Influence of Mg ions and spermine on ATP-dependent Ca2+ transport in myometrial intracellular structures. II. Comparative study of spermine, Mg ions and cyclosporin A effects on Ca2+ transport in mitochondria

In experiments carried out with the use of the radioactive label (45Ca2+) on suspension of the rat uterus myocytes processed by digitonin solution (0.1 mg/ml), influence of spermine and cyclosporin A on Mg2+, ATP-dependent Ca2+ transport in mitochondria at different Mg2+ concentration were investigated. Ca2+ accumulation in mitochondria was tested as such which was not sensitive to thapsigargin (100 nM) and was blocked by ruthenium red (10 microM). It has been shown, that spermine (1 mM) stimulates Mg2+, ATP-dependent Ca2+ accumulation in mitochondria irrespective of Mg2+ concentration (3 or 7 mM) in the incubation medium. At the same time cyclosporin A (5 microM) effects on Ca2+ accumulation in mitochondria depend on Mg2+ concentration in the incubation medium: at 3 mM Mg2+ the stimulating effect was observed, and at 7 mM Mg2+ - the inhibitory one. In conditions which led to the increase of nonspecific mitochondrial permeability and, accordingly, to dissipation of electrochemical potential (it was reached by 5 min. preincubation of myocytes suspension in the medium that contained 10 microM Ca2+, 2 mM phosphate and 3 or 7 mM Mg2+, but not ATP) significant inhibition of Mg2+, ATP-dependent Ca2+ accumulation in mitochondria was observed. The inhibition to the greater degree was observed when medium ATP and Mg2+ were absent simultaneously in the preincubation. Thus the quality of spermine effects on Ca2+ accumulation was kept: stimulation in the presence both of 3 mM and 7 mM Mg2+. Ca2+ accumulation did not reach the control level when 3 mM Mg2+ and 1 mM spermine was present and ATP absent in the preincubation medium. However, in the presence of 7 mM Mg2+ and 1 mM spermine practically full restoration (up to a control level) of Ca2+ accumulation was observed. At the same time with other things being equal such restoration was not observed at simultaneous absence of ATP and Mg2+ in the preincubation medium. The quality of cyclosporin A effects on Ca2+ accumulation in mitochondria was also kept: stimulation - in the presence of 3 mM Mg2+, inhibition - in the presence of 7 mM Mg2+ in the preincubation medium. And, at last, in the presence of cyclosporin A irrespective of the fact which preincubation medium was used, Ca2+ accumulation level practically did not depend on Mg2+ concentration.     

Utilization of purified myometrium cell plasma membrane Ca2+, Mg2+-ATPase for comparative estimation of the efficiency of energy-dependent Ca2+-transport inhibitors

With the aim of comparative estimation of efficacy of well-known inhibitors of energy-dependent Ca(2+)-transporting systems their effects were investigated on the activity of purified Ca2+, Mg(2+)-ATPase of the myometrium cell plasma membranes. From the approved inhibitors (eosin Y, o-vanadate, thapsigargin, cyclopiazonic acid, ruthenium red, sodium azide) only eosin Y and o-vanadate are potent inhibitors of myometrium sarcolemma Ca(2+)-pump: the values of Ki equal 0.8 and 4.7 microM, respectively. Thapsigargin and cyclopiazonic acid as well as ruthenium red in concentrations inhibiting, respectively, endo(sarco)plasmic reticulum Ca(2+)-pump and energy-dependent Ca(2+)-transport in mitochondria had no effect on the Ca2+, Mg(2+)-ATPase of the uterus smooth muscle cell plasma membrane. Sodium azide (10 mM) blocking completely Ca(2+)-transport in mitochondria inhibited activity of the plasma membrane Ca(2+)-transporting ATPase by 14%.     

Single channel and 45Ca2+ flux measurements of the cardiac sarcoplasmic reticulum calcium channel.

Purified canine cardiac sarcoplasmic reticulum vesicles were passively loaded with 45CaCl2 and assayed for Ca2+ releasing activity according to a rapid quench protocol. Ca2+ release from a subpopulation of vesicles was found to be activated by micromolar Ca2+ and millimolar adenine nucleotides, and inhibited by millimolar Mg2+ and micromolar ruthenium red. 45Ca2+ release in the presence of 10 microM free Ca2+ gave a half-time for efflux of 20 ms. Addition of 5 mM ATP to 10 microM free Ca2+ increased efflux twofold (t1/2 = 10 ms). A high-conductance calcium-conducting channel was incorporated into planar lipid bilayers from the purified cardiac sarcoplasmic reticulum fractions. The channel displayed a unitary conductance of 75 +/- 3 pS in 53 mM trans Ca2+ and was selective for Ca2+ vs. Tris+ by a ratio of 8.74. The channel was dependent on cis Ca2+ for activity and was also stimulated by millimolar ATP. Micromolar ruthenium red and millimolar Mg2+ were inhibitory, and reduced open probability in single-channel recordings. These studies suggest that cardiac sarcoplasmic reticulum contains a high-conductance Ca2+ channel that releases Ca2+ with rates significant to excitation-contraction coupling.     

Adenine nucleotide stimulation of Ca2+-induced Ca2+ release in sarcoplasmic reticulum

Rabbit skeletal muscle sarcoplasmic reticulum was fractionated into a "Ca2+-release" and "control" fraction by differential and sucrose gradient centrifugation. External Ca2+ (2-20 microM) caused the release of 40 nmol of 45Ca2+/mg of protein/s from Ca2+-release vesicles passively loaded at pH 6.8 with an internal half-saturation Ca2+ concentration of 10-20 mM. Ca2+-induced Ca2+ release had an approximate pK value of 6.6 and was half-maximally inhibited at an external Ca2+ concentration of 2 X 10(-4) M and Mg2+ concentration of 7 X 10(-5) M. 45Ca2+ efflux from control vesicles was slightly inhibited at external Ca2+ concentrations that stimulated the rapid release of Ca2+ from Ca2+-release vesicles. Adenine, adenosine, and derived nucleotides caused stimulation of Ca2+-induced Ca2+ release in media containing a "physiological" free Mg2+ concentration of 0.6 mM. At a concentration of 1 mM, the order of effectiveness was AMP-PCP greater than cAMP approximately AMP approximately ADP greater than adenine greater than adenosine. Other nucleoside triphosphates and caffeine were minimally effective in increasing 45Ca2+ efflux from passively loaded Ca2+-release vesicles. La3+, ruthenium red, and procaine inhibited Ca2+-induced Ca2+ release. Ca2+ flux studies with actively loaded vesicles also indicated that a subpopulation of sarcoplasmic reticulum vesicles contains a Ca2+ permeation system that is activated by adenine nucleotides.     

Rapid filtration studies of Ca2+-induced Ca2+ release from skeletal sarcoplasmic reticulum. Role of monovalent ions

We have developed a rapid filtration technique for the measurement of Ca2+ release from isolated sarcoplasmic reticulum vesicles. Using this technique, we have studied the Ca2+-induced Ca2+ release of sarcoplasmic reticulum vesicles from rabbit skeletal muscle passively loaded with 5 mM Ca2+. The effect of known effectors (adenine nucleotides and caffeine) and inhibitors (Mg2+ and ruthenium red) of this release were investigated. In a medium composed of 100 mM KCl buffered at pH 6.8 with 20 mM K/3-(N-morpholino)propanesulfonic acid the Ca2+ release rate was maximal (500 nmol of Ca2+ released.(mg of protein)-1.s-1) at 1 micron external Ca2+ and 5 mM ATP. We also observed a rapid Ca2+ release induced by micromolar Ag+ in the presence of ATP (at 1 nM Ca2+). The Ag+-induced Ca2+ release was totally inhibited by 5 micron ruthenium red. We have also investigated the effect of monovalent ions on the Ca2+ release elicited by Ca2+ or Ag+. We show that the Ca2+ release rate: 1) was dependent upon the presence of K+ or Na+ in the release medium and 2) was influenced by a K+ gradient created across the sarcoplasmic reticulum membrane. These results directly support the idea of the involvement of an influx of K+ (through K+ channels) during the Ca2+ release and allow to reconsider a possible influence of the membrane potential of the sarcoplasmic reticulum on the Ca2+ release.     

Kinetic model of spermine effect on the Mg2+, ATP-dependent transport of Ca ions in the smooth muscle mitochondria

In experiments, carried out with the use of a radioactive label (45Ca2+) on suspension of rat uterus myocytes treated with digitonin solution (0.1 mg/ml), influence of spermine on the Mg2+, ATP-dependent Ca2+ transport in the mitochondria was investigated. Ca2+ accumulation in the mitochondria was tested as such which was blocked by ruthenium red (10 microM) and was not sensitive to thapsigargin (100 nM). It was shown, that dependence of initial speed of Ca ions accumulation in the mitochondria on spermine concentration (0.1-10 mm) is described by a bell-shaped curve. Spermine concentration being increased in the range of 0.1-1 mM the stimulation of Ca2+ accumulation was observed, at the further increase in polyamine concentration up to 10 mM the suppression of this process took place. On the basis of the analysis of the authors' experimental results and the literature data the model of complex spermine action on Ca2+ accumulation in mitochondria was proposed and analyzed. The existence of two spermine binding sites on mitochondrial membrane--S1 and S2 occupation of which is connected to activation and inhibition of Ca(2+)-unipoter, accordingly, was taken into account. The kinetic analysis of the model which has been made in an equilibrium mode, allowed to calculate some important quantitative parameters describing spermine influence on Ca ions accumulation in mitochondria. It is supposed, that the proposed model can be useful in the further research of polyamine influence on transmembrane exchange of Ca ions in mitochondria.     

Ca2+-transporting properties of myometrial plasma cell membrane Ca2+, Mg2+-ATPase reconstituted in liposomes

Purified myometrium cells plasma membrane Ca2+, Mg(2+)-ATPase was reconstitute in liposomes in functionally active state by the method of cholate dialysis: it showed ATP-hydrolase activity increased by 0.8 microM A23187 average 4 times and it showed Mg2+, ATP-dependent Ca(2+)-transporting activity. Reconstituted system transported Ca2+ at an initial rate of 114.4 +/- 16.3 nmol.min-1.mg-1 with the stoichiometry Ca2+: ATP = 1: (3.2-3.7). Calmodulin increased by 30% the initial rate of Ca(2+)-accumulation by the proteoliposomes with reconstituted Ca2+, Mg(2+)-ATPase; 0.1 mM orthovanadate decreased by 80% Ca(2+)-accumulation by this system. Ca2+, Mg(2+)-ATPase reconstituted in liposomes is just Ca(2+)-transporting ATPase of the plasma membrane. Obtained enzyme preparate can be utilised for study of the properties of this important energy-dependent Ca(2+)-transporting system of smooth muscle cell.     

Purified ryanodine receptor from skeletal muscle sarcoplasmic reticulum is the Ca2+-permeable pore of the calcium release channel

The ryanodine receptor of rabbit skeletal muscle sarcoplasmic reticulum was purified by immunoaffinity chromatography as a single approximately 450,000-Da polypeptide and it was shown to mediate single channel activity identical to that of the ryanodine-treated Ca2+ release channel of the sarcoplasmic reticulum. The purified receptor had a [3H]ryanodine binding capacity (Bmax) of 280 pmol/mg and a binding affinity (Kd) of 9.0 nM. [3H]Ryanodine binding to the purified receptor was stimulated by ATP and Ca2+ with a half-maximal stimulation at 1 mM and 8-9 microM, respectively. [3H]Ryanodine binding to the purified receptor was inhibited by ruthenium red and high concentrations of Ca2+ with an IC50 of 2.5 microM and greater than 1 mM, respectively. Reconstitution of the purified receptor in planar lipid bilayers revealed the Ca2+ channel activity of the purified receptor. Like the native sarcoplasmic reticulum Ca2+ channels treated with ryanodine, the purified receptor channels were characterized by (i) the predominance of long open states insensitive to Mg2+ and ruthenium red, (ii) a main slope conductance of approximately 35 pS and a less frequent 22 pS substate in 54 mM trans-Ca2+ or Ba2+, and (iii) a permeability ratio PBa or PCa/PTris = 8.7. The approximately 450,000-Da ryanodine receptor channel thus represents the long-term open "ryanodine-altered" state of the Ca2+ release channel from sarcoplasmic reticulum. We propose that the ryanodine receptor constitutes the physical pore that mediates Ca2+ release from the sarcoplasmic reticulum of skeletal muscle.     

Properties of the smooth muscle cell endoplasmic reticulum calcium pump

In experiments with 45Ca2+ conducted on digitonin-treated (0.1 mg/ml) myometrium cells suspension, the properties of ruthenium red-insensitive, oxalate- or phosphate-stimulated and thapsigargin- or cyclopiasonic acid-suppressed Mg2+, ATP-dependent calcium pump of myometrium sarcoplasmic reticulum was studied. The Ca2+ accumulation increased linearly in time up to 10 min, the average initial rate was 80-130 pmol Ca2+/10(6) cells per min. In the presence of 10 mM oxalate the values of the activation constant KMg for Mg2+ and K(m) for ATP were 0.6 and 1.0 mM, respectively. The relative efficiency of the different cations in insuring of the ATP-dependent Ca2+ accumulation was Mg2+ > Mn2+ = Co2+ > Ni2+; the Ca2+ accumulation was not observed in the presence of 3 mM Zn2+ or Cu2+. We observed the suppression of calcium pump activity by different inhibitors such as thapsigargin, cyclopiazonic acid, p-chloromercuribenzoic acid, eosin Y ad Na3 VO4: the values of K0.5 were 2.0 nM, 0.3 microM, 0.6 microM, 0.8 microM and 45 microM respectively. The conclusion was made that suspension of myometrial cells treated with digitonin represent a suitable experimental model for studying the properties of myometrium sarcoplasmic reticulum calcium pump.     

Calcium homeostasis in the thymocyte apoptosis II. Accumulation of calcium in mitochondria and endoplasmic reticulum

The activity of energy-dependent Ca2+-accumulation systems in rat thymocytes mitochondria and endoplasmic reticulum (ER) in control and at the early stage of X-irradiation or H2O2-induced apoptosis were determined in experiments using the model of digitonin-permeabilized cells with addition of thapsigargin and ruthenium red. The mitochondrial Ca2+-transporting system proved to be more sensitive to both apoptotic stimuli. The stationary level of Ca2+, accumulated in mitochondria and initial rate of Ca2+ accumulation in ER were reduced 15 min after H2O2 treatment. The parameters of mitochondrial Ca2+-accumulation system in irradiated cells were decreased 30 min after irradiation. Cyclosporin A almost completely inhibited DNA fragmentation in irradiated and partly--in peroxide-treated cells. The mitochondrial calcium homeostasis imbalance is suggested to be an early event in thymocytes apoptosis initiation.     

The enhancement of Ca2+ efflux from sarcoplasmic reticulum vesicles by urea.

Urea, in nondenaturing concentrations, inhibited Ca2+ uptake by sarcoplasmic reticulum vesicles with no concomitant effect on ATP hydrolysis. This inhibition was antagonized by 5 mM oxalate and 20 mM orthophosphate. At concentrations of 0.2 to 1.0 M, urea induced an increase in the Ca2+ efflux from preloaded vesicles diluted in a medium at pH 7.0 containing 2 mM ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid, 0.1 mM orthophosphate, and 0.1 mM MgCl2. The urea-induced efflux was arrested by ligands of the (Ca(2+)-Mg2+) ATPase, namely, K+, Mg2+, Ca2+, and ADP, and by ruthenium red and the polyamines spermine, spermidine, and putrescine. In the case of polyamines a dissociation between the effect on the efflux and the net Ca2+ uptake was observed, as only the efflux could be blocked by the drugs. Glycine betaine, trimethylamine-N-oxide, and sucrose antagonized the effects of urea on both the net Ca2+ uptake and the rate of Ca2+ efflux.     

The effect of spermine on transport of Ca2+ ions in brain mitochondria

The effect of spermine (50-400 microM) on the Ca-transporting system of brain mitochondria was studied. In a medium containing Mg2+ and ATP, spermine facilitates the accumulation of Ca2+ by decreasing Km of the uniporter. Spermine inhibits Na-stimulated Ca2+ efflux; this effect is dependent on the ionic strength of the medium--it is decreased when KCl concentration is increased from 20 to 120 mM. Spermine (200 microM) decreases (by 50%) the steady state concentration of Ca2+ maintained by mitochondria. The importance of spermine as a regulator of Ca2+-transport in brain mitochondria is discussed.     

Calixarene C-91 stimulates Ca2+ accumulation in the myometrium mitochondria

Calixarenes, owing to the ability to form supramolecular complexes with biologically important molecules and ions, can influence a course of biochemical processes and, accordingly, be considered as perspective molecular platforms for creation of physiologically active compounds. The work purpose is to study calixarene C-91 influence on systems of active Ca ions transport which are localized in subcellular membrane structures (mitochondria, sarcoplasmic reticulum, plasma membrane) of myometrial cells. It has been shown, that calixarene C-91 addition to incubation medium led to an increase in Ca2+ accumulation level in mitochondria. The maximal stimulating effect was 173% and it was observed at 100 microM concentration. It is suggested, that calixarene C-91 can enter mitochondria with the subsequent precipitation of Ca ions in a matrix therefore calcium capacity increases, and as a consequence, higher Ca2+ accumulation in these structures is observed. In a wide range of concentration (1-100 microM) calixarene C-91 did not influence a level of Ca2+ accumulation in sarcoplasmic reticulum of myometrial cells. Titration of solubilized Ca2+, Mg2+-ATPase by calixarene C-91 (0,1-100 microM) did not cause changes in its activity. Thus, calixarene C-91 increases Ca2+ accumulation level in mitochondria, but practically does not influence calcium pumps activity of a plasma membrane and sarcoplasmic reticulum of myometrial cells.     

Interactions between spermine and Mg2+ on mitochondrial Ca2+ transport

The effects of the polyamine spermine on the regulation of Ca2+ transport by subcellular organelles from rat liver, heart, and brain were investigated using ion-sensitive minielectrodes and a 45Ca2+ tracer method. Spermine stimulated Ca2+ uptake by mitochondria but not by microsomes. In the presence of spermine, isolated mitochondria could maintain a free extramitochondrial Ca2+ concentration of 0.3-0.2 microM. Stimulation of the initial rates of Ca2+ uptake and 45Ca2+ cycling of mitochondria by spermine shows that this was accomplished through a decrease of the apparent Km for Ca2+ uptake by the Ca2+ uniporter. The half maximally effective concentration of spermine (50 microM) was in the range of physiological concentrations of this polyamine in the cell. Spermidine was five times less effective. Putrescine was ineffective. The stimulation of mitochondrial Ca2+ uptake by spermine was inhibited by Mg2+ in a concentration-dependent manner. However, the diminished contribution of the mitochondria to the regulation of the free extraorganellar Ca2+ concentration could mostly be compensated for by microsomal Ca2+ uptake. Spermine also reversed ruthenium red-induced Ca2+ efflux from mitochondria. It is concluded that spermine is an activator of the mitochondrial Ca2+ uniporter and Mg2+ an antagonist. By this mechanism, the polyamines can confer to the mitochondria an important role in the regulation of the free cytoplasmic Ca2+ concentration in the cell and of the free Ca2+ concentration in the mitochondrial matrix.     

Effects of spermine on mitochondrial Ca2+ transport and the ranges of extramitochondrial Ca2+ to which the matrix Ca2+-sensitive dehydrogenases respond.

1. Spermine has previously been reported to be an activator of mitochondrial Ca2+ uptake [Nicchitta & Williamson (1984) J. Biol. Chem. 259, 12978-12983]. This is confirmed in the present studies on rat heart, liver and kidney mitochondria by using the activities of the Ca2+-sensitive intramitochondrial dehydrogenases (pyruvate, NAD+-isocitrate and 2-oxoglutarate dehydrogenases) as probes for matrix Ca2+, and also, for the heart mitochondria, by using entrapped fura-2. 2. As also found previously [Damuni, Humphreys & Reed (1984) Biochem. Biophys. Res. Commun. 124, 95-99], spermine activated extracted pyruvate dehydrogenase phosphate phosphatase. However, it was found to have no effects at all on the extracted NAD+-isocitrate or 2-oxoglutarate dehydrogenases. It also had no effects on activities of the enzymes in mitochondria incubated in the absence of Ca2+, or on the Ca2+-sensitivity of the enzymes in uncoupled mitochondria. 3. Spermine clearly activated 45Ca uptake by coupled mitochondria, but had no effect on Ca2+ egress from mitochondria previously loaded with 45Ca. 4. Spermine (with effective Km values of around 0.2-0.4 mM) caused an approx. 2-3-fold decrease in the effective ranges of extramitochondrial Ca2+ in the activation of the Ca2+-sensitive matrix enzymes in coupled mitochondria from all of the tissues. The effects of spermine appeared to be largely independent of the other effectors of mitochondrial Ca2+ transport, such as Mg2+ (inhibitor of uptake) and Na+ (promoter of egrees). 5. In the most physiological circumstance, coupled mitochondria incubated with Na+ and Mg2+, the presence of saturating spermine (2 mM) resulted in an effective extramitochondrial Ca2+ range for matrix enzyme activation of from about 30-50 nM up to about 800-1200 nM, with half-maximal effects around 250-400 nM-Ca2+. The implications of these findings for the regulation of matrix and extramitochondrial Ca2+ are discussed.     

Effects of local anesthetics on the passive permeability of sarcoplasmic reticulum vesicles to Ca2+ and Mg2+

Sarcoplasmic reticulum vesicles are used here as model membrane system to question the hypothesis of enhancement of permeability of cations by anesthetics, particularly that of Ca2+ and of Mg2+. The effects of dibucaine (up to 800 microM), tetracaine (up to 2 mM), lidocaine (up to 10 mM) and procaine (up to 10 mM) on the permeability of these membranes to Ca2+ and Mg2+ have been measured. We have used an experimental approach based on the light scattering method (Kometani, T. and Kasai, M. (1978) J. Membrane Biol. 41, 295-308). It has been found that all the local anesthetics cited above markedly increase the permeability of sarcoplasmic reticulum vesicles to Mg2+ and, in the concentration range tested herein, only dibucaine and tetracaine increase the permeability to Ca2+. The kinetic analysis of the time dependence of the light-scattering data after the osmotic shock shows that, in the absence of local anesthetics, the Mg2+ influx can be described as proceeding through a unique type of channel. However, Ca2+ influx appears to involve two channel of different kinetic properties. Because the relative fraction of both types of Ca2+ channel is similar to the average ratio between light and heavy vesicles in unfractionated sarcoplasmic reticulum, we suggest that each type of channel can be preferentially located in one of these fractions. The determined rate constants for Ca2+ permeability through both types of channel are 0.77 +/- 0.08 min-1 (fast channels) and 0.025 +/- 0.005 min-1 (slow channels) and that for Mg2+ is 0.08 +/- 0.02 min-1. These results agree with data obtained by other groups using different experimental approaches. Dibucaine and tetracaine significantly alter the rate of Mg2+ and Ca2+ influx through the slow channels. In addition, these two local anesthetics also produce the effect that the Mg2+ influx cannot be described with only one exponential process, thus suggesting a differential effect on vesicles of different density. The increase of Ca2+ and Mg2+ permeability by dibucaine and by tetracaine is found at concentrations of these drugs that do not produce a noticeable inhibition of the (Ca2+ + Mg2+)-ATPase activity of sarcoplasmic reticulum vesicles.     

Characterization of Ca2+ release from heterogeneous Ca2+ stores in sarcoplasmic reticulum isolated from arterial and gastric smooth muscle

Ca2+ transport was investigated in vesicles of sarcoplasmic reticulum subfractionated from bovine main pulmonary artery and porcine gastric antrum using digitonin binding and zonal density gradient centrifugation. Gradient fractions recovered at 15-33% sucrose were studied as the sarcoplasmic reticulum component using Fluo-3 fluorescence or 45Ca2+ Millipore filtration. Thapsigargin blocked active Ca2+ uptake and induced a slow Ca2+ release from actively loaded vesicles. Unidirectional 45Ca2+ efflux from passively loaded vesicles showed multicompartmental kinetics. The time course of an initial fast component could not be quantitatively measured with the sampling method. The slow release had a half-time of several minutes. Both components were inhibited by 20 microM ruthenium red and 10 mM Mg2+. Caffeine, inositol 1,4,5-trisphosphate, ATP, and diltiazem accelerated the slow component. A Ca2+ release component activated by ryanodine or cyclic adenosine diphosphate ribose was resolved with Fluo-3. Comparison of tissue responses showed that the fast Ca2+ release was significantly smaller and more sensitive to inhibition by Mg2+ and ruthenium red in arterial vesicles. They released more Ca2+ in response to inositol 1,4,5-trisphosphate and were more sensitive to activation by cyclic adenosine diphosphate ribose. Ryanodine and caffeine, in contrast, were more effective in gastric antrum. In each tissue, the fraction of the Ca2+ store released by sequential application of caffeine and inositol 1,4,5-trisphosphate depended on the order applied and was additive. The results indicate that sarcoplasmic reticulum purified from arterial and gastric smooth muscle represents vesicle subpopulations that retain functional Ca2+ channels that reflect tissue-specific pharmacological modulation. The relationship of these differences to physiological responses has not been determined.     

Distances between functional sites of the Ca2+ + Mg2(+)-ATPase from sarcoplasmic reticulum using Co2+ as a spectroscopic ruler

Cobalt ion inhibits the Ca2+ + Mg2(+)-ATPase activity of sealed sarcoplasmic reticulum vesicles, of solubilized membranes and of the purified enzyme. To use Co2+ appropriately as a spectroscopic ruler to map functional sites of the Ca2+ + Mg2(+)-ATPase, we have carried out studies to obtain the kinetic parameters needed to define the experimental conditions to conduct the fluorimetric studies. 1. The apparent K0.5 values of inhibition of this ATPase are 1.4 mM, 4.8 mM and 9.5 mM total Co2+ at pH 8.0, 7.0 and 6.0, respectively. The inhibition by Co2+ is likely to be due to free Co2+ binding to the enzyme. Millimolar Ca2+ can fully reverse this inhibition, and also reverses the quenching of the fluorescence of fluorescein-labeled sarcoplasmic reticulum membranes due to Co2+ binding to the Ca2+ + Mg2(+)-ATPase. Therefore, we conclude that Co2+ interacts with Ca2+ binding sites. 2. Co2+.ATP can be used as a substrate by this enzyme with Vmax of 2.4 +/- 0.2 mumol ATP hydrolyzed min-1 (mg protein)-1 at 20-22 degrees C and pH 8.0, and with a K0.5 of 0.4-0.5 mM. 3. Co2+ partially quenches, about 10 +/- 2%, the fluorescence of fluorescein-labeled sarcoplasmic reticulum Ca2+ + Mg2(+)-ATPase upon binding to this enzyme at pH 8.0. From the fluorescence data we have estimated an average distance between Co2+ and fluorescein in the ATPase of 1.1-1.8 nm or 1.3-2.1 nm for one or two equidistant Co2+ binding sites, respectively. 4. Co2+.ATP quenches about 20-25% of the fluorescence of fluorescein-labeled Ca2+ + Mg2(+)-ATPase, from which we obtain a distance of 1.1-1.9 nm between Co2+ and fluorescein located at neighbouring catalytic sites.     

Mechanism of passive Ca2+ permeability of vesicular sarcolemmal preparations from rat hearts

Vesicular sarcolemmal preparations isolated from rat hearts were characterized by high total ATPase (4.32 +/- 0.57 mumol/min per mg), adenylate cyclase (121 +/- 11 pmol/min per mg) and creatine kinase (1.73 +/- 0.35 mumol/min per mg) activities as well as Na-Ca exchange specific to sodium. ATPase activity was inhibited with digitoxigenin by 50-70% and was not changed by ouabain, ionophore A23187 or oligomycin. Sarcolemmal vesicles bound [3H]digitoxigenin and [3H]ouabain in isotonic medium in the presence of Pi and Mg2+. The number of binding sites for hydrophobic digitoxigenin (N = 237 pmol/mg) was several-times higher than that for hydrophilic ouabain (N = 32.7 pmol/mg). These data show that sarcolemmal preparations were not significantly contaminated by mitochondria and sarcoplasmic reticulum and consisted mostly of inside-out vesicles. Incubation of these vesicles with 45Ca2+ (0.5-10 mM) led to penetration of the latter into the vesicles with the following binding characteristics: number of binding sites (N = 20.5 +/- 4.6 nmol/mg, Kd approximately equal to 2.0 mM). Ca2+ binding to the inner surface of vesicles was proved by the following facts: (1) Ca2+ ionophore A23187 increased slightly total intravesicular Ca2+ content but markedly accelerated Ca2+ efflux along its concentration gradient; (2) gramicidin and osmotic shock showed a similar accelerating effect. Ca2+ efflux from the vesicles along its concentration gradient ([Ca2+]i/[Ca2+]e = 2.0 mM/0.1 microM) was inhibited by Mn2+, Co2+, and verapamil when they acted inside the vesicles. The rate of Ca2+ efflux was hyperbolically dependent on intravesicular Ca2+ concentration (Km approximately equal to 2.9 mM). These data reveal that Ca2+ efflux from sarcolemmal vesicles is controlled by Ca2+ binding to the sarcolemmal membrane. Ca2+ efflux from the vesicles was stimulated 1.7--times after incubation of vesicles with 0.2 mM MgATP or MgADP and 15-times after treatment with 0.2 mM adenylyl beta, gamma-imidodiphosphate. Enhancement in the rate of Ca2+ efflux correlated with the increase in the intravesicular Ca2+ content. ATP-stimulated Ca2+ efflux was suppressed by verapamil and was nonmonotonically dependent upon the transmembrane potential created by the K+ concentration gradient in the presence of valinomycin, Ca2+ efflux being slower at extreme values of membrane potential (+/- 80 mV).     

Kinetics of rapid Ca2+ release by sarcoplasmic reticulum. Effects of Ca2+, Mg2+, and adenine nucleotides

A radioisotope flux-rapid-quench-Millipore filtration method is described for determining the effects of Ca2+, adenine nucleotides, and Mg2+ on the Ca2+ release behaviour of "heavy" sarcoplasmic reticulum (SR) vesicles. Rapid 45Ca2+ efflux from passively loaded vesicles was blocked by the addition of Mg2+ and ruthenium red. At pH 7 and 10(-9) M Ca2+, vesicles released 45Ca2+ with a low rate (k = 0.1 s-1). An increase in external Ca2+ concentration to 4 microM or the addition of 5 mM ATP or the ATP analogue adenosine 5'-(beta,gamma-methylenetriphosphate) (AMP-PCP) resulted in intermediate 45Ca2+ release rates. The maximal release rate was observed in media containing 4 microM Ca2+ and 5 mM AMP-PCP and had a first-order rate constant of 30-100 s-1. Mg2+ partially inhibited Ca2+- and nucleotide-induced 45Ca2+ efflux. In the absence of AMP-PCP, 45Ca2+ release was fully inhibited at 5 mM Mg2+ or 5 mM Ca2+. The composition of the release media was systematically varied, and the flux data were expressed in the form of Hill equations. The apparent n values of activation of Ca2+ release by ATP and AMP-PCP were 1.6-1.9. The Hill coefficient of Ca2+ activation (n = 0.8-2.1) was dependent on nucleotide and Mg2+ concentrations, whereas the one of Mg2+ inhibition (n = 1.1-1.6) varied with external Ca2+ concentration. These results suggest that heavy SR vesicles contain a "Ca2+ release channel" which is capable of conducting Ca2+ at rates comparable with those found in intact muscle. Ca2+, AMP-PCP (ATP), and Mg2+ appear to act at noninteracting or interacting sites of the channel.