, enhances cardiac SR Ca2+ release and causes spontaneous SR Ca2+ waves. Isoprostanes have become a recognized marker of oxidative stress in rodents and humans. γ-ketoaldehydes(γ-KAs) are the most reactive product of the isoprostane pathway. Recently, we found that lipophilic pyridoxamine analogues, salicylamine(SA) scavenge γ-KAs and thereby prevent formation of γ-KA protein adducts in response to oxidative stress. We hypothesized that γ-KAs are potential mediators of oxidant-induced RyR2 channel dysfunction and spontaneous SR Ca2+ waves, and that SA would prevent oxidant-induced spontaneous SR Ca2+ waves(SCW) in the ventricular myocytes. • We compared the effect of γ-KAs(1uM) or H2O2(10uM) and the effect of SA on Ca-current induced Ca release(CICR) in murine ventricular myocytes loaded with Fura-2AM or Fluo-4. All data are expressed relative to vehicle(Mean±SEM, n=15-50 per group). • Acute exposure(3 min) to γ-KAs(1 uM) or H2O2(10 uM) increased the amplitude of Ca2+ transients, and the fraction of Ca2+ released from the SR(γ-KAs130±10%∗, H2O2120±10%, ∗p<0.05) during each beat. Furthermore, the rate of SCW was significantly increased(γ-KAs 42%∗, H2O233%∗, ∗p<0.05) and SR Ca2+ content was reduced. In voltage-clamped myocytes, dialysis with γ-KAs enhanced Ca2+ release without changing L-type Ca2+ current, demonstrating that the effect of γ-KAs is the result of RyR2 modification. However, after chronic exposure(30 min) to γ-KAs(1 uM) or H2O2(10 uM), Ca2+ transients(γ-KAs 0.53±0.1∗, H2O2 0.7±0.1∗, ∗p<0.05) and SR Ca2+ contents decreased, and SCW remained elevated. Pre-treatment(3 days) of salicylamine reduced H2O2-induced spontaneous Ca2+ waves(SCWs/sec, H2O21.2±0.3∗, SA-H2O20.4±0.2∗, ∗p<0.05) preserved with SR Ca2+ content in ventricular myocytes. • We found that H2O2 and γ-KAs have analogous biphasic effects on SR Ca2+ release in ventricular myocytes. The protective effect of γ-KA scavengers suggests that γ-KAs are possible mediators of oxidant-induced RyR2 channel dysfunction.

H2O2‐induced Ca2+ influx and its inhibition by N‐(p‐amylcinnamoyl) anthranilic acid in the β‐cells: involvement of TRPM2 channels

Type 2 melastatin-related transient receptor potential channel (TRPM2), a member of the melastatin-related TRP (transient receptor potential) subfamily is a Ca2+-permeable channel activated by hydrogen peroxide (H2O2). We have investigated the role of TRPM2 channels in mediating the H2O2-induced increase in the cytoplasmic free Ca2+ concentration ([Ca2+]i) in insulin-secreting cells. In fura-2 loaded INS-1E cells, a widely used model of β-cells, and in human β-cells, H2O2 increased [Ca2+]i, in the presence of 3 mM glucose, by inducing Ca2+ influx across the plasma membrane. H2O2-induced Ca2+ influx was not blocked by nimodipine, a blocker of the L-type voltage-gated Ca2+ channels nor by 2-aminoethoxydiphenyl borate, a blocker of several TRP channels and store-operated channels, but it was completely blocked by N-(p-amylcinnamoyl)anthranilic acid (ACA), a potent inhibitor of TRPM2. Adenosine diphosphate phosphate ribose, a specific activator of TRPM2 channel and H2O2, induced inward cation currents that were blocked by ACA. Western blot using antibodies directed to the epitopes on the N-terminal and on the C-terminal parts of TRPM2 identified the full length TRPM2 (TRPM2-L), and the C-terminally truncated TRPM2 (TRPM2-S) in human islets. We conclude that functional TRPM2 channels mediate H2O2-induced Ca2+ entry in β-cells, a process potently inhibited by ACA.

Three-dimensional Reconstruction Using Transmission Electron Microscopy Reveals a Swollen, Bell-shaped Structure of Transient Receptor Potential Melastatin Type 2 Cation Channel

Transient receptor potential melastatin type 2 (TRPM2) is a redox-sensitive, calcium-permeable cation channel activated by various signals, such as adenosine diphosphate ribose (ADPR) acting on the ADPR pyrophosphatase (ADPRase) domain, and cyclic ADPR. Here, we purified the FLAG-tagged tetrameric TRPM2 channel, analyzed it using negatively stained electron microscopy, and reconstructed the three-dimensional structure at 2.8-nm resolution. This multimodal sensor molecule has a bell-like shape of 18 nm in width and 25 nm in height. The overall structure is similar to another multimodal sensor channel, TRP canonical type 3 (TRPC3). In both structures, the small extracellular domain is a dense half-dome, whereas the large cytoplasmic domain has a sparse, double-layered structure with multiple internal cavities. However, a unique square prism protuberance was observed under the cytoplasmic domain of TRPM2. The FLAG epitope, fused at the C terminus of the ADPRase domain, was assigned by the antibody to a position close to the protuberance. This indicates that the agonist-binding ADPRase domain and the ion gate in the transmembrane region are separately located in the molecule.

Spatial variation in H2O2 response of Arabidopsis thaliana root epidermal Ca2+ flux and plasma membrane Ca2+ channels

Hydrogen peroxide is an important regulatory agent in plants. This study demonstrates that exogenous H2O2 application to Arabidopsis thaliana root epidermis results in dose-dependent transient increases in net Ca2+ influx. The magnitude and duration of the transients were greater in the elongation zone than in the mature epidermis. In both regions, treatment with the cation channel blocker Gd3+ prevented H2O2-induced net Ca2+ influx, consistent with application of exogenous H2O2 resulting in the activation of plasma membrane Gd3+-sensitive Ca2+-influx pathways. Application of 10 mm H2O2 to the external plasma membrane face of elongation zone epidermal protoplasts resulted in the appearance of a hyperpolarization-activated Ca2+-permeable conductance. This conductance differed from that previously characterized as being responsive to extracellular hydroxyl radicals. In contrast, in mature epidermal protoplasts a plasma membrane hyperpolarization-activated Ca2+-permeable channel was activated only when H2O2 was present at the intracellular membrane face. Channel open probability increased with intracellular [H2O2] and at hyperpolarized voltages. Unitary conductance decreased thus: Ba2+ > Ca2+ (14.5 pS) > Mg2+ > Zn2+ (20 mM external cation, 1 mM H2O2). Lanthanides and Zn2+ (but not TEA+) suppressed the open probability without affecting current amplitude. The results suggest spatial heterogeneity and differential sensitivity of Ca2+ channel activation by reactive oxygen species in the root that could underpin signalling.

Inhibition of TRPM2 cation channels by N‐(p‐amylcinnamoyl)anthranilic acid

1. TRPM2 is a Ca2+ -permeable nonselective cation channel activated by intracellular ADP-ribose (ADPR) and by hydrogen peroxide (H2O2). We investigated the modulation of TRPM2 activity by N-(p-amylcinnamoyl)anthranilic acid (ACA). ACA has previously been reported to inhibit phospholipase A2 (PLA2). 2. Using patch-clamp and calcium-imaging techniques, we show that extracellular application of 20 microM ACA completely blocked ADPR-induced whole-cell currents and H2O2-induced Ca2+ signals (IC50 = 1.7 microM) in HEK293 cells transfected with human TRPM2. Two other PLA2 inhibitors, p-bromophenacyl bromide (BPB; 100 microM) and arachidonyl trifluoromethyl ketone (20 microM), had no significant effect on ADPR-stimulated TRPM2 activity. 3. Inhibition of TRPM2 whole-cell currents by ACA was voltage independent and accelerated at decreased pH. ACA was ineffective when applied intracellularly. The single-channel conductance was not changed during ACA treatment, suggesting a reduction of TRPM2 open probability by modulating channel gating. 4. ACA (20 microM) also blocked currents through human TRPM8 and TRPC6 expressed in HEK293 cells, while BPB (100 microM) was ineffective. TRPC6-mediated currents (IC50 = 2.3 microM) and TRPM8-induced Ca2+ signals (IC50 = 3.9 microM) were blocked in a concentration-dependent manner. 5. ADPR-induced currents in human U937 cells, endogeneously expressing TRPM2 protein, were fully suppressed by 20 microM ACA. 6. Our data indicate that ACA modulates the activity of different TRP channels independent of PLA2 inhibition. Owing to its high potency and efficacy ACA can serve, in combination with other blockers, as a useful tool for studying the unknown function of TRPM2 in native cells.

A Critical Role of TRPM2 in Neuronal Cell Death by Hydrogen Peroxide

A brief exposure to hydrogen peroxide (H2O2) induces severe deterioration of primary cultured neurons in vitro. We have investigated a link between the H2O2-induced neuronal death and Ca2+-permeable TRPM2 channels regulated by ADP-ribose (ADPR). In cultured cerebral cortical neurons from fetal rat, TRPM2 proteins were detected at cell bodies and neurite extensions. Application of H2O2 to the cultured neurons elicited an increase in intracellular Ca2+ concentration ([Ca2+]i) caused by Ca2+ influx and the Ca2+-dependent neuronal death in a similar concentration range. Molecular cloning of TRPM2 cDNA from rat brain revealed several differences in amino acid sequences within the Nudix box region as compared with those of human and mouse TRPM2. ADPR-induced current responses, H2O2-induced Ca2+ influx, and H2O2-induced cell death were induced in human embryonic kidney cells heterologously expressing rat TRPM2. Treatment of cultured neurons with small interfering RNA against rat TRPM2,which efficiently suppressed immunoreactive TRPM2 content and the H2O2-induced Ca2+ influx,significantly inhibited H2O2-induced neuronal death. These results suggest that TRPM2 plays a pivotal role in H2O2-induced neuronal death as redox-sensitive Ca2+-permeable channels expressed in neurons.

Extracellular-Added ADP-Ribose Increases Intracellular Free Ca2+ Concentration Through Ca2+ Release From Stores, but Not Through TRPM2-Mediated Ca2+ Entry, in Rat β-Cell Line RIN-5F

Intracellular ADP-ribose is an activator of TRPM2, which is a Ca2+-permeable channel and mediates H2O2-induced cell death, in the TRPM2-expressing rat beta-cell line RIN-5F. We examined the effect of extracellular-added ADP-ribose on intracellular Ca2+ concentration in RIN-5F cells. ADP-ribose induced Ca2+ release from the thapsigargin-sensitive Ca2+ store, but not Ca2+ entry across the plasma membrane. A phospholipase C (PLC) inhibitor and a non-specific IP3 receptor inhibitor blocked its Ca2+ release. H2O2-induced Ca2+ entry through TRPM2 was not affected by extracellular ADP-ribose. These findings suggest that extracellular-added ADP-ribose induces Ca2+ release through the PLC-IP3 pathway and does not act as a TRPM2 activator.

H(2)O(2)-induced Ca(2+) overload in NRVM involves ERK1/2 MAP kinases: role for an NHE-1-dependent pathway.

Generation of reactive oxygen species (ROS) and intracellular Ca(2+) overload are key mechanisms involved in ischemia-reperfusion (I/R)-induced myocardial injury. The relationship between I/R injury and Ca(2+) overload has not been fully characterized. The increase in Na(+)/H(+) exchanger (NHE-1) activity observed during I/R injury is an attractive candidate to link increased ROS production with Ca(2+) overload. We have shown that low doses of H(2)O(2) increase NHE-1 activity in an extracellular signal-regulated kinase (ERK)-dependent manner. In this study, we examined the effect of low doses of H(2)O(2) on intracellular Ca(2+) in fura 2-loaded, spontaneously contracting neonatal rat ventricular myocytes. H(2)O(2) induced a time- and concentration-dependent increase in diastolic intracellular Ca(2+) concentration that was blocked by inhibition of ERK1/2 activation with 5 microM U-0126 (88%) or inhibition of NHE-1 with 5 microM HOE-642 (50%). Increased NHE activity was associated with phosphorylation of the NHE-1 carboxyl tail that was blocked by U-0126. These results suggest that H(2)O(2) induced Ca(2+) overload is partially mediated by NHE-1 activation secondary to phosphorylation of NHE-1 by the ERK1/2 MAP kinase pathway.

Hydrogen Peroxide Activates Na+-Dependent Ca2+ Influx in Coronary Endothelial Cells

The purpose of the present study was to examine the effect of short duration H2O2 exposure on coronary artery endothelial cell [Ca2+]i regulation. Freshly dispersed cells from porcine coronary artery were exposed to H2O2 (300 μmol/L) for 3 min while monitoring [Ca2+]i using fura-2 microfluorometry. H2O2 increased [Ca2+]i from 0.86 ± 0.03 to 2.19 ± 0.41 ratio units at 3 min of H2O2 (P < 0.05). Intracellular Ca2+ remained elevated 3 min following removal of H2O2, yet H2O2 had no effect on the subsequent [Ca2+]i response to bradykinin (0.1 μmol/L). The H2O2-induced [Ca2+]i increase was completely abolished either by removal of extracellular Ca2+ or lowering extracellular Na+. Cells exposed to the Na+ ionophore, monensin, showed an increase in [Ca2+]i with a time course similar to that seen with H2O2. Furthermore, H2O2-induced Ca2+ influx was not attenuated by either Ni2+ (300 μmol/L) or econazole (10 μmol/L), excluding Ca2+ influx via the agonist-sensitive pathway. Thus, in coronary arterial endothelial cells, H2O2 increases Ca2+ influx in an extracellular Na+-dependent manner via an agonist-insensitive pathway.

Effect of hydrogen peroxide on the membrane currents of sinoatrial node cells from rabbit heart.

The effects of H(2)O(2) on pacemaker activity and underlying membrane currents were studied in isolated rabbit sinoatrial (SA) node cells using perforated patch current- and voltage-clamp methods. Short-term exposure (<10 min) of the nodal cells to H(2)O(2) (200 microM) resulted in an initial shortening of spontaneous action potential cycle length (from 445 +/- 60 to 398 +/- 56 ms; P < 0.05) and a prolongation of action potential duration. H(2)O(2) (100 microM) significantly increased peak L-type Ca(2+) current (I(Ca,L)) from -384 +/- 77 to -439 +/- 84 pA (116 +/- 2%, n = 6). Additionally, the persistent or non-inactivating component of I(Ca,L) was increased from -52 +/- 3 to -88 +/- 14 pA (174 +/- 19%, n = 6). The hyperpolarization-activated current (I(f)) was decreased from -228 +/- 62 to -161 +/- 72 pA after exposure to H(2)O(2) (n = 7). There were no changes in the delayed rectifier K(+) current (I(K)) (n = 7). H(2)O(2)-induced Ca(2+) currents were blocked by 2 microM nicardipine (n = 6), 2 mM Ni(2+) (n = 2), and the protein kinase C (PKC) inhibitor bisindolylmaleimide (10(-7) M; n = 4) but not by 20 microM tetrodotoxin. These results suggest that H(2)O(2) can increase the spontaneous pacing rate in rabbit SA node cells by enhancing I(Ca,L) and that this effect is mediated by a PKC-dependent pathway.

H2O2 induces DNA repair in mononuclear cells: Evidence for association with cytosolic Ca2+ fluxes

Cellular DNA repair systems are induced whenever DNA is damaged. Reactive oxygen species (ROS) are generated, in vivo, in the tissues as a result of regular cellular metabolism or after exposure to oxidizing agents, such as ultraviolet (UV) irradiation. It has been suggested that ROS mediate DNA damage. The objectives of the study were as follows: (1) to investigate whether hydrogen peroxide (H2O2), the commonly occurring cellular ROS, induces DNA repair as a response to the damage it probably causes; (2) to evaluate whether H2O2-induced DNA repair, if present, is signaled through a Ca2+-dependent pathway via the tyrosine kinase signal transduction. H2O2 was found to induce DNA repair in human peripheral blood mononuclear cells (PBMCs) in a dose-dependent manner. The recovery of RNA synthesis, which occurred after DNA repair, confirmed that transcribable DNA was repaired. the inhibition of tyrosine kinase activity by genistein reduced the DNA repair significantly. Furthermore, H2O2 caused a dose-dependent significant rise in cytosolic calcium ((Ca2+)i). H2O2 also induced a small rise in (Ca2+)i of cytosolic Ca2+-depleted cells, probably reflecting the release of Ca2+ from internal stores. Genistein inhibited both Ca2+ influx and Ca2+ release from internal stores. In summary, H2O2 induced a DNA repair synthesis that was in part Ca2+ dependent and signaled via tyrosine kinase. The changes in DNA repair paralleled changes in (Ca2+)i. The H2O2-induced (Ca2+)i rise was mostly the result of influx, but to some degree it was also due to the translocation of Ca2+ from internal stores.

Role of intracellular signalling pathways in hydrogen peroxide-induced injury to rat glomerular mesangial cells.

1. Brief exposure of cultured rat glomerular mesangial cells (GMC) to H2O2 in nominally bicarbonate-free solution induced a rapid dose dependent, dantrolene-inhibitable increase in intracellular free Ca2+ from 65 +/- 6 to 203 +/- 14 nmol/L and a prolonged release of [14C]-arachidonic acid [14C]-AA which preceded the onset of cell membrane damage assessed by trypan-blue uptake. 2. Ca2+ responses were potentiated in HCO3-/CO2 containing buffers and reached values of 1145 +/- 100 nmol/L at 1 mmol/L H2O2. In HCO3-/CO2 solutions, but not HEPES buffer, H2O2-induced Ca2+ increases were markedly attenuated by verapamil (100 mumol/L) or removal of extracellular calcium. 3. Enhanced release of [14C]-AA was partially attenuated by inhibitors of key intracellular signalling mechanisms including the phospholipase-A2 (PLA2) inhibitor mepacrine (100 mumol/L), the NADPH oxidase inhibitor diphenyliodonium (10 mumol/L), the mitochondrial calcium-cycling inhibitor ruthenium red (10 mumol/L) and the iron chelator dipyridyl (100 mumol/L). Release was unaffected by protein kinase C inhibition with H7 (100 mumol/L), inositol triphosphate antagonism with neomycin (1 mmol/L) or overnight treatment with the G-protein antagonist pertussis toxin (5 micrograms/mL). 4. Several structurally diverse lipoxygenase inhibitors, including esculetin, baicalein and phenidone, over the dose range 1-100 mumol/L, also prevented [14C]-AA release and markedly protected against cell membrane damage. No drug directly scavenged H2O2 assessed by UV absorption. 5. These results indicate that H2O2 activates in GMC a complex series of interrelated pathological mechanisms which in turn contribute to a prolongation of oxidative damage beyond the time of the initial exposure. These include an increase in intracellular calcium which, depending upon conditions, appears to be mediated by release from intracellular stores as well as Ca2+ entry from the extracellular space. In turn there is a sustained release of arachidonic acid, which may partly depend on prolonged activation of PLA2 but not phospholipase C. 6. Release of [14C]-AA could be attenuated by inhibitors of NADPH oxidase, mitochondrial calcium-cycling, iron chelators and a structurally diverse range of lipoxygenase inhibitors in association with protection from H2O2-mediated cell membrane damage.

Increase vs. decrease of calcium uptake by isolated heart cells induced by H2O2 vs. HOCl

Adult rat heart myocytes were labeled rapidly with exogenous [45Ca2+]. Addition of 2.5 mM H2O2 to the heart cell suspension raised the content of rapidly exchangeable intracellular Ca2+ twofold, whereas addition of 1-30 mM HOCl decreased the Ca2+ content. The H2O2-induced increase in Ca2+ content was dependent on the medium Na+, pH, and temperature but was not significantly affected by addition of verapamil, diltiazem, amiloride, or 3-aminobenzamide. The [3H]ouabain binding to myocytes was suppressed by H2O2, whereas the Ca2+ efflux from myocytes was not influenced. An uncoupler, carbonyl cyanide m-chlorophenylhydrazone, reduced Ca2+ content, implying that the H2O2-induced change in Ca2+ content was not directly related to ATP depletion. On the other hand, the H2O2-induced Ca2+ accumulation in myocytes was prevented by deferoxamine or o-phenanthroline. These results suggest that H2O2 inhibited Na+-K+-ATPase, resulting in an increase in intracellular Na+ concentration and stimulation of sarcolemmal Na+-Ca2+ exchange activity, which caused a transient net Ca2+ influx into myocytes. By contrast, HOCl decreased the Ca2+ content of the rapidly exchangeable pool below control levels and this action of HOCl was antagonized by 1,4-dithiothreitol. HOCl accelerated Ca2+ efflux from myocytes. Ca2+ uptake and Ca2+-ATPase of the isolated sarcoplasmic reticular (SR) fraction were highly sensitive to the action of HOCl. Ca2+ uptake by intracellular sites, studied with myocytes permeabilized with digitonin, was inhibited by both H2O2 and HOCl. Thus these results suggest that HOCl inhibits the SR Ca2+ pump, resulting in the observed acceleration of Ca2+ efflux from and decline in Ca2+ content of myocytes.