Calcium transport by sarcoplasmic reticulum of vascular smooth muscle: II. Effects of calmodulin and calmodulin inhibitors.

The role of calmodulin (CaM) in modulating calcium (Ca) uptake by sarcoplasmic reticulum (SR) of vascular smooth muscle was studied in saponin skinned strips of rat caudal artery. Exogenous CaM concentrations ranging from 0.3-1.8 microM did not statistically change the steady state MgATP-dependent Ca content, the MgATP-independent Ca content, or the oxalate-stimulated Ca influx. Calmidazolium (CDZ), W-7, and trifluoperazine (TFP) were used to examine the potential effect of an endogenous CaM pool on inward Ca transport. The IC50 of these antagonists for inhibition of Ca-CaM-stimulated phosphodiesterase activity and Ca-activated superprecipitation of canine aortic actomyosin was measured and found to be in the low micromolar range with a rank order of potency for inhibition of CDZ greater than TFP greater than W-7. In skinned tissues, micromolar concentrations of antagonists that inhibited CaM-mediated reactions in isolated enzyme systems did not reduce Ca content or oxalate-stimulated Ca influx. At higher concentrations of 100-200 microM, the MgATP-dependent Ca content was significantly reduced by TFP and W-7 but not by CDZ. The order of potency for inhibition of Ca uptake was TFP greater than W-7 greater than CDZ. The MgATP-independent Ca content was significantly decreased only by 200 microM TFP. Although none of these inhibitors significantly altered Ca efflux at concentrations up to 100 microM, Ca release was significantly stimulated by all three at 200 microM. The TFP-stimulated Ca release was partially inhibited by ruthenium red. The results indicate that neither exogenous CaM nor an endogenous CaM pool directly modulates inward Ca transport by the SR of saponin skinned caudal artery. The inhibition of Ca uptake produced by hundred micromolar concentrations of CaM antagonists fails to correlate with the order of and with the potency of inhibition measured in isolated enzyme systems. This suggests that the inhibition of Ca uptake produced by high concentrations of these antagonists may be independent of a specific interaction with CaM. The activation of Ca release by high concentrations of CaM antagonists may involve a nonspecific increase in membrane permeability as well as modulation of a membrane Ca channel.     

MgATP-independent and MgATP-dependent exocytosis. Evidence that MgATP primes adrenal chromaffin cells to undergo exocytosis

The MgATP dependency of secretion was investigated in digitonin-permeabilized adrenal chromaffin cells. Shortly after permeabilization there is a component of Ca2+-dependent secretion that occurs in the absence of MgATP in the medium. This secretion occurs from cells which are permeable to Ca2+/[ethylene-bis(oxyethylenenitrilo)]tetraacetic acid buffers, to nucleotides, and to proteins. It is prevented by treatment of cells with metabolic inhibitors to reduce cellular ATP prior to permeabilization. The rate of MgATP-independent secretion is rapid and terminates by approximately 2 min after introduction of Ca2+. MgATP-independent secretion is labile and is lost unless Ca2+ is introduced within 8 min of permeabilization. MgATP-dependent secretion occurs at a slower rate than MgATP-independent secretion and continues at a constant rate for 12 min. Preincubation of permeabilized cells with MgATP enhances Ca2+-dependent secretion during a subsequent incubation in the absence of MgATP. Similar MgATP sensitivities are observed when MgATP is present only prior to or only during stimulation with Ca2+ with half-maximal stimulation occurring at 0.4-0.5 and 0.6 mM MgATP, respectively. The data indicate that intact cells are primed by intracellular ATP so that immediately upon permeabilization, there is a component of secretion which is independent of medium MgATP. MgATP partially maintains the primed state after permeabilization by acting before Ca2+ in the secretory pathway.     

Spermine. A regulator of mitochondrial calcium cycling

Steady-state free Ca2+ concentrations have been measured with a Ca2+ electrode using suspensions of isolated rat liver mitochondria or saponin-treated hepatocytes. Mitochondria, when incubated in the presence of Mg2+ and MgATP2-, maintain a steady-state pCa2+ (-log [Ca2+]) of approximately 6.1 (0.8 microM). Addition of spermine lowered this value to a pCa2+ of 6.6 (0.25 microM). Spermine was the most effective polyamine, giving half-maximal effects at 170 microM and maximal effects at 400 microM. With saponin-permeabilized hepatocytes, spermine addition similarly showed that the mitochondria buffered the steady-state medium-free Ca2+ at a level approximating the cytosolic free Ca2+ concentration of intact hepatocytes. The initial rate of Ca2+ uptake by the mitochondrial Ca2+ uniporter was investigated using Ca2+-depleted mitochondria incubated in the presence of succinate and 0.3 mM free Mg2+. Under control conditions, Ca2+ uptake was not observed at free Ca2+ concentrations below 0.5 microM. Spermine (350 microM) increased the rate of Ca2+ uptake at all Ca2+ concentrations below 4.5 microM, but at higher Ca2+ concentrations, it was inhibitory. Spermine also affected mitochondrial Ca2+ efflux by decreasing the apparent Km from 16 to 3.8 nmol of Ca2+/mg of mitochondrial protein with no change of Vmax. Experiments with 45Ca2+ confirmed that spermine increased mitochondrial Ca2+ cycling at 0.2 microM free Ca2+. Hepatic spermine contents are reported to be about 1 mumol/g, wet weight, suggesting that this polyamine may have an important physiological role in intracellular calcium homeostasis.     

Uptake and release of calcium by canine gastric corpus smooth muscle plasma membrane enriched fraction

Calcium movements across plasma membrane enriched vesicles isolated from canine gastric corpus smooth muscle were investigated. The ATP-dependent Ca2+ uptake increased with time up to 10 min. The uptake for the initial 2-min period was approximately linear with time. The apparent initial velocity of the ATP-dependent Ca2+ uptake increased monotonically with free Ca2+ concentration from 0.1 to 2 microM, and further increases in free Ca2+ concentration did not increase the Ca2+ uptake. The free Ca2+ dependence curve could be described with a Hill coefficient of approximately 1.0 and Km of 0.85 +/- 0.01 microM for free Ca2+ concentration. Passive Ca2+ uptake (reaction time = 1 h) also increased with increasing free Ca2+ concentrations from 0.02 to 4.0 mM. Dilution of loaded vesicles in isotonic media containing EGTA led to initial rapid loss (less than 1 min) followed by a slower release which showed simple exponential decay. The t 1/2 values of the slower Ca2+ loss from these vesicles were 16.1 +/- 0.9 min (actively loaded n = 5) and 18.4 +/- 0.9 min (passively loaded n = 3), respectively. Dilution in isotonic medium containing both EGTA and A23187 released all the sequestered Ca2+ from these loaded vesicles.     

Calcium accumulation and release by the sarcoplasmic reticulum of digitonin-lysed adult mammalian ventricular cardiomyocytes.

We have developed a model for characterizing calcium handling by the intact cardiac sarcoplasmic reticulum (SR) that yields data consistent with both mathematical simulations of in situ SR Ca2+ uptake and deduced behavior of the Ca2(+)-induced Ca2+ efflux channels in mechanically skinned single cardiac cells. In Na(+)-based media (37 degrees C, pH 7.2, 50 mM Pi, 10 mM MgATP, pMg 3.3, 10 mM phosphocreatine), SR 45Ca2+ uptake by digitonin-lysed rat myocytes as a function of free [Ca2+] peaked at pCa 6.2, declined until pCa 5.6 and increased again at lower pCa. When Ca2(+)-induced Ca2+ efflux was inhibited with 30 microM ruthenium red and 10 mM procaine, uptake was saturable with a Vmax of 160 +/- 5 nmol.min-1.mg-1, K0.5 of 500 nM free [Ca2+] and slope factor of 1.6. In K(+)-based media, maximum Pi- and oxalate-supported uptake increased to 220 and 260 nmol.min-1.mg-1, respectively. Without phosphocreatine, 45Ca2+ uptake declined under all conditions; this was correlated with a decrease in ATP/ADP. Vmax for 45Ca2+ uptake was increased 20% in hyperthyroid myocytes but depressed 30% in myocytes from heart failure-prone rats. In canine myocytes, Vmax was the same as in normal rat cells, but K0.5 was 830 nM. Without efflux inhibitors, ryanodine caused a concentration-dependent decline in net Pi-supported 45Ca2+ uptake at pCa 6.3 (K0.5 = 1 microM), while 10 microM ryanodine depressed uptake at all pCa between 7.2 and 5.6. Ruthenium red/procaine fully reversed this effect.     

The bulk of Ca2+ released to the myoplasm is free in the sarcoplasmic reticulum and does not unbind from calsequestrin

Calsequestrin (CS) is the major Ca2+ binding protein contained in the lumen of sarcoplasmic reticulum (SR). Ca2+ binding properties and tissue concentration of CS of frog skeletal muscle were measured. At equilibrium, maximal Ca2+ binding capacity of purified CS was about 1.2 mumol Ca2+/mg protein. Apparent Kds for Ca2+ were around 50 microM in the absence of salts, around 0.9 mM in the presence of 100 mM KCl, and around 1.1 mM under 'physiological' conditions. Quantitation of CS in homogenates was accomplished by three methods (Stains-all staining, immunoblotting and 45Ca ligand overlay). Frog muscle contained about 0.5 mg of CS/g wet weight, that is 6.1 mM CS inside the SR. At rest the in situ free [Ca2+] of SR was calculated to be 3.6 mM, and, thus, CS is largely saturated with Ca2+. Moreover, computer simulations of Ca2+ release indicated that about 75% of Ca2+ released during a twitch is free in the SR and does not unbind from CS.     

Quantification of calsequestrin 2 (CSQ2) in sheep cardiac muscle and Ca2+-binding protein changes in CSQ2 knockout mice

Calsequestrin 2 (CSQ2) is generally regarded as the primary Ca2+-buffering molecule present inside the sarcoplasmic reticulum (SR) in cardiac cells, but findings from CSQ2 knockout experiments raise major questions about its role and necessity. This study determined the absolute amount of CSQ2 present in cardiac ventricular muscle to gauge its likely influence on SR free Ca2+ concentration ([Ca2+]) and maximal Ca2+ capacity. Ventricular tissue from hearts of freshly killed sheep was examined by SDS-PAGE without any fractionation, and CSQ2 was detected by Western blotting; this method avoided the >90% loss of CSQ2 occurring with usual fractionation procedures. Band intensities were compared against those for purified CSQ2 run on the same blots. Fidelity of quantification was verified by demonstrating that CSQ2 added to homogenates was detected with equal efficacy as purified CSQ2 alone. Ventricular tissue from sheep (n=8) contained 24±2 μmol CSQ2/kg wet wt. Total Ca2+ content of the ventricular tissue, measured by atomic absorption spectroscopy, was 430±20 μmol/kg (with SR Ca2+ likely<250 μmol/kg) and displayed a linear correlation with CSQ2 content, with gradient of ～10 Ca2+ per CSQ2. The large amount of CSQ2 bestows the SR with a high theoretical maximal Ca2+-binding capacity (～1 mmol Ca2+/kg ventricular tissue, assuming a maximum of ～40 Ca2+ per CSQ2) and would keep free [Ca2+] within the SR relatively low, energetically favoring Ca2+ uptake and reducing SR leak. In mice with CSQ2 ablated, histidine-rich Ca2+-binding protein was upregulated ～35% in ventricular tissue, possibly in compensation.     

Stimulatory effect of glucose 6-phosphate on the non-mitochondrial Ca2+ uptake in permeabilized hepatocytes and Ca2+ release by inositol trisphosphate

The relationships between Ca2+ transport and glucose-6-phosphatase activity, previously studied in isolated liver microsomes, were investigated in permeabilized hepatocytes in the presence of mitochondrial inhibitors. It was found that the addition of glucose 6-phosphate to the cells markedly stimulates the MgATP-dependent Ca2+ uptake. A progressive increase in the stimulation of Ca2+ uptake was seen with increasing amounts of glucose 6-phosphate up to 5 mM concentrations. Vanadate, when added in adequate concentrations (20-40 microM) to the hepatocytes inhibits both the glucose-6-phosphatase activity and the stimulation of Ca2+ uptake by glucose 6-phosphate, while not affecting the MgATP-dependent Ca2+ uptake. The addition of inositol 1,4,5-trisphosphate to permeabilized hepatocytes in which Ca2+ had been accumulated in the presence of MgATP and glucose 6-phosphate, results in a rapid release of Ca2+.     

Inactivation of a Ca2+-induced Ca2+ release channel from skeletal muscle sarcoplasmic reticulum during active Ca2+ transport

ATP-dependent Ca2+ uptake by subfractions of skeletal muscle sarcoplasmic reticulum (SR) was studied with the Ca2+ indicator dye, antipyrylazo III. Ca2+ uptake by heavy SR showed two phases, a slow uptake phase and a fast uptake phase. By contrast, Ca2+ uptake by light SR exhibited a monophasic time course. In both fractions a steady state of Ca2+ uptake was observed when the concentration of free Ca2+ outside the vesicles was reduced to less than 0.1 microM. In the steady state, the addition of 5 microM Ca2+ to the external medium triggered rapid Ca2+ release from heavy SR but not from light SR, indicating that the heavy fraction contains a Ca2+-induced Ca2+ release channel. During Ca2+ uptake, heavy SR showed a constant Ca2+-dependent ATPase activity (1 mumol/mg protein X min) which was about 150 times higher than the rate of Ca2+ uptake in the slow uptake phase. Ruthenium red, an inhibitor of Ca2+-induced Ca2+ release, enhanced the rate of Ca2+ uptake during the slow phase without affecting Ca2+-dependent ATPase activity. Adenine nucleotides, activators of Ca2+ release, reduced the Ca2+ uptake rate. These results suggest that the rate of Ca2+ accumulation by heavy SR is not proportional to ATPase activity during the slow uptake phase due to the activation of the channel for Ca2+-induced Ca2+ release. In addition, they suggest that the release channel is inactivated during the fast Ca2+ uptake phase.     

Effects of MgATP, MgADP, and Pi on actin movement by smooth muscle myosin.

To test the idea that the in vitro motility assay is a simplified model system for muscle contraction, the MgATP-dependent movement of actin filaments by thiophosphorylated smooth muscle myosin was characterized in the presence of the products MgADP and inorganic phosphate. The dependence of actin filament velocity on MgATP concentration was hyperbolic with a maximum velocity of 0.6 micron/s and an apparent Km = 40 microM (30 degrees C). MgADP competitively inhibited actin movement by MgATP with a Ki = 0.25 mM. Inorganic phosphate did not affect actin filament velocity in the presence of 1 mM MgATP, but competitively inhibited movement in the presence of 50 microM MgATP with a Ki = 9.5 mM. The effects of ADP and Pi on velocity agree with fiber mechanical studies, confirming that the motility assay is an excellent system to investigate the molecular mechanisms of force generation and shortening in smooth muscle. The rate at which rigor cross-bridges can be recruited to move actin filaments was observed by initiating cross-bridge cycling from rigor by flash photolysis of caged MgATP. Following the flash, which results in a rapid increase in MgATP concentration, actin filaments experienced a MgATP-dependent delay prior to achieving steady state velocity. The delay at low MgATP concentrations was interpreted as evidence that motion generating cross-bridges are slowed by a load due to a transiently high percentage of rigor cross-bridges immediately following MgATP release.     

Assessment of intra-SR free [Ca] and buffering in rat heart.

To measure the free intrasarcoplasmic reticulum [Ca] ([Ca]SR) in isolated rat cardiac microsomes, ventricular tissue was homogenized in the presence of the low-affinity Ca indicator furaptra. Stepwise increases in cuvette [Ca] ([Ca]c) in the presence of ATP caused progressive increases in steady-state intravesicular fluorescence ratio to a maximum (Rmax). Steady-state [Ca]SR/[Ca]c was approximately 7000. Therefore the resting [Ca]SR may approach 700 microM in the rat cardiac myocyte at [Ca]c = 100 nM. The sarcoplasmic reticulum (SR) Ca pump requires a free energy of deltaG approximately 44 kJ x mol(-1) to generate this [Ca] gradient (e.g., approximately 74% of deltaG(ATP)). Total SR 45Ca uptake was also measured in digitonin-permeabilized myocytes as a function of [Ca]c in the absence of precipitating ions. The steady-state SR Ca content at 100 nM [Ca]c was approximately 400 micromol/liter cytosolic volume. Used together, these data allowed evaluation of the in situ SR Ca-buffering properties. The SR Ca-binding site concentration was approximately 14 mM, and Kd(Ca) approximately 0.638 mM [Ca]SR.     

Abnormal rapid Ca2+ release from sarcoplasmic reticulum of malignant hyperthermia susceptible pigs.

Using the rapid filtration technique to investigate Ca2+ movements across the sarcoplasmic reticulum (SR) membrane, we compare the initial phases of Ca2+ release and Ca2+ uptake in malignant hyperthermia susceptible (MHS) and normal (N) pig SR vesicles. Ca2+ release is measured from passively loaded SR vesicles. MHS SR vesicles present a 2-fold increase in the initial rate of calcium release induced by 0.3 microM Ca2+ (20.1 +/- 2.1 vs. 6.3 +/- 2.6 nmol mg-1 s-1). Maximal Ca2+ release is obtained with 3 microM Ca2+. At this optimal concentration, rate of Ca2+ efflux in absence of ATP is 55 and 25 nmol mg-1 s-1 for MHS and N SR, respectively. Ca(2+)-induced Ca2+ release is inhibited by Mg2+ in a dose-dependent manner for both MHS and N pig SR vesicles (K1/2 = 0.2 mM). Caffeine (5 mM) and halothane (0.01% v/v) increase the Ca2+ sensitivity of Ca(2+)-induced Ca2+ release. ATP (5 mM) strongly enhances the rate of Ca2+ efflux (to about 20-40-fold in both MHS and N pig SR vesicles). Furthermore, both types of vesicles do not differ in their high-affinity site for ryanodine (Kd = 12 nM and Bmax = 6 pmol/mg), lipid content, ATPase activity and initial rate of Ca2+ uptake (0.948 +/- 0.034 vs. 0.835 +/- 0.130 mumol mg-1 min-1 for MHS and N SR, respectively). Our results show that MH syndrome is associated to a higher rate of Ca2+ release in the earliest phase of the calcium efflux.     

Intrinsic cytosolic calcium buffering properties of single rat cardiac myocytes.

Intracellular passive Ca2+, buffering was measured in voltage-clamped rat ventricular myocytes. Cells were loaded with indo-1 (K+ salt) to an estimated cytosolic concentration of 44 +/- 5 microM (Mean +/- SEM, n = 5), and accessible cell volume was estimated to be 24.5 +/- 3.6 pl. Ca2+ transport by the sarcoplasmic reticulum (SR) Ca-ATPase and sarcolemmal Na-Ca exchange was inhibited by treatment with thapsigargin and Na-free solutions, respectively. Extracellular [Ca2+] was maintained at 10 mM and, in some experiments, the mitochondrial uncoupler "1799" was used to assess the degree of mitochondrial Ca2+ uptake. To perform single cell titrations, intracellular Ca2+ ([Ca2+]i) was increased progressively by a train of depolarizing voltage clamp pulses from -40 to +10 mV. The total Ca2+ gain with each pulse was calculated by integration of the Ca current and then analyzed as a function of the rapid change in [Ca2+]i during the pulse. In the range of [Ca2+]i from 0.1 to 2 microM, overall cell buffering was well described as a single lumped Michaelis-Menten type species with an apparent dissociation constant, KD, of of 0.63 +/- 0.07 microM (n = 5) and a binding capacity, Bmax, of 162 +/- 15 mumol/l cell H2O. Correction for buffering attributable to cytosolic indo-1 gives intrinsic cytosolic Ca2+ buffering parameters of KD = 0.96 +/- 0.18 microM and Bmax = 123 +/- 18 mumol/l cell H2O. The fast Ca2+ buffering measured in this manner agrees reasonably with the characteristics of known rapid Ca buffers (e.g., troponin C, calmodulin, and SR Ca-ATPase), but is only about half of the total Ca2+ buffering measured at equilibrium. Inclusion of slow Ca buffers such as the Ca/Mg sites on troponin C and myosin can account for the differences between fast Ca2+ buffering in phase with the Ca current measured in the present experiments and equilibrium Ca2+ buffering. The present data indicate that a rapid rise of [Ca2+]i from 0.1 to 1 microM during a contraction requires approximately 50 microM Ca2+ to be added to the cytosol.     

MgATP-dependent accumulation of calcium ions and inorganic phosphate in a liver reticular pool.

1. MgATP-dependent Ca2+ uptake by rat liver microsomal preparations and permeabilized hepatocytes was measured in the presence or absence of Pi. 2. Monitoring of free Ca2+ in incubation systems with a Ca2+ electrode in the presence of Pi (2-7 mM) revealed a biphasic Ca2+ uptake, with the onset of a second, Pi-dependent, Ca2+ accumulation. 3. Increasing Pi concentrations (up to 10 mM) caused a progressive enlargement of 45Ca2(+)-loading capacity of microsomal fractions. 4. As a result of Pi stimulation of active Ca2+ uptake, [32P]Pi and 45Ca2+ were co-accumulated. 5. Experiments with permeabilized hepatocytes revealed that the amount of Ca2+ releasable by myo-inositol 1,4,5-trisphosphate is unaffected by Pi.     

Inositol trisphosphate does not release Ca2+ from permeabilized cardiac myocytes and sarcoplasmic reticulum

The possibility that inositol 1,4,5-trisphosphate (IP3) may act as a Ca2+-mobilizing second messenger in cardiac muscle in a manner analogous to its actions in other cell types has been examined using saponin-permeabilized myocytes and isolated cardiac sarcoplasmic reticulum. Myocytes permeabilized in the presence of MgATP2- sequestered Ca2+ to a level of about 200 nM, similar to the cytosolic free Ca2+ concentration of intact cells, but addition of IP3 was ineffective in causing Ca2+ release from intracellular stores. Similarly, IP3 (up to 50 microM) was unable to inhibit Ca2+ uptake or cause Ca2+ release from isolated canine cardiac sarcoplasmic reticulum vesicles in the presence of either EGTA or sodium vanadate. These results indicate that IP3 is unlikely to mediate mobilization of intracellular Ca2+ stores in myocardial cells.     

Effects of 4-h ischemia and 1-h reperfusion on rat muscle sarcoplasmic reticulum function

To investigate the hypothesis that ischemia and reperfusion would impair sarcoplasmic reticulum (SR) Ca(2+) regulation in skeletal muscle, Sprague-Dawley rats (n = 20) weighing 290 +/- 3.5 g were randomly assigned to either a control control (CC) group, in which only the effects of anesthetization were studied, or to a group in which the muscles in one hindlimb were made ischemic for 4 h and allowed to recover for 1 h (I). The nonischemic, contralateral muscles served as control (C). Measurements of Ca(2+)-ATPase properties in homogenates and SR vesicles, in mixed gastrocnemius and tibialis anterior muscles, indicated no differences between groups on maximal activity, the Hill coefficient, and Ca(50), defined as the Ca(2+) concentration needed to elicit 50% of maximal activity. In homogenates, Ca(2+) uptake was lower (P < 0.05) by 20-25%, measured at 0.5 and 1.0 microM of free Ca(2+) ([Ca(2+)](f)) in C compared with CC. In SR vesicles, Ca(2+) uptake was lower (P < 0.05) by 30-38% in I compared with CC at [Ca(2+)](f) between 0.5 and 1.5 microM. Silver nitrate induced Ca(2+) release, assessed during both the initial, early rapid (phase 1), and slower, prolonged late (phase 2) phases, in homogenates and SR vesicles, indicated a higher (P < 0.05) release only in phase 1 in SR vesicles in I compared with CC. These results indicate that the alterations in SR Ca(2+) regulation, previously observed after prolonged ischemia by our group, are reversed within 1 h of reperfusion. However, the lower Ca(2+) uptake observed in long-term, nonischemic homogenates suggests that altered regulation may occur in the absence of ischemia.     

Ca2+ uptake in bovine adrenocortical microsomes: formation of phosphorylated intermediate of Ca2+ dependent ATPase

Bovine adrenocortical microsomes were prepared and partially purified by discontinuous sucrose density gradient. Light fractions of the microsomes at the interface between 15 and 30% sucrose solution, exhibited ATP dependent Ca2+ uptake. The Ca2+ uptake was dependent on temperature and stimulated by free Ca2+ (the concentration for half maximal activation = 1.0 microM) and Mg2+. The Ca2+ uptake was inhibited by ADP but not affected by 10 mM NaN3 or 0.5 mM ouabain. Calcium release from the microsomes was accelerated by a Ca2+ ionophore, A23187, but not by a Ca2+ antagonist, diltiazem. A microsomal protein with a molecular weight of 100-110 kDa was phosphorylated by [gamma-32P]ATP in the presence of Ca2+, and the Ca2+ dependency was over the same range as the Ca2+ uptake (the concentration for half maximal activation = 3.0 microM). The phosphorylated protein (EP) was stable at acidic pH but labile at alkaline pH and sensitive to hydroxylamine. The rate of EP formation at 0 degrees C in the presence of 1 microM ATP and 10 microM Ca2+ (half time = 0.2 s) was less than that in the sarcoplasmic reticulum (SR) of rabbit skeletal muscle (half time = 0.1 s). The rate of EP decomposition at 0 degrees C after adding EGTA was about 6.7 times slower (rate constant: kd = 4.3 X 10(-3) s-1) than that of SR. It was suggested that adrenocortical microsomes contain a Ca2+ dependent ATPase which function as a Ca2+ pump with similar properties to that of SR.     

Calcium homeostasis in procyclic and bloodstream forms of Trypanosoma brucei. Lack of inositol 1,4,5-trisphosphate-sensitive Ca2+ release.

When Trypanosoma brucei procyclic trypomastigotes were permeabilized with digitonin in a reaction medium containing MgATP, succinate, and 3.5 microM free Ca2+, they lowered the medium Ca2+ concentration to the submicromolar level (0.05-0.1 microM), a range that correlates favorably with that detected in the intact cells with fura-2. The carbonyl cyanide p-trifluoromethoxyphenylhydrazone-insensitive Ca2+ uptake, certainly represented by the endoplasmic reticulum, was completely inhibited by 500 microM vanadate. When vanadate instead of carbonyl cyanide p-trifluoromethoxyphenylhydrazone was present, the Ca2+ set point was increased to 0.6-0.7 microM. The succinate dependence and carbonyl cyanide p-trifluoromethoxyphenylhydrazone sensitivity of the later Ca2+ uptake indicate that it may be exerted by the mitochondria. When bloodstream trypomastigotes were used, neither succinate nor alpha-glycerophosphate stimulated the mitochondrial Ca2+ uptake. The mitochondrial Ca2+ transport could be measured only in the presence of ATP and 500 microM vanadate to inhibit the endoplasmic reticulum uptake. Bloodstream trypomastigotes have a lower cytosolic Ca2+ concentration, as detected with fura-2 and a smaller extramitochondrial Ca2+ pool than procyclic trypomastigotes. Despite the presence of inositol phosphates, as determined by [3H]inositol incorporation, and the large extramitochondrial Ca2+ pool of procyclic trypomastigotes (61.7 nmol of Ca2+/mg of protein), no inositol 1,4,5-trisphosphate-sensitive Ca2+ release could be detected in these parasites.     

Tricyclic antidepressant amitriptyline alters sarcoplasmic reticulum calcium handling in ventricular myocytes

Tricyclic antidepressants such as amitriptyline (AMT) have been reported to have adverse side effects on cardiac performance. AMT effects on Ca handling in ventricular myocytes, however, are not well understood. Therefore, we investigated AMT action on sarcoplasmic reticulum (SR) Ca release in ventricular myocytes, ryanodine receptor (RyR) activity, and Ca uptake by SR microsomes. In permeabilized myocytes, AMT transiently increased free luminal Ca concentration ([Ca]) followed by marked depletion. AMT (10 microM) caused a rapid and a transient increase of Ca spark frequency, followed by a significant suppression of spark activity. The latter was associated with a decrease of Ca spark amplitude and SR Ca load to 87 and 60%, respectively. AMT (10 microM) completely abolished propagation of spontaneous Ca waves. Higher concentrations of AMT (0.1-1 mM) evoked SR Ca release reminiscent of the effect of caffeine (20 mM) and caused almost complete depletion of SR Ca content. Studies on single calsequestrin-free RyR channels revealed that AMT increased the mean open time and open probability (Po) in a dose-dependent fashion (dissociation constant = 4.2 microM). High concentrations of AMT (> 25 microM) evoked frequent long openings with Po reaching very high levels (> 0.70). In studies with cardiac SR microsomes, AMT slowed the rate of ATP-dependent Ca uptake. We conclude that AMT affects SR Ca handling in ventricular myocytes by multiple mechanisms, including direct stimulation of RyRs and inhibition of SR Ca uptake. These effects could contribute to AMT cardiotoxicity.     

Involvement of the 60 kDa phosphoprotein in the regulation of Ca2+ release from sarcoplasmic reticulum of normal and malignant hyperthermia susceptible pig muscles

Junctional sarcoplasmic reticulum (SR) vesicles isolated from back muscles of normal and malignant hyperthermia susceptible (MHS) pigs were phosphorylated by addition of MgATP in the presence of 5 mM Ca2+ and 1 microM calmodulin (CaM). The major site of phosphorylation was a 60 kDa protein both in normal and MHS SR. The maximal amount of phosphorylation in MHS SR (5 pmol P/mg SR) was significantly lower than that in the normal SR (12 pmol P/mg SR). The phosphorylated 60 kDa protein was spontaneously dephosphorylated both in normal and MHS SR. Ca2+ release from the passively loaded SR was induced by a Ca2+-jump, and monitored by stopped-flow fluorometry using chlorotetracycline. In the absence of preincubation with MgATP, no significant difference was found in any of the kinetic parameters of Ca2+ release between normal and MHS SR. Upon addition of 20 microM MgATP to the passively loaded SR to phosphorylate the 60 kDa protein, the initial rate of Ca2+ release in normal SR significantly decreased from 659 +/- 102 to 361 +/- 105 nmol Ca2+/mg SR per s, whereas in MHS SR the rate decreased from 749 +/- 124 to 652 +/- 179 nmol Ca2+/mg SR per s. Addition of 20 microM adenosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppA) did not significantly alter the initial rate of Ca2+ release both in normal and MHS SR. These results suggest that the previously reported higher Ca2+ release rate in MHS SR (Kim et al. (1984) Biochim. Biophys. Acta 775, 320-327) is at least partly due to the reduced extent of the Ca2+/CaM-dependent phosphorylation of the 60 kDa protein. Two-dimensional gel electrophoresis study showed that amount of a protein with Mr = 55,000 was significantly lower in MHS SR than in normal SR suggesting that the abnormally lower amount of 55 kDa protein would cause the lower amount of phosphorylation of the 60 kDa protein in MHS SR.