Epigallocatechin-3-gallate has dual, independent effects on the cardiac sarcoplasmic reticulum/endoplasmic reticulum Ca2+ ATPase.

We determined the effects of epigallocatechin-3-gallate (EGCG) and epicatechin (EC), on pump turnover and Ca2+ transport by the cardiac form of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA). Fluorescence spectroscopy was used to directly measure SERCA ATPase activity and to measure Ca2+ uptake into cardiac sarcoplasmic reticulum (SR) vesicles and microsomes derived from human embryonic kidney (HEK) cells expressing human cardiac SERCA2a. We found that EGCG reduces the maximum velocity of Ca2+ uptake into cardiac SR vesicles and increases the Ca2+-sensitivity of uptake in a concentration-dependent manner. EC is less potent than EGCG in increasing the Ca2+-sensitivity of uptake and does not affect maximum uptake velocity. The EGCG-dependent reduction in Ca2+ uptake velocity is well correlated with direct inhibition of SERCA. The effect of EGCG on the Ca2+-sensitivity of Ca2+ uptake into cardiac SR vesicles is affected by the phosphorylation status of phospholamban (PLB). When cardiac SERCA2a is expressed in HEK cells without PLB, EGCG reduces the maximum velocity of Ca2+ uptake but does not affect the Ca2+-sensitivity of uptake into microsomes derived from these cells indicating that the effect of EGCG on Ca2+-sensitivity requires the presence of PLB. Our results show that EGCG has dual effects on SERCA function in cardiac SR vesicles: it directly affects SERCA by reducing maximum uptake velocity; it increases the Ca2+-sensitivity of Ca2+ uptake in a manner that appears to depend on the interaction between SERCA and PLB.

Effects of anti-oestrogens and beta-estradiol on calcium uptake by cardiac sarcoplasmic reticulum.

1. Tamoxifen and a group of structurally similar non-steroidal, triphenolic compounds inhibit the oestrogen receptor. In addition to this action, these anti-oestrogens are known to inhibit some types of plasma membrane ion channels and other proteins through mechanisms that do not appear to involve their interactions with the estrogen receptor but could be the result of their effect on membrane lipid structure or fluidity. 2. We studied the effects of beta-estradiol and three anti-oestrogens (tamoxifen, 4-hydroxytamoxifen and clomiphene) on Ca(2+) uptake into sarcoplasmic reticulum (SR) vesicles isolated from canine cardiac ventricular tissue. 3. The antiestrogens all inhibit SR Ca(2+) uptake in a concentration-dependent manner (order of potency: tamoxifen > 4-hydroxytamoxifen > or = clomiphene). Although these compounds rapidly inhibit net Ca(2+) uptake they do not have a similar rapid effect on the ATPase activity of the SR Ca pump. beta-estradiol has no effect on Ca(2+) uptake nor does it alter the inhibitory action of tamoxifen on the SR. 4. The differences in the effects of beta-estradiol and the anti-oestrogens on cardiac SR Ca(2+) uptake do not correlate with differences in the ways in which these compounds have been reported to interact with membrane lipids. Our results are consistent, however, with direct effects on a membrane protein (possibly an SR Cl(-) or K(+) channel).

Iodide and bromide inhibit Ca(2+) uptake by cardiac sarcoplasmic reticulum.

Recent studies indicate that the Ca(2+) permeability of the sarcoplasmic reticulum (SR) can be affected by its anionic environment. Additionally, anions could directly modulate the SR Ca(2+) pump or the movement of compensatory charge across the SR membrane during Ca(2+) uptake or release. To examine the effect of anion substitution on cardiac SR Ca(2+) uptake, fluorometric Ca(2+) measurements and spectrophotometric ATPase assays were used. Ca(2+) uptake into SR vesicles was inhibited in a concentration-dependent manner when Br(-) or I(-) replaced extravesicular Cl(-) (when Br(-) completely replaced Cl(-), uptake velocity was approximately 70% of control; when I(-) completely replaced Cl(-), uptake velocity was approximately 39% of control). Replacement of Cl(-) with SO(2)(-4) had no effect on SR uptake. Although both I(-) and Br(-) inhibited net Ca(2+) uptake, neither anion directly inhibited the SR Ca(2+) pump nor did they increase the permeability of the SR membrane to Ca(2+). Our results support the hypothesis that an anionic current that occurs during SR Ca(2+) uptake is reduced by the substitution of Br(-) or I(-) for Cl(-).

Tamoxifen inhibits Ca2+ uptake by the cardiac sarcoplasmic reticulum.

Ca2+ transients in isolated cardiac ventricular myocytes and the amount of Ca2+ that could be released from the sarcoplasmic reticulum (SR) in these cells by caffeine were reduced in the presence of tamoxifen. To examine the effects of tamoxifen on the cardiac muscle SR directly, isolated SR vesicles and fluorimetry methods were used to measure the uptake of Ca2+ by the SR and the ATPase activity of the SR Ca2+ pump. SR Ca2+ uptake was inhibited by tamoxifen at concentrations greater than 2.4 microM. Half-maximal inhibition was seen at approximately 5 microM. Inhibition of uptake was not due to the development of a substantial tamoxifen-dependent leak of Ca2+ from the SR or to a direct inhibitory effect of tamoxifen on the ATPase activity of the SR Ca2+ pump. In addition to its effect on SR Ca2+ uptake, tamoxifen also reduced the rate at which stored Ca2+ could be released from the SR by the Ca2+ ionophore 4-bromo A23187. Our results are consistent with the hypothesis that tamoxifen inhibits an ion current that accompanies Ca2+ movement across the SR membrane. This possibility is also consistent with the known inhibitory action of tamoxifen on some types of Cl- and K+ channels.

Chloride channel blockers inhibit Ca2+ uptake by the smooth muscle sarcoplasmic reticulum.

Despite the fact that Ca2+ transport into the sarcoplasmic reticulum (SR) of muscle cells is electrogenic, a potential difference is not maintained across the SR membrane. To achieve electroneutrality, compensatory charge movement must occur during Ca2+ uptake. To examine the role of Cl- in this charge movement in smooth muscle cells, Ca2+ transport into the SR of saponin-permeabilized smooth muscle cells was measured in the presence of various Cl- channel blockers or when I-, Br-, or SO42- was substituted for Cl-. Calcium uptake was inhibited in a dose-dependent manner by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and by indanyloxyacetic acid 94 (R(+)-IAA-94), but not by niflumic acid or 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS). Smooth muscle SR Ca2+ uptake was also partially inhibited by the substitution of SO42- for Cl-, but not when Cl- was replaced by I- or Br-. Neither NPPB nor R(+)-IAA-94 inhibited Ca2+ uptake into cardiac muscle SR vesicles at concentrations that maximally inhibited uptake in smooth muscle cells. These results indicate that Cl- movement is important for charge compensation in smooth muscle cells and that the Cl- channel or channels involved are different in smooth and cardiac muscle cells.

Ruthenium red reduces the Ca2+ sensitivity of Ca2+ uptake into cardiac sarcoplasmic reticulum.

Ruthenium red inhibits mitochondrial Ca2+ uptake and is widely used as an inhibitor of ryanodine-sensitive Ca2+ channels that function to release Ca2+ from the sarcoplasmic reticulum (SR) of muscle cells. It also has effects on other Ca2+ channels and ion transporters. To study the effects of ruthenium red on Ca2+ transport into the SR of cardiac muscle cells, fluorescence measurements of Ca2+ uptake into cardiac SR vesicles were made. Ruthenium red significantly decreased the Ca2+ sensitivity of SR uptake in a dose-dependent manner at concentrations ranging from 5 microM to 20 microM. There were no significant effects of ruthenium red on the maximum velocity or the Hill coefficient of SR Ca2+ uptake.

Anti-phospholamban and protein kinase A alter the Ca2+ sensitivity and maximum velocity of Ca2+ uptake by the cardiac sarcoplasmic reticulum.

The activity of the SERCA2a Ca2+ pump in the sarcoplasmic reticulum (SR) of cardiac muscle is inhibited by phospholamban. When phospholamban is phosphorylated by cyclic-AMP-dependent protein kinase (PKA) this inhibition is relieved. It is generally agreed that this results in an increase in the Ca2+ sensitivity of the SR Ca2+ pump; however, some investigators have also reported an increase in the maximum velocity of the pump. We have used a sensitive fluorescence method to measure net Ca2+ uptake by native cardiac SR vesicles and compared the effects of a constitutively active subunit of PKA (cPKA) with those of a monoclonal antibody (A1) that binds to phospholamban and is thought to mimic the effect of phosphorylation. Both the Ca2+ sensitivity and the maximum velocity of uptake were increased by cPKA and by A1. The effects of cPKA and A1 on uptake velocity were only slightly additive. No changes in uptake were detected with denatured cPKA or denatured A1. These results indicate that the functional effect of phospholamban phosphorylation is to increase both the Ca2+ sensitivity and the maximum velocity of net Ca2+ uptake into the SR.

Predicted changes in concentrations of free and bound ATP and ADP during intracellular Ca2+ signaling.

High Ca2+ concentrations can develop near Ca2+ sources during intracellular signaling and might lead to localized regulation of Ca2+-dependent processes. By shifting the amount of Ca2+ and other cations associated with ATP, local high Ca2+ concentrations might also alter the substrate available for membrane-associated and cytoplasmic enzymes. To study this, simultaneous equations were solved over a range of Ca2+ and Mg2+ concentrations to determine the general effects of Ca2+ on the concentrations of free and Ca2+- and Mg2+-bound forms of ATP. To obtain a more specific picture of the changes that might occur in smooth muscle cells, mathematical models of Ca2+ diffusion and regulation were used to predict the magnitude and time course of near-membrane Ca2+ transients and their effects on the free and bound forms of ATP near the membrane. The results of this work indicate that changes in free Ca2+ concentration over the range of 50 nM-100 microM would result in significant changes in free ATP concentration, MgATP concentration, and the CaATP-to-MgATP concentration ratio.

Hypomagnesemia: characterization of a model of sudden cardiac death.

OBJECTIVES: We sought to compare the incidence of sudden death in rats treated with magnesium-deficient and control diets and to address the electrophysiologic characteristics associated with these end points. BACKGROUND: Although magnesium deficiency is associated with an increased incidence of sudden cardiac death in patients, there has been no clear cause and effect relation because of a number of covariables, including diuretic use, hypokalemia, digitalis use and left ventricular dysfunction. METHODS: Hypomagnesemic rats and their paired control rats underwent in vivo electrophysiologic studies and measurements of the total calcium and magnesium content of their cardiac ventricles RESULTS: Serum magnesium levels were 0.5 +/- 0.3 mEq/liter (mean +/- SD) in hypomagnesemic animals and 1.2 +/- 0.9 mEq/liter in control animals. A modest but significant prolongation of the repolarization time was seen at the apical epicardial site (83 +/- 8 ms in hypomagnesemic rats vs. 68 +/- 13 ms in control rats, p < 0.05), but not at the other sites studied. Bradyarrhythmias and tachyarrhythmias were observed in 82% of the hypomagnesemic rats during the in vivo electrophysiologic studies, compared with 0% in the control group. During these studies, sudden, unexpected asystolic deaths were observed in 4 of 11 hypomagnesemic rats and 0 of 8 control rats. Polymorphic nonsustained ventricular tachycardia was provoked by rapid pacing in 5 to 11 hypomagnesemic rats and 0 of 8 control rats. Three of six hypomagnesemic rats exposed to auditory stimuli developed seizures, followed immediately by sudden deaths-two due to asystole and one due to ventricular fibrillation-although no end points occurred in the control animals. CONCLUSIONS: In this model, magnesium deficiency results in sudden cardiac death. The presence of startle induction of sudden death preceded by seizures suggests that sudden cardiac death results from a neurologic trigger.

The sarcoplasmic reticulum calcium pump is functionally altered in dystrophic muscle.

In Duchenne muscular dystrophy, muscle cells, which lack the protein dystrophin, have been reported to have elevated resting intracellular calcium levels. It has also been noted that, compared to normal muscle, intracellular [Ca2+] in dystrophic muscle returns more slowly to its resting level following contractile stimulation. Consistent with this, it has been suggested that dystrophin is directly involved in the regulation of Ca2+ influx. A secondary alteration in the sarcoplasmic reticulum Ca2+ pump, however, could also contribute to, or be responsible for, the abnormal Ca2+ handling seen. To determine whether the Ca2+ pump is functionally altered in dystrophic muscle, we examined Ca2+ uptake by vesicles derived from skeletal muscle sarcoplasmic reticulum of normal and dystrophic (mdx) mice. The Hill coefficient and the Ca2+ sensitivity of the Ca2+- ATPase were the same in both cases. The maximum velocity of Ca2+ uptake, however, normalized to the ATPase content of the vesicles, was less for mdx muscle.

Direct measurement of Ca2+ uptake and release by the sarcoplasmic reticulum of saponin permeabilized isolated smooth muscle cells.

To make direct measurements of Ca2+ uptake and release by the sarcoplasmic reticulum (SR) of isolated smooth muscle cells, a fluorometric method for monitoring Ca2+ uptake by striated muscle SR vesicles (Kargacin, M.E., C.R. Scheid, and T.W. Honeyman. 1988. American Journal of Physiology. 245:C694-C698) was modified. With the method, it was possible to make continuous measurements of SR function in saponin-skinned smooth muscle cells in suspension. Calcium uptake by the SR was inhibited by thapsigargin and sequestered Ca2+ could be released by Br-A23187 and thapsigargin. From the rate of Ca2+ uptake by the skinned cells and the density of cells in suspension, it was possible to calculate the Ca2+ uptake rate for the SR of a single cell. Our results indicate that the SR Ca2+ pump in smooth muscle cells can remove Ca2+ at a rate that is 45-75% of the rate at which Ca2+ is removed from the cytoplasm of intact cells during transient Ca2+ signals. From estimates of SR volume reported by others and our measurements of the amount of Ca2+ taken up by the skinned cells, we conclude that the SR of a single cell can store greater than 10 times the amount of Ca2+ needed to elicit a single transient contractile response.

Effects of intracellular calcium on sodium current density in cultured neonatal rat cardiac myocytes.

1. Na+ channel mRNA levels in the heart can be modulated by changes in intracellular Ca2+ ([Ca2+]i). We have investigated whether this regulation of Na+ channel biosynthesis by cytosolic Ca2+ translates into functional Na+ channels that can be detected electrophysiologically. 2. Whole-cell Na+ currents (INa) were recorded using patch-clamp techniques from single ventricular myocytes isolated from neonatal rats and maintained in tissue culture for 24 h. Na+ current density, measured at a membrane potential of -10 mV, was significantly decreased in the cells which were exposed for 24 h to culture medium containing 10 mM of both external Ca2+ and K+ in order to raise [Ca2+]i compared with control cells which were maintained in culture medium containing 2 and 5 mM of Ca2+ and K+, respectively. In contrast, Na+ current density (at -10 mV) was significantly increased in cells exposed for 24 h to 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetraacetoxymethyl ester (BAPTA AM; a cell membrane-permeable Ca2+ chelator) which lowered the average [Ca2+]i compared with control. 3. Changes in current density were not associated with changes in the voltage dependence of activation and inactivation of INa. There were no changes in single-channel conductances. 4. It is concluded that Na+ current density in neonatal rat cardiac myocytes is modulated by [Ca2+]i. The findings suggest that the differences in current density are attributable to a change in Na+ channel numbers rather than to changes in single-channel conductance or gating. These changes are consistent with the previously documented modulation of Na+ channel biosynthesis by cytosolic Ca2+.

Methods for determining cardiac sarcoplasmic reticulum Ca2+ pump kinetics from fura 2 measurements.

We explored the use of four methods for analyzing real and simulated fura 2 measurements of Ca2+ uptake by membrane vesicles derived from the sarcoplasmic reticulum (SR) of cardiac muscle. Uptake velocity was calculated 1) directly from the raw data, 2) after segmenting the raw data and averaging the data points in each segment, 3) after smoothing of the raw data by moving-window averaging, and 4) by Savitsky-Golay convolution. Methods 2, 3, and 4 could be used to determine maximum uptake velocity, the Hill coefficient, and the Ca2+ concentration at half-maximal pump velocity from Ca2+ concentration vs. time and velocity curves that were too noisy to analyze directly. Data analysis using these methods should have general applicability to biological experiments, especially those in which large numbers of measurements are made. The fluorometric method we describe for measuring Ca2+ uptake by cardiac SR vesicles opens up the possibility of studying SR function from very small starting tissue samples.

The effects of alpha-adrenoceptor agonists on intracellular Ca2+ levels in freshly dispersed single smooth muscle cells from rat tail artery.

1. The presence of functional alpha-adrenoceptors in freshly dispersed single smooth muscle cells from rat tail arteries was investigated by use of selective alpha-adrenoceptor agonists and antagonists. 2. Cirazoline, a selective alpha 1-adrenoceptor agonist, caused a prazosin-sensitive, rapid but transient increase in intracellular Ca2+, which was partially inhibited by the voltage-dependent Ca2+ channel blocker, nifedipine. 3. TL99, an alpha 2-adrenoceptor agonist, in the presence of prazosin, initiated a slow and sustained increase in intracellular Ca2+ which was partially inhibited by yohimbine and almost completely blocked by nifedipine. 4. In rat tail artery, the effects (dose-response and time-response curves) of cirazoline and TL99 on intracellular Ca2+ levels in freshly dispersed single smooth muscle cells were comparable with those obtained with organ bath studies of ring preparations of artery. 5. In freshly dispersed single smooth muscle cells, the time-course response curves induced by the selective alpha 1-adrenoceptor agonist, phenylephrine and the selective alpha 2-adrenoceptor agonist, UK14304, were similar to those observed with cirazoline and TL99, respectively. 6. These results indicate that: (a) functional alpha 1- and alpha 2-adrenoceptors are present in freshly dispersed single smooth muscle cells from rat tail artery and (b) alpha 1- and alpha 2-adrenoceptors are coupled to different cellular processes that lead to an increase in intracellular Ca2+.

Ligand binding and G protein coupling of muscarinic receptors in airway smooth muscle.

Ligand binding properties of muscarinic receptors were examined in membranes and isolated cells prepared from bovine trachea. The binding of the muscarinic antagonist [3H]quinuclidinyl benzilate (QNB) to both membranes and isolated cells was saturable, reversible, and of high affinity [dissociation constant (KD) = 100-200 pM]. The binding constants of three selective antagonists, pirenzepine, AF-DX 116, and 4-DAMP, were examined, and the results indicate that the smooth muscle cells contain at least two receptor subtypes. The majority of receptors exhibit binding constants for these selective antagonists similar to those of the M2-subtype. AF-DX 116 binding curves indicated the presence of a small population of receptors with binding constants similar to those reported for the M3-subtype. These findings suggest that the smooth muscle cells may contain both M2- and M3-receptors and are in agreement with evidence of the presence of mRNAs coding for these two subtypes in tracheal extracts (A. Maeda, T. Kubo, M. Mishina, and S. Numa. FEBS Lett. 239: 339-342, 1988). [3H]QNB displacement curves of the muscarinic agonist oxotremorine were best described as a sum of binding to high- and low-affinity sites with KD values of 3.8 nM and 2.2 microM. Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) shifted the high-affinity sites to low affinity, suggesting that the high-affinity sites may represent receptors coupled to G proteins. Pertussis toxin catalyzed the ADP ribosylation of a 40- to 41-kDa protein band present in the membranes but had no significant effect on high-affinity agonist binding, suggesting that most of the receptors are coupled to G proteins in a toxin-insensitive manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Continuous monitoring of Ca2+ uptake in membrane vesicles with fura-2.

The Ca2+-selective, fluorescent dye, fura-2, was used to monitor ATP-dependent Ca2+ uptake by membrane vesicles isolated from rabbit skeletal muscle. Micromolar fura-2 concentrations, added outside the vesicles, served as a high-affinity, low-capacity Ca2+ buffer. Changes in fura-2 fluorescence resulted from the decline in free Ca2+ concentration [( Ca2+]free) owing to active Ca2+ accumulation by the vesicles. Ca2+ uptake (delta[Ca2+]total) was calculated from changes in [Ca2+]free and from the Kd value for the fura-2-Ca2+ complex. The velocity of Ca2+ uptake determined in this manner had an apparent [Ca2+]0.5 of approximately 200 nM. The Hill coefficient for dependence of uptake velocity on [Ca2+]free was congruent to 2. Changes in [Ca2+]free and Ca2+ uptake expected for Ca2+ transport by skeletal muscle sarcoplasmic reticulum were determined theoretically from known kinetic parameters and found to be similar to experimental values. This method of directly monitoring Ca2+ uptake can be used to determine the kinetic parameters for Ca2+ transport with small amounts of vesicles and with greater precision than possible with radiometric techniques.