Differential effects of chronic reserpine exposure on Ca2+ sequestering mechanisms in rat submandibular gland vesicles

To determine the effects of exposure to reserpine on subcellular Ca2+ transporting systems, active Ca2+ uptake was measured with and without ruthenium red in submandibular gland vesicles obtained from rats after chronic treatment with reserpine. The properties of ruthenium red-sensitive Ca2+ uptake were similar to those measured in submandibular gland vesicles from untreated rats: it was abolished by the dye, was relatively low at 1 microM Ca2+ but increased markedly at millimolar Ca2+ levels and was positively and significantly correlated with the mitochondrial membrane marker, cytochrome-C oxidase activity, in membrane subfractions obtained by differential centrifugation (r = 0.67, p = 0.0005, n = 29). On the other hand, ruthenium red-insensitive Ca2+ uptake, though stimulated at submicromolar Ca2+ concentrations, was reduced by a mean of 54% compared to preparations from untreated animals and particulate RNA content was 18% of that found in control preparations. Moreover, the distributions of ruthenium red-insensitive Ca2+ uptake and particulate RNA (which are closely correlated in vesicles from untreated rats) were not significantly related when measured in vesicles of submandibular glands from reserpine treated rats. Other membrane markers and overall membrane protein content were not significantly altered after chronic reserpine exposure. We conclude that reserpine treatment has little effect on mitochondrial Ca2+ uptake capacity but abolishes or drastically reduces the high affinity Ca2+-sequestering activity which, in submandibular gland vesicles from untreated rats, is apparently associated with the endoplasmic reticulum.     

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.     

Oxidation induced by phthalocyanine dyes causes rapid calcium release from sarcoplasmic reticulum vesicles

The copper containing phthalocyanine dyes, alcian blue, copper phthalocyanine tetrasulfonic acid, and Luxol fast blue MBSN are found to induce rapid calcium efflux from actively loaded sarcoplasmic reticulum (SR) vesicles. Alcian blue (5 microM), with 1 mM free Mg2+ triggered Ca2+ efflux at rates greater than 20 nmol/mg of SR/s. As in the case of Ca2+ efflux induced by calcium, heavy metals, or SH oxidation with Cu2+/cysteine, efflux induced by phthalocyanines is also stimulated by adenine containing nucleotides and inhibited by millimolar Mg2+ and submicromolar ruthenium red (RR). In addition, analogs of RR, such as hexamminecobalt(III) chloride or hexammineruthenium(III) chloride also inhibit Ca2+ efflux but are effective at somewhat higher concentrations (approximately 50 microM). Calcium release stimulated by phthalocyanines is specific for SR derived from the terminal cisternae region rather than longitudinal SR. Preincubation of alcian blue with the reducing agents, sodium dithionite, dithiothreitol, or cysteine causes complete loss of Ca2+ release activity from SR vesicles. Reoxidation of the alcian blue leads to return of the Ca2+ release activity of the phthalocyanine dye. The copper containing phthalocyanine dyes appear to cause rapid Ca2+ release from SR vesicles by oxidizing sulfhydryl groups associated with the calcium release channel. Moreover, phthalocyanines appear to act by oxidizing a pair of neighboring sulfhydryls to a disulfide because subsequent additions of the reducing agent dithiothreitol promote the closure of the Ca2+ channel and calcium re-uptake.     

Effects of Mg2+ on calcium accumulation by two fractions of sarcoplasmic reticulum from rabbit skeletal muscle

Calcium accumulation by two fractions of sarcoplasmic reticulum presumably derived from longitudinal tubules (light vesicles) and terminal cisternae (heavy vesicles) was examined radiochemically in the presence of various free Mg2+ concentrations. Both fractions of sarcoplasmic reticulum exhibited a Mg2+-dependent increase in phosphate-supported calcium uptake velocity, though half-maximal velocity in heavy vesicles occurred at a much higher free Mg2+ concentration than that in light vesicles (i.e., approx. 0.90 mM vs. approx. 0.02 mM Mg2+). Calcium uptake velocity in light vesicles correlated with Ca2+-dependent ATPase activity, suggesting that Mg2+ stimulated the calcium pump. Calcium uptake velocity in heavy vesicles did not correlate with Ca2+-dependent ATPase activity, although a Mg2+-dependent increase in calcium influx was observed. Thus, Mg2+ may increase the coupling of ATP hydrolysis to calcium transport in heavy vesicles. Analyses of calcium sequestration (in the absence of phosphate) showed a similar trend in that elevation of Mg2+ from 0.07 to 5 mM stimulated calcium sequestration in heavy vesicles much more than in light vesicles. This difference between the two fractions of sarcoplasmic reticulum was not explained by phosphoenzyme (EP) level or distribution. Analyses of calcium uptake, Ca2+-dependent ATPase activity, and unidirectional calcium flux in the presence of approx. 0.4 mM Mg2+ suggested that ruthenium red (0.5 microM) can also increase the coupling of ATP hydrolysis to calcium transport in heavy vesicles, with no effect in light vesicles. These functional differences between light and heavy vesicles suggest that calcium transport in terminal cisternae is regulated differently from that in longitudinal tubules.     

Uncoupler-stimulated release of Ca2+ from Ehrlich ascites tumor cell mitochondria

Ruthenium red-insensitive, uncoupler-stimulated release of Ca2+ from Ehrlich ascites tumor cell mitochondria is much slower than from rat liver mitochondria under comparable conditions. In the presence of Pi and at moderate or high Ca2+ loads, ruthenium red-insensitive Ca2+ efflux elicited with uncoupler is approximately 20 times more rapid for rat liver than Ehrlich cell mitochondria. This is attributed to resistance of tumor mitochondria to damage by Ca2+ due to a high level of endogenous Mg2+ that also attenuates Ca2+ efflux. Calcium release from rat liver and tumor mitochondria is inhibited by exogenous Mg2+. This applies to ruthenium red-insensitive spontaneous Ca2+ efflux associated with Ca2+ uptake and uncoupling, and (b) ruthenium red-insensitive Ca2+ release stimulated by uncoupling agent. The endogenous Mg2+ level of Ehrlich tumor mitochondria is approximately three times that of rat liver mitochondria. Endogenous Ca2+ is also much greater (six fold) in Ehrlich tumor mitochondria compared to rat liver. Despite the quantitative difference in endogenous Mg2+, the properties of internal Mg2+ are much the same for rat liver and Ehrlich cell mitochondria. Ehrlich ascites tumor mitochondria exhibit slow, metabolically dependent Mg2+ release and rapid limited release of Mg2+ during Ca2+ uptake. Both have been observed with rat liver and other types of mitochondria. The proportions of apparently "bound" and "free" Mg2+ (inferred from release by the ionophore, A23187) do not differ significantly between tumor and liver mitochondria. Thus, the endogenous Mg2+ of tumor mitochondria has no unusual features but is simply elevated substantially. Ruthenium red-insensitive Ca2+ efflux, when expressed as a function of the intramitochondrial Ca2+/Mg2+ ratio, is quite similar for tumor and rat liver. It is proposed, therefore, that endogenous Mg2+ is a major regulatory factor responsible for differences in the sensitivity to damage by Ca2+ and Ca2+ release by Ehrlich ascites tumor mitochondria compared to mitochondria from normal tissues.     

Ruthenium red-insensitive Ca2+ uptake and release by mitochondria

Calcium uptake by rat liver mitochondria driven by an artificial pH gradient is ruthenium red insensitive, electrically neutral, and inhibited by the local anesthetic, nupercaine. This pH-driven Ca2+ transport is also inhibited by NH3, Pi, and acetate. Direct measurements of Pi indicate it is not translocated with Ca2+ during pH-driven Ca2+ uptake. Calcium is therefore not transported by a Ca2+-Pi symport mechanism. Ruthenium red-insensitive Ca2+ efflux is similar in its inhibition by nupercaine and its kinetics, and is also electroneutral. This suggests that the Ca2+ uptake described here occurs via reversal of the principal pathway of mitochondrial Ca2+ release. From the available data, pH-driven Ca2+ uptake (and presumably Ca2+ efflux) is hypothesized to occur by Ca2+ symport with unidentified anions. Protons may move counter to Ca2+ or reversibly dissociate from cotransported anions, which therefore couples Ca2+ transport to the pH gradient.     

Drug-induced Ca2+ release from isolated sarcoplasmic reticulum. II. Releases involving a Ca2+-induced Ca2+ release channel

Calcium ions that have been preloaded into isolated sarcoplasmic reticulum subfractions in the presence of ATP and pyrophosphate may be released upon addition of a large number of diverse pharmacologic substances. We report here that not only caffeine, but also Ca2+ ions, thymol, quercetin, menthol, halothane, chloroform, 1-ethyl-2-methylbenzimidazole, ryanodine, tetraphenylboron, ketoconazole, miconazole, clotrimazole, W-7, doxorubicin, 5,5'-dithiobis-(2-nitrobenzoic acid), p-chloromercuribenzoic acid, and low concentrations of Ag+ induce Ca2+ release from such triadic sarcoplasmic reticulum. All these drugs induce increased undirectional Ca2+ efflux. We believe all these drug-induced Ca2+ releases are mediated by Ca2+ efflux through the same ion channel since these releases are all greatly attenuated when light sarcoplasmic reticulum is substituted for triads and are even more pronounced when transverse tubule-free terminal cisternae are substituted for triads, and all these forms of drug-induced Ca2+ release are inhibited by submicromolar concentrations of ruthenium red, and by submillimolar concentrations of tetracaine, 9-aminoacridine, and Ba2+, yet they are not affected by nifedipine even at a concentration of 50 microM.     

Drug-induced Ca2+ release from isolated sarcoplasmic reticulum. I. Use of pyrophosphate to study caffeine-induced Ca2+ release

A demonstration is made of pyrophosphate's use as a precipitating anion in studies of Ca2+ release from isolated sarcoplasmic reticulum (SR). Not only does pyrophosphate speed up the rate at which Ca2+ can be preloaded into SR, but it also allows the accumulated Ca2+ to be released in response to agents such as caffeine. Because so much Ca2+ can be preloaded into SR with pyrophosphate present, more experiments can be performed with a given amount of SR material, and even rapid Ca2+ release rates (greater than 1 mumol/mg X min) are maintained for many seconds. These rates can easily be quantified using conventional spectrophotometric and isotopic methods, without the need for expensive rapid mixing equipment. Caffeine-induced Ca2+ release is exhibited by triadic and terminal cisterna SR subfractions but not by light SR. Caffeine specifically increases the rate of unidirectional 45Ca2+ efflux. This increased efflux is blocked by ruthenium red at submicromolar concentrations and by tetracaine, 9-aminoacridine, or Ba2+ at submillimolar concentrations.     

Effects of alkaline pH on sarcoplasmic reticulum Ca2+ release and Ca2+ uptake.

Alkalinization-induced Ca2+ release from isolated frog or rabbit sarcoplasmic reticulum vesicles appears to consist of two distinct components: 1) a direct activation of ruthenium red-sensitive Ca2+ release channels in terminal cisternae and 2) an increased ruthenium red-insensitive Ca2+ efflux through some other efflux pathway distributed throughout the sarcoplasmic reticulum. The first of these releases exhibits an alkalinization-induced inactivation process and does not depend on the ruthenium red-insensitive form of Ca2+ release as a triggering agent for secondary Ca(2+)-induced Ca2+ release. Both releases are inhibited when the extravesicular (i.e. cytoplasmic) free [Ca2+] is reduced. This may reflect an increased sensitivity of the Ca2+ release channels to Ca2+ at alkaline pH. The pH sensitivity of the ruthenium red-sensitive Ca2+ release channels could be of significance during excitation-contraction coupling. The ruthenium red-insensitive form of Ca2+ release is less likely to be physiologically relevant, but it probably has contributed greatly to reports of alkalinization-induced decreases in net sarcoplasmic reticulum Ca2+ uptake, particularly under conditions where oxalate supported Ca2+ uptake is much less affected, as here.     

The high affinity (Ca2+-Mg2+)-ATPase in liver plasma membranes is a Ca2+ pump. Reconstitution of the purified enzyme into phospholipid vesicles

The purified (Ca2+-Mg2+)-ATPase from rat liver plasma membranes (Lotersztajn, S., Hanoune, J., and Pecker, F. (1981) J. Biol. Chem. 256, 11209-11215) was incorporated into soybean phospholipid vesicles, together with its activator. In the presence of millimolar concentrations of Mg2+, the reconstituted proteoliposomes displayed a rapid, saturable, ATP-dependent Ca2+ uptake. Half-maximal Ca2+ uptake activity was observed at 13 +/- 3 nM free Ca2+, and the apparent Km for ATP was 16 +/- 6 microM. Ca2+ accumulated into proteoliposomes (2.8 +/- 0.2 nmol of Ca2+/mg of protein/90 s) was totally released upon addition of the Ca2+ ionophore A-23187. Ca2+ uptake into vesicles reconstituted with enzyme alone was stimulated 2-2.5-fold by the (Ca2+-Mg2+)-ATPase activator, added exogenously. The (Ca2+-Mg2+)-ATPase activity of the reconstituted vesicles, measured using the same assay conditions as for ATP-dependent Ca2+ uptake activity (e.g. in the presence of millimolar concentrations of Mg2+), was maximally activated by 20 nM free Ca2+, half-maximal activation occurring at 13 nM free Ca2+. The stoichiometry of Ca2+ transport versus ATP hydrolysis approximated 0.3. These results provide a direct demonstration that the high affinity (Ca2+-Mg2+)-ATPase identified in liver plasma membranes is responsible for Ca2+ transport.     

Effect of compound 48/80 and ruthenium red on the Ca2+-ATPase of sarcoplasmic reticulum

The effects of the condensation product of N-methyl-p-methoxyphenethylamine with formaldehyde (compound 48/80) and ruthenium red on the partial reactions of the catalytic cycle of the sarcoplasmic reticulum Ca2+-ATPase of skeletal muscle were studied. The ATPase activity and both Ca2+ and Sr2+ uptake were inhibited by compound 48/80 when oxalate was used as a precipitating agent. The degree of inhibition decreased when oxalate was replaced by orthophosphate as the precipitating anion. Both the fast Ca2+ efflux and the synthesis of ATP observed during reversal of the Ca2+ pump were inhibited by compound 48/80. Inhibition of the reversal of the Ca2+ pump was caused by a competition between compound 48/80 and orthophosphate for the phosphorylation site of the enzyme. The fast Ca2+ release promoted by arsenate was impaired by compound 48/80. Ruthenium red competes with Ca2+ for the high affinity binding site of the Ca2+-ATPase, but did not interfere with the binding of Ca2+ to the low affinity binding site of the enzyme. In presence of Ca2+ concentrations higher than 5 microM, ruthenium red in concentrations up to 200 microM had no effect on both ATPase activity and Ca2+ uptake. However, the fast Ca2+ efflux promoted by arsenate and the fast Ca2+ efflux coupled with the synthesis of ATP observed during the reversal of the Ca2+ pump were inhibited by ruthenium red, half-maximal inhibition being attained in presence of 10-20 microM ruthenium red. In contrast to the effect of compound 48/80, ruthenium red did not inhibit the phosphorylation of the enzyme by orthophosphate. The ATP in equilibrium with Pi exchange catalyzed by the Ca2+-ATPase in the absence of transmembrane Ca2+ gradient was also inhibited by ruthenium red.     

Block by ruthenium red of the ryanodine-activated calcium release channel of skeletal muscle

The effects of ruthenium red and the related compounds tetraamine palladium (4APd) and tetraamine platinum (4APt) were studied on the ryanodine activated Ca2+ release channel reconstituted in planar bilayers with the immunoaffinity purified ryanodine receptor. Ruthenium red, applied at submicromolar concentrations to the myoplasmic side (cis), induced an all-or-none flickery block of the ryanodine activated channel. The blocking effect was strongly voltage dependent, as large positive potentials that favored the movement of ruthenium red into the channel conduction pore produced stronger block. The half dissociation constants (Kd) for ruthenium red block of the 500 pS channel were 0.22, 0.38, and 0.62 microM, at +100, +80, and +60 mV, respectively. Multiple ruthenium red molecules seemed to be involved in the inhibition, because a Hill coefficient of close to 2 was obtained from the dose response curve. The half dissociation constant of ruthenium red block of the lower conductance state of the ryanodine activated channel (250 pS) was higher (Kd = 0.82 microM at +100 mV), while the Hill coefficient remained approximately the same (nH = 2.7). Ruthenium red block of the channel was highly asymmetric, as trans ruthenium red produced a different blocking effect. The blocking and unblocking events (induced by cis ruthenium red) can be resolved at the single channel level at a cutoff frequency of 2 kHz. The closing rate of the channel in the presence of ruthenium red increased linearly with ruthenium red concentration, and the unblocking rate of the channel was independent of ruthenium red concentrations. This suggests that ruthenium red block of the channel occurred via a simple blocking mechanism. The on-rate of ruthenium red binding to the channel was 1.32 x 10(9) M-1 s-1, and the off-rate of ruthenium red binding was 0.75 x 10(3) s-1 at +60 mV, in the presence of 200 nM ryanodine. The two related compounds, 4APd and 4APt, blocked the channel in a similar way to that of ruthenium red. These compounds inhibited the open channel with lower affinities (Kd = 170 microM, 4APd; Kd = 656 microM, 4APt), and had Hill coefficients of close to 1. The results suggest that ruthenium red block of the ryanodine receptor is due to binding to multiple sites located in the conduction pore of the channel.     

Ba2+ ions inhibit the release of Ca2+ ions from rat liver mitochondria

The release of Ca2+ from respiring rat liver mitochondria following the addition of either ruthenium red or an uncoupler was measured by a Ca2+-selective electrode or by 45Ca2+ technique. Ba2+ ions are asymmetric inhibitors of both Ca2+ release processes. Ba2+ ions in a concentration of 75 microM inhibited the ruthenium red and the uncoupler induced Ca2+ release by 80% and 50%, respectively. For the inhibition, it was necessary that Ba2+ ions entered the matrix space: Ba2+ ions did not cause any inhibition of Ca2+ release if addition of either ruthenium red or the uncoupler preceded that of Ba2+. The time required for the development of the inhibition of the Ca2+ release and the time course of 140Ba2+ uptake ran in parallel. Ba2+ accumulation is mediated through the Ca2+ uniporter as 140Ba2+ uptake was competitively inhibited by extramitochondrial Ca2+ and prevented by ruthenium red. Due to the inhibition of the ruthenium red insensitive Ca2+ release, Ba2+ shifted the steady-state extramitochondrial Ca2+ concentration to a lower value. Ba2+ is potentially a useful tool to study mitochondrial Ca2+ transport.     

Effect of ruthenium red upon Ca2+ and Mn2+ uptake in Saccharomyces cerevisiae. Comparison with the effect of La3+

The initial rate of both Ca2+ and Mn2+ uptake is inhibited by ruthenium red to about the same extent as by equivalent concentrations of La3+. The inhibition of Ca2+ uptake, however, is relieved during further incubation with ruthenium red. On preincubating the cells with ruthenium red even a stimulation of divalent cation uptake can be found. Relieve of the inhibition of divalent cation uptake is accompanied by K+ efflux. Both ruthenium red and La3+ displace Ca2+ very effectively from binding sites at the cell surface. The inhibition of initial Ca2+ uptake is accompanied by a reduction in the binding of Ca2+.     

Subcellular fractionation of pig coronary artery smooth muscle

A detailed procedure for subcellular fractionation of the smooth muscle from pig coronary arteries based on dissection of the proper tissue, homogenization, differential centrifugation and sucrose density gradient centrifugation is described. A number of marker enzymes and Ca2+ uptake in presence or absence of oxalate, ruthenium red and azide were studied. The ATP-dependent oxalate-independent azide- or ruthenium red-insensitive Ca2+ uptake, and the plasma membrane markers K+-activated ouabain-sensitive p-nitrophenylphosphatase, 5'-nucleotidase and Mg2+-ATPase showed maximum enrichment in the F2 fraction (15-28% sucrose) which was also contaminated with the endoplasmic reticulum marker NADPH: cytochrome c reductase, and to a small extent with the inner mitochondrial marker cytochrome c reductase, and also showed a small degree of oxalate stimulation of the Ca2+ uptake. F3 fraction (28-40% sucrose) was maximally enriched in the ATP- and oxalate-dependent azide-insensitive Ca2+ uptake and the endoplasmic reticulum marker NADPH: cytochrome c reductase but was heavily contaminated with the plasma membrane and the inner mitochondrial markers. The mitochondrial fraction was enriched in cytochrome c oxidase and azide- or ruthenium red-sensitive ATP-dependent Ca2+ uptake but was heavily contaminated with other membranes. Electron microscopy showed that F2 contained predominantly smooth surface vesicles and F3 contained smooth surface vesicles, rough endoplasmic reticulum and mitochondria. The ATP-dependent azide-insensitive oxalate-independent and oxalate-stimulated Ca2+ uptake comigrated with the plasma membrane and the endoplasmic reticulum markers, respectively, and were preferentially inhibited by digitonin and phosphatidylserine, respectively. This study establishes a basis for studies on receptor distribution and further Ca2+ uptake studies to understand the physiology of coronary artery vasodilation.     

Characterisation of an ATP-dependent Ca2+ transport system in a plasma membrane enriched fraction from rat parotid

A membrane fraction enriched in plasma membrane marker enzymes K+-dependent p-nitrophenyl phosphatase, 5'-nucleotidase and alkaline phosphatase was prepared from rat parotid glands using Percoll self-forming gradient. This fraction contained an ATP-dependent CA2+ transport system which was distinct from those located on the endoplasmic reticulum and mitochondria of parotid glands. The Km for ATP was 0.57 +/- 0.07 mM (n = 3). Nucleotides other than ATP such as ADP, AMP, GTP, CTP, UTP or ITP were unable to support significant Ca2+ uptake. ATP-dependent Ca2+ uptake displayed sigmoidal kinetics with respect to free Ca2+ concentration with a Hill coefficient of 2.02. The K0.5 for Ca2+ was 44 +/- 3.1 nM (n = 3) and the average Vmax was 13.5 +/- 1.1 nmol/min per mg of protein. The pH optimum was 7.2. Trifluorperazine inhibited Ca2+ transport with half maximal inhibition observed at 30.8 microM. Complete inhibition was observed at 70 microM trifluorperazine. Exogenous calmodulin however had no effect on the rate of transport. Na+ and K+ ions activated Ca2+ transport at 20 to 30 mM ion concentrations. Higher concentrations of Na+ or K+ were inhibitory.     

Ruthenium red and caffeine affect the Ca2+-ATPase of the sarcoplasmic reticulum

Effects of ruthenium red and caffeine (a Ca2+ release blocker and an inducer, respectively) on Ca2+ uptake by sarcoplasmic reticulum (SR) vesicles and formation of the phosphorylated intermediate (EP) of the Ca2+-ATPase were studied using fast-kinetic techniques. Ruthenium red increased the rate and the maximum level of EP formation, while caffeine decreased both. Similarly, ruthenium red accelerated rapid Ca2+ uptake, while caffeine inhibited it. These drugs affected EP formation also with detergent solubilized Ca2+-ATPase. The concentrations required for half maximal effects on these functions (0.2 microM ruthenium red, 1.0 mM caffeine) are about the same as those for altering Ca2+ release. These results indicate that these reagents affect both the Ca2+-pump as well as the Ca2+ release mechanism, suggesting that the Ca2+-pump and Ca2+ release have some mechanisms in common.     

High affinity, calcium-stimulated adenosine triphosphatase activity in the particulate fraction of rat pancreatic acini

Adenosine triphosphatase activity which is Mg2+-dependent and stimulated by submicromolar concentrations of Ca2+ (as Ca . ATP) was identified in the total particulate fraction of rat pancreatic acini. Half-maximal activity (V0.5) is obtained at 100.1 +/- 6 nM Ca . ATP with a Hill coefficient of 2.2 +/- 0.1 (mean +/- S.E.; n = 4). Maximal activity was 75 +/- 19 pmol of Pi released from ATP minute-1 microgram of membrane protein-1 (mean +/- S.E.; n = 7). High affinity Ca2+-ATPase activity was unaffected by ouabain, Na+, K+, La3+, and added calmodulin. Activity was slightly reduced by ruthenium red (0.1 mM) and by oligomycin (80 micrograms/ml) but was reduced almost 50% by the phenothiazine derivative fluphenazine in a dose-related and Ca2+-dependent manner. Hydrolysis of p-nitrophenyl phosphate was 9% of the rate of ATP hydrolysis and was independent of Ca2+ concentration. However, ADP, GTP, UTP, and ITP were hydrolyzed at 76-93% the rate that ATP was hydrolyzed with V0.5 values and Hill coefficients similar to those of Ca . ATP. We conclude that rat pancreatic acini contain an enzyme for active Ca2+ translocation: ATPase activity that is Mg2+-dependent and stimulated by submicromolar concentrations of Ca . ATP. Substrate hydrolysis appears to involve positive cooperative interactions of multiple ligand-binding sites and may be regulated in part by calmodulin.     

Regulated release of Ca2+ from respiring mitochondria by Ca2+/2H+ antiport.

Simultaneous measurements of oxygen consumption and transmembrane transport of Ca2+, H+, and phosphate show that the efflux of Ca2+ from respiring tightly coupled rat liver mitochondria takes place by an electroneutral Ca2+/2H+ antiport process that is ruthenium red-insensitive and that is regulated by the oxidation-reduction state of the mitochondrial pyridine nucleotides. When mitochondrial pyridine nucleotides are kept in a reduced steady state, the efflux of Ca2+ is inhibited; when they are in an oxidized state, Ca2+ efflux is activated. These processes were demonstrated by allowing phosphate-depleted mitochondria respiring on succinate in the presence of rotenone to take up Ca2+ from the medium. Upon subsequent addition of ruthenium red to block Ca2+ transport via the electrophoretic influx pathway, and acetoacetate, to bring mitochondrial pyridine nucleotides into the oxidized state, Ca2+ efflux and H+ influx ensued. The observed H+ influx/Ca2+ efflux ratio was close to the value 2.0 predicted for the operation of an electrically neutral Ca2+/2H+ antiport process.     

Effects of Taurine and Mitochondrial Metabolic Inhibitors on ATP-Dependent Ca2+ Uptake in Synaptosomal and Mitochondrial Subcellular Fractions of Rat Retina

ATP-dependent Ca2+ uptake was investigated at low Ca2+ concentrations (10 microM) in rat retinal synaptosomal and mitochondrial preparations obtained by differential centrifugation on Ficoll gradients. Ca2+ uptake in the synaptosomal and mitochondrial subcellular preparations was stimulated by ATP and additionally stimulated by ATP plus taurine. The ATP-dependent and taurine-stimulated ATP-dependent Ca2+ uptakes were inhibited by mitochondrial metabolic inhibitors (atractyloside, oligomycin, and ruthenium red). These metabolic inhibitors had a greater effect on the ATP-dependent and taurine-stimulated ATP-dependent Ca2+ uptake activities in the mitochondrial preparation than in the synaptosomal preparation. ATP-dependent Ca2+ uptake in a synaptosomal subfraction obtained by osmotic shock was only partially inhibited by atractyloside. ATP-dependent Ca2+ uptake in the synaptosomal subfraction was also stimulated by taurine but to a lesser extent than in either the synaptosomal or mitochondrial preparation. These studies suggest that mitochondria are primarily responsible for taurine-stimulated ATP-dependent Ca2+ uptake in synaptosomal preparations.