Cardiac Sarcolemma Research Articles

The effect of hypercholesterolemia on the sodium inward currents in cardiac myocyte

To determine whether chronic hypercholesterolemia affects sodium inward currents in cardiac myocytes, whole-cell clamp recordings were made in single cardiac myocytes isolated from normo- and hypercholesterolemic rabbits. Modification of the serum cholesterol was accomplished by feeding ten 3-month-old male New Zealand white rabbits with control diet (group I) and ten with 0.5% cholesterol-enriched diet (group II), for 3 months. The serum cholesterol levels of group II were much higher than those of group I (2042 +/- 231 v 82 +/- 9 mg/dl, P < 0.001). The cholesterol-ester and free cholesterol component of cardiac sarcolemma of group II were also significantly higher than those of group I (26.6 +/- 12.4 v 10.8 +/- 4.5 nmole/dl, P < 0.001, and 50.9 +/- 14.8 v 27.5 +/- 6.2 nmole/dl, P < 0.001, respectively). The cell capacitance of hyperlipidemic myocytes seemed larger than that of normolipidemic ones (157.4 +/- 6.4 pF v 103.6 +/- 3.0 pF, P < 0.05). However, the sodium current density on hypercholesterolemic ventricular sarcolemma was significantly lower than that of normolipidemic sarcolemma. This effect was associated with a leftward shift in the inactivation potential and a slowing of the time course of recovery. In conclusion, hypercholesterolemia has important effects on the sodium inward currents in ventricular myocytes, which may be due to a decrease in current density and an alteration in channel functional state.

Ion specificity and stoichiometry of the cardiac inositol transporter

Cardiac myocytes have a high affinity, Mg 2+-dependent, electrogenic, Na-inositol co-transporter on the plasma or sarcolemma (SL) membrane (Rubin and Hale, 1993). The ion specificity and stoichiometry of this transport process is unknown. Using bovine cardiac SL vescies, we have determined that transmembrane movement of myo-[ 3H]inositol requires Na + and is not supported by K + or Li +. Furthermore, replacing Cl − with a non-permeant anion has no effect on inositol transport. Carrier-mediated inositol efflux indicated that efflux was Na +-dependent and electrogenic but independent of extravesicular Mg 2+ in the efflux media. The transport stoichiometry of 1 Na + for one inositol was derived by determining the null point of myo-[ 3H]inositol flux under conditions where the inside and outside concentration (ratio) of myo-inositol and Na + were controlled. The results suggest that Na +, inositol, and Mg 2+ bind to the same side of the membrane for transport to occur and that the stoichiometry of Na + and inositol transport is 1:1.

Ouabain-insensitive, Na + -stimulated ATPase activity in rabbit cardiac sarcolemma

The rabbit cardiac sarcolemma shows an ouabain, Na,K-stimulated ATPase activity and an ouabain-insensitive, Na-stimulated ATPase activity. The Na-ATPase has the following characteristics: 1. (i) It is also stimulated by other monovalent cations. 2. (ii) It is inhibited by 2 mM Furosemide and by 2 mM ethacrynic acid. 3. (iii) It reaches maximal values ( V max ) at around 20 mM Na +. 4. (iv) The apparent K m is around 5 mM. Except for the monovalent cation stimulation, the main characteristics of this ATPase are very similar to those of the ouabain-insensitive, Na-stimulated ATPase of mammalian kidneys.

Inhibition of cardiac sarcolemma Na(+)-K+ ATPase by oxyradical generating systems.

The Na(+)-K+ ATPase activity and SH group content were decreased whereas malondialdehyde (MDA) content was increased upon treating the porcine cardiac sarcolemma with xanthine plus xanthine oxidase, which is known to generate superoxide and other oxyradicals. Superoxide dismutase either alone or in combination with catalase and mannitol fully prevented changes in SH group content but the xanthine plus xanthine oxidase-induced depression in Na(+)-K+ ATPase activity as well as increase in MDA content were prevented partially. The Lineweaver-Burk plot analysis of the data for Na(+)-K+ ATPase activity in the presence of different concentrations of MgATP or Na+ revealed that the xanthine plus xanthine oxidase-induced depression in the enzyme activity was associated with a decrease in Vmax and an increase in Km for MgATP; however, Ka value for Na+ was decreased. Treatment of sarcolemma with H2O2 plus Fe2+, an hydroxyl and other radical generating system, increased MDA content but decreased both Na(+)-K+ ATPase activity and SH group content; mannitol alone or in combination with catalase prevented changes in SH group content fully but the depression in Na(+)-K+ ATPase activity and increase in MDA content were prevented partially. The depression in the enzyme activity by H2O2 plus Fe2+ was associated with a decrease in Vmax and an increase in Km for MgATP. These results indicate that the depressant effect of xanthine plus xanthine oxidase on sarcolemmal Na(+)-K+ ATPase may be due to the formation of superoxide, hydroxyl and other radicals. Furthermore, the oxyradical-induced depression in Na(+)-K+ ATPase may be due to the formation of superoxide, hydroxyl and other radicals.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of linoleic acid hydroperoxide and hydroxide on calcium transport and Na, K-ATPase activity in cardiac sarcolemma

Effects of linoleic acid hydroperoxide and hydroxide on calcium transport and Na, K-ATPase activity in cardiac sarcolemma

Ca 2+-ATPase distributes differently in cardiac sarcolemma than dihydropyridine receptor α1 subunit and Na +/Ca 2+ exchanger

We have investigated the distribution of the sarcolemmal Ca 2+ transporters in hamster and dog ventricular myocytes by immunocytochemical and membrane fractionation techniques. The data suggest that the DHP receptor α1 subunit and the Na +/Ca 2+ exchanger are present in surface sarcolemma as well as T-tubule membranes located at the cardiac dyads. Compared with these Ca 2+ transporters, the sarcolemmal Ca 2+-ATPase is much less abundant in the latter fraction. Thus the sarcolemmal Ca 2+-ATPase seems to be located predominantly in surface sarcolemma.

The action of Na+ as a cofactor in the inhibition by cytoplasmic protons of the cardiac Na(+)-Ca2+ exchanger in the guinea-pig.

1. Na(+)-Ca2+ exchange current was activated in giant excised patches of guinea-pig cardiac sarcolemma by raising the intracellular sodium concentration ([Na+]i). When the pHi was simultaneously acidified to 6.4, the current was transient, dropping by 80% in 30 s. 2. Pre-exposure to a pHi of 6.4 for 15 s reduced the peak Na(+)-Ca2+ exchange current without altering the decay rate or steady-state current. Recovery from proton inhibition was seen when [Na+]i was removed for 9 s. 3. A mathematical model of Na(+)-Ca2+ exchange function reproduced the experimental results. In addition, two model-dependent predictions were seen experimentally. (i) [Na+]i-dependent 'inactivation' of Na(+)-Ca2+ exchange may arise from pHi effects. We observed experimentally that pre-exposure to acidic pHi can remove the transient current component attributed to [Na+]i-dependent 'inactivation'. (ii) self-exchange should be inhibited by acidification. This has been observed by other investigators. 4. We have hypothesized that there are two components to inhibition of the Na(+)-Ca2+ exchanger by intracellular protons, and that one is enhanced by increased [Na+]i (Doering & Lederer, 1993b). This hypothesis is supported by the data presented here and by a model of Na(+)-Ca2+ exchange behaviour in which binding of intracellular sodium to the exchanger enhances the affinity of the exchanger for inhibitory intracellular protons.

Melatonin prevents the suppression of cardiac Ca(2+)-stimulated ATPase activity induced by alloxan.

The effects of melatonin treatment on cardiac sarcolemmal membrane function were investigated in alloxan-injected rats. Ca(2+)-stimulated adenosine-triphosphatase (ATPase, Ca2+ pump) and Mg(2+)-ATPase activities were depressed significantly in sarcolemmal preparations from alloxan-injected rats compared with levels in control rats. These deficits were observed 2 days after alloxan injection, and they were accompanied by an increase in the density of voltage-sensitive calcium channels, as measured by the [3H]nitrendipine-binding assay. In a dose-dependent manner, treatment of rats with melatonin before alloxan injection significantly overcame the suppression of Ca(2+)-stimulated ATPase in cardiac sarcolemma. Melatonin (1, 5, and 10 mg/kg) overcame Ca(2+)-stimulated ATPase suppression by 13, 35, and 70%, respectively. In addition, melatonin at a dose of 10 mg/kg also prevented the suppression of the Mg(2+)-ATPase by 31%. The number of [3H]nitrendipine-binding sites was not influenced by melatonin. The patent Na(+)-K(+)-ATPase and ouabain-sensitive Na(+)-K(+)-ATPase activities were not different between the control and experimental groups. The results indicate that Ca2+ pump activity is suppressed by acute alloxan treatment, whereas the density of voltage-sensitive calcium channels is increased. These changes may be a consequence of alloxan toxicity to the cardiac sarcolemma. Melatonin, likely because of its antioxidant capacity, exerts a protective effect on heart sarcolemmal membrane function in alloxan-injected rats.

Modification of Cardiac Membrane Adenylate Cyclase Activity and GSα by NAD and Endogenous ADP-ribosyltransferase

The mechanism by which NAD stimulates cardiac adenylate cyclase was investigated. In highly purified canine cardiac sarcolemma, NAD stimulated adenylate cyclase activity in the presence of agents which activate GS (i.e. 5 mM AlF4-, 10 μM GTPγS. 10 μM GppNHp or isoproterenol plus 2 nM GTPγS). Furthermore the EC50 of isoproterenol of stimulate adenylate cyclase was reduced in the presence of NAD. In membranes incubated with [32P]-NAD, AlF4-, 10 μM GTPγS or isoproterenol plus 2 nM GTPγS produced a selective increase in the radiolabeling of a single 45-kDA protein which was identified as GSα by immunoprecipitation. Cholera toxin catalysed radiolabeling of the same protein. Neutral hydroxylamine released [32P]-ADP-ribose from GSα prelabeled in the presence of AlF4- and [32P]-NAD indicating that an arginine residue on GSα was modified by an endogenous ADP-ribosyltransferase. ADP-ribosyltransferase inhibitors, novobiocin, vitamin K1 or 3-aminobenzamide, inhibited AlF4- stimulated ADP-ribosylation of GSα and NAD potentiation of adenylate cyclase with similar efficacies. The activity responsible for NAD potentiation of adenylate cyclase and ADP-ribosylation of GSα was not removed under hypotonic or hypotonic conditions and therefore appears to be tightly membrane bound. Collectively, these observations indicate that canine cardiac sarcolemma possess and ADP-ribosyltransferase which may constitutively catalyse transfer of an ADP-ribose to activated GSα.

Differential Sensitivity of Rat Cardiac Sarcolemma and Mitochondria to Damage Induced by Lipid Peroxidation

It is well known that the sarcolemma is the organelle most susceptible to lipid peroxidative attack in the isolated membrane preparations. To determine whether this also occurs in the intact heart, we studied the effect of cumene hydroperoxide, an agent capable of initiating lipid peroxidation, on the ultrastructure and lanthanum (La) staining of isolated rat hearts perfused with HEPES buffer (pH 7.4) containing: 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 3 mM HEPES, 1.5 mM CaCl2 and 11 mM glucose. No ultrastructural alterations or intracellular deposits of La were observed in myocytes of rats perfused with HEPES buffer. Perfusion with cumene hydroperoxide (0.5 mM) for 30 min induced a release of malondialdehyde-like substance in the perfusate and a spectrum of myocardial ultrastructural alterations. La was always observed only outside the sarcolemma in myocytes with moderate damage consisting of clearing of the mitochondrial matrix and slight margination of chromatin in the nuclei. Intracellular La was found in myocytes with severe and irreversible damages consisting of fragmentation of cristae and electron-dense amorphous particles in mitochondria. La was deposited on the outer surface of the mitochondrial membranes, lipid droplets and myofilaments. These data suggest that mitochondria are more susceptible than is the sarcolemma to lipid peroxidation induced by cumene hydroperoxide in the beating rat heart.

Immunocytochemical localization of sodium-calcium exchanger in canine nephron.

The sodium-calcium exchanger was localized in tissue sections of canine kidneys with two different polyclonal antisera raised against purified sodium-calcium exchanger protein derived from dog cardiac sarcolemma. The sodium-calcium exchanger was prominent in the basolateral plasmalemma of the majority of cells in all connecting tubules. In contrast, it was observed rarely and inconspicuously over the basolateral aspects of proximal tubular cells. The sodium-calcium exchanger was undetectable in all other portions of the nephron.

Characterization of a Mg-Dependent, Na-Inositol Co-Transport Process in Cardiac Sarcolemmal Vesicles

[ 3H]- myo-Inositol transport into cardiac sarcolemmal vesicles is both a time- and Na +-dependent process. Transport was stimulated 3-fold by 1 μM valinomycin suggesting the process is electrogenic. myo-Inositol transport was dependent upon the presence of Mn 2+ or Mg 2+ but not Ca 2+. Kinetic analysis revealed a high affinity transport process that was saturable (V max, 650 ± 71 pmoles myo-inositol/mg protein/min; K m, 37.7 ± 1.3 μM) and a low affinity process that was unsaturable up to 1.0 mM substrate. Transport was not inhibited by 1.0 mM of either L-glucose, D- or L-galactose. However, D-glucose and L-fucose at 1.0 mM were inhibitory. Higher concentrations (30 mM) of each of the sugars inhibited transport. myo -Inositol transport was also inhibited by the inositol isomers (1.0 mM), D- chiro-inositol, epi-inositol and scyllo-inositol with scyllo-inositol being most effective. Kinetic analysis established sycllo -inisitol as a competitive inhibitor of cardiac myo -inositol transport, increasing the K m for substrate three-fold (122 ± 21 μM). These data indicate that inositol transport across cardiac sarcolemma is a Mg 2+-dependent Na + co-transport process that is electrogenic and stereospecific.

The cardiac Na+-Ca2+ exchanger binds to the cytoskeletal protein ankyrin

Na+-Ca2+ exchange is the major pathway of Ca2+ efflux during excitation-contraction coupling in cardiac muscle. The Na+-Ca2+ exchanger is present in cardiac transverse tubules with an apparent high density (Frank, J.S., Mottino, G., Reid, D., Molday, R. S., and Philipson, K.D. (1992) J. Cell Biol. 117, 337-345). The mechanism for this localization is unknown but may involve interactions with the cytoskeleton. In the present study, we examined the interaction of the Na+-Ca2+ exchanger with the cytoskeletal protein ankyrin. On immunoblots of isolated canine cardiac sarcolemma, an antibody raised against purified rabbit red blood cell-ankyrin (RBC-ankyrin) recognized a 220-kDa protein, which is the same size as RBC-ankyrin. Alkaline extraction of sarcolemma removed this protein. The Na+-Ca2+ exchange protein, purified from recombinant baculovirus-infected insect cells, bound 125I-labeled-RBC-ankyrin with a KD of 42 +/- 3 nm. 125I-RBC-ankyrin was co-precipitated by antibodies to the Na+-Ca2+ exchanger after preincubation with solubilized cardiac sarcolemma. Myocardial ankyrin could be localized to both surface and T-tubular sarcolemma by immunofluorescence techniques. These results demonstrate that the cardiac Na+-Ca2+ exchanger binds ankyrin with high affinity. This interaction may be important for localizing the Na+-Ca2+ exchanger to specific domains of the sarcolemma.

In vivo and in vitro effects of the pineal gland and melatonin on [Ca(2+) + Mg2+]-dependent ATPase in cardiac sarcolemma.

The possible diurnal variation in cardiac [Ca(2+) + Mg2+]-dependent ATPase (Ca2+ pump) activity and the influence of pinealectomy and melatonin on this enzyme in rat heart have been studied. Lowest levels of cardiac sarcolemmal membrane [Ca(2+) + Mg2+]-dependent ATPase activity were measured in late afternoon in rats kept under a 14:10 light:dark cycle. Late in the dark phase the enzyme activity began to increase with the rise continuing until 0900, 3 hr after light onset. These time-dependent changes in [Ca(2+) + Mg2+]-dependent ATPase activity did not occur in either pinealectomized or light-exposed rats suggesting that melatonin, secreted from the pineal gland during the night, induces the change in [Ca(2+) + Mg2+]-dependent ATPase activity, In vitro studies in which cardiac tissue was incubated in the presence of melatonin over a wide range of doses showed that this indole stimulated the Ca2+ pump. The half-maximal effect of melatonin was observed at a melatonin concentration of 28 ng/ml. These findings suggest that the daily change in [Ca(2+) + Mg2+]-dependent ATPase activity in the sarcolemma of heart tissue is a result of the circadian rhythm in pineal melatonin production and secretion. These findings may be applicable to normal cardiac physiology.

Selectivity of inhibition of Na(+)-Ca2+ exchange of heart mitochondria by benzothiazepine CGP-37157.

The objective was to determine if the benzothiazepine compound CGP-37157 selectively inhibits the Na(+)-Ca2+ exchanger of cardiac mitochondria without affecting the L-type voltage-dependent calcium channel, the Na(+)-Ca2+ exchanger, or the Na(+)-K(+)-ATPase of the cardiac sarcolemma, or the Ca(2+)-ATPase of the cardiac sarcoplasmic reticulum. Mitochondrial Na(+)-Ca2+ exchange activity was determined by monitoring intramitochondrial free [Ca2+] in isolated heart mitochondria loaded with the Ca(2+)-sensitive fluorophore fura-2. CGP-37157 inhibited the activity of mitochondrial Na(+)-Ca2+ exchange in a dose-dependent manner (IC50 0.36 microM). Calcium currents were recorded by whole-cell voltage clamp in isolated neonatal ventricular myocytes. Diltiazem was able to block the recorded current completely, thus confirming the current to be exclusively L-type. CGP-37157 had no effect on the calcium current recorded under identical conditions. CGP-37157, at concentrations < or = 10 microM, had no effect on the activities of the Na(+)-Ca2+ exchanger and Na(+)-K(+)-ATPase in isolated cardiac sarcolemmal vesicles or on activity of the Ca(2+)-ATPase in isolated cardiac sarcoplasmic reticulum vesicles. The data suggest that CGP-37157 is a potent, selective, and specific inhibitor of mitochondrial Na(+)-Ca2+ exchange at concentrations < or = 10 microM.

Sodium-potassium pump and sodium-calcium exchange in adult and neonatal canine cardiac sarcolemma

The purpose of this study was to determine whether the Na(+)-K+ pump and the Na(+)-Ca2+ exchanger, two systems thought to be important in excitation-contraction coupling in the heart, change during postnatal development. In adult and neonatal canine cardiac sarcolemmal preparations, Na(+)-K(+)-adenosine-triphosphatase (ATPase) activity and specific [3H]ouabain binding were found to be higher in the adult compared with the neonate, although Na(+)-K(+)-ATPase turnover numbers were not significantly different. Furthermore, ouabain association and dissociation rate constants, examined under a variety of conditions, were the same for both the neonate and adult preparations. These results suggest that the number of pump units per milligram sarcolemmal protein changes during postnatal development. In contrast, the initial rate of Na(+)-dependent-45Ca2+ influx, an index of Na(+)-Ca2+ exchange activity, was not significantly different in the two age groups. Additionally, the sensitivity of the exchanger to extravesicular Ca2+ and K+ was the same. In summary, during postnatal development sarcolemmal Na(+)-K(+)-ATPase and [3H]ouabain binding increase from neonate to adult, whereas Na(+)-Ca2+ exchange activity remains the same. This may result in a greater importance of Ca2+ flux through the Na(+)-Ca2+ exchange system in the neonate compared with the adult.

Kinetic and thermodynamic properties of membrane bound Ca-ATPase with low affinity to calcium in cardiac sarcolemma; response to global ischemia of the heart

Thermodynamic and kinetic properties of membrane bound Ca-ATPase with low affinity to calcium in cardiac sarcolemma were investigated with respect to the effect of global ischemia on the heart. Energy barrier for ATP hydrolysis catalyzed by the Ca-ATPase was slightly higher in hearts subjected to ischemia, as it was evident from increased values of activation energy. Ischemia also induced a time dependent decrease in the activity and maximum velocity (Vmax) value of Ca-ATPase. The depression of enzyme activity was evident as early as 15 minutes after the onset of ischemia. After 30 minutes of ischemia the decrease in Vmax slowed down, probably due to an “adaptational” decrease of the Km value for Ca-ATPase. This phenomenon may be interpreted as a mechanism by which the enzyme attempts to keep working in a situation when the supply of ATP is insufficient.

Phe-Met-Arg-Phe-NH2 (FMRFa)-related peptides inhibit Na(+)-Ca2+ exchange in cardiac sarcolemma vesicles.

The molluscan cardioexcitatory tetrapeptide FMRF-amide (Phe-Met-Arg-Phe-NH2) and related peptides inhibit Na(+)-Ca2+ exchange in calf cardiac sarcolemma vesicles. FMRFa itself has a low inhibitory potency (IC50 = 750 microM) which completely resides in its COOH-terminal RFa portion. The physiologically active analog FLRFa is 10-fold more potent (IC50 = 60 microM). Two other substitutions of the Met2 in FMRFa, by either Ile or Lys increase inhibitory potency 7- and 50-fold, respectively. The inhibitory potency increases 300-500-fold if the NH2-terminal Phe1 in FMRFa is substituted by either Val or His (IC50 = 1-2 microM). The inhibitory activity of WnLRFa (IC50 = 40 microM) is lost when either the NH2-terminal amino group is acylated or the NH2-terminal Trp1 is deleted. These data suggest that the COOH-terminal portion is essential for the basic low potency inhibition of Na(+)-Ca2+ exchange, whereas the NH2-terminal portion is important for the potentiation of the inhibitory activity. Although the IC50 values of various peptides range widely (10(-6)-10(-3) M), all of them induce a complete inhibition. The dose-response pattern of the peptide-induced inhibition is identical for the Na(+)-Ca2+ exchange and its partial reaction, the Ca(2+)-Ca2+ exchange. The inhibitory effect is reversible and affects both Nai(or Cai)-dependent 45Ca uptake and Nao-dependent 45Ca efflux, suggesting that the bidirectional movements of ions are altered. A mild pretreatment of vesicles with trypsin augments the Na(+)-Ca2+ exchange 1.5-fold but diminishes the inhibitory potency 3-4-fold, suggesting that the inhibition is mediated by an extravesicular membrane protein. The characteristics of the peptide-induced inhibition resemble the effect of opiates on Na(+)-Ca2+ exchange. FLRFa and dextrorphan (a non-opioid stereoisomer of an opiate agonist) are mutually exclusive inhibitors, suggesting that they may bind to the same site. This putative site lacks the pharmacological properties of opiate receptors and may be located either on the Na(+)-Ca2+ exchanger or at its vicinity. Endogenous analogs of FMRFa may regulate intracellular calcium via Na(+)-Ca2+ exchange.

Solubilization and purification of the prostaglandin E 2 receptor from cardiac sarcolemma

A prostaglandin E 2 (PGE 2) receptor was solubilized and isolated from cardiac sarcolemma membranes. Its binding characteristics are almost identical to those of the membrane bound receptor. [ 3H]PGE 2 binding to solubilized and membrane bound receptor was sensitive to elevated temperature and no binding was observed in the absence of NaCl. No significant effects of DTT, ATP, Mg 2+, Ca 2+ or of changes in buffer pH were observed on [ 3H]PGE 2 binding to either solubilized or membrane-bound receptor. Unlabelled PGE 1 displaced over 90% of [ 3H]PGE 2 from the CHAPS-solubilized receptor. PGD 2, PGI 2, PGF 2α and 6-keto-PGF 1α were not effective in displacing [ 3H]PGE 2 from the receptor. Scatchard analysis of [ 3H]PGE 2 binding to CHAPS-solubilized receptor revealed the presence of two types of PGE 2 binding sites with K d of 0.33 ± 0.05 nM and 3.00 ± 0.27 nM and B max of 0.5 ± 0.04 and 2.0 ± 0.1 pmol/mg of protein. The functional PGE 2 receptor was isolated from CHAPS-solubilized SL membrane using two independent methods: first by a WGA-Sepharose chromatography and second by sucrose gradient density centrifugation. Receptor isolated by these two methods bound [ 3H]PGE 2. Unlabelled PGE 1 and PGF 2 displaced [ 3H]PGE 2 from the purified receptor. Scatchard analysis of [ 3H]PGE 2 binding to purified receptor revealed the presence of the two bindingsites as observed for the membrane bound and CHAPS-solubilized receptor. SDS-polyacrylamide gel electrophoresis of the purified receptor fractions revealed the presence of a protein band of M r of approx. 100 000. This 100-kDa was photolabelled with [ 3H]azido-PGE 2, a photoactive derivative of PGE 2. We propose that this 100-kDa protein is a cardiac PGE 2 receptor.

Protein kinase C mediated activation and phosphorylation of Ca(2+)-pump in cardiac sarcolemma.

The effects of purified protein kinase C (PKC) on the Ca(2+)-pumping ATPase of cardiac sarcolemma were investigated. The addition of PKC to sarcolemmal vesicles resulted in a significant increase in ATP-dependent Ca2+ uptake, by increasing the calcium affinity by 2.8-fold (Km 0.14 vs. 0.4 microM for control) and by increasing Vmax from 5 to 6.8 nmol.mg protein-1.min-1. The addition of PKC also stimulated Ca2+ ATPase activity in sarcolemmal preparations. This activity was increased further upon the addition of calmodulin. These results suggest that PKC stimulates Ca2+ ATPase through a kinase-directed phosphorylation. The addition of PKC to a purified preparation of Ca2+ ATPase in the presence of [gamma-32P]ATP resulted in a 100% increase in phosphorylation that was dependent on the presence of Ca2+, phosphatidylserine, and phorbol 12,13-dibutyrate. These results demonstrate that the Ca2+ ATPase of canine cardiac muscle can be phosphorylated by PKC in vitro, resulting in increased affinity of the Ca2+ ATPase for Ca2+ and increase in the Ca2+ pump pumping rate. The results suggest that the Ca(2+)-pumping ATPase in heart tissue can be stimulated by PKC, thereby regulating the intracellular Ca2+ levels in whole heart.