The effect of verdazyl radicals on Ca2+ metabolism in preparations of heavy sarcoplasmic reticulum

Three verdazyl radicals were studied for their effect on calcium accumulation and outflux (passive and Ca- or caffeine-induced) and conformational state of the Ca-ATPase. All three compounds differently affected the ATP-dependent Ca-accumulation. Their effect on the Ca-release from the sarcoplasmic reticulum vesicles could not be explained by their influence on the Ca-accumulation system. The Ca2+ amount liberated by the calcium or caffeine addition was equal in both cases but was modified differently by the used verdazyl compounds. The data obtained suggest that Ca-induced and caffeine-induced calcium release is realized by different mechanisms.     

Conformational changes of the Ca2+-ATPase as early events of Ca2+ release from sarcoplasmic reticulum

Rabbit skeletal sarcoplasmic reticulum vesicles were loaded with Ca2+ by ATP-dependent Ca2+ accumulation in the presence of low [Mg2+] (0.2-0.5 mM), and Ca2+ release was induced by addition of caffeine or ADP or by means of a Ca2+ jump. The levels of the phosphorylated intermediate (EP) and the tryptophan fluorescence of the Ca2+-ATPase were monitored during both the Ca2+ accumulation and the induced Ca2+ release using fast kinetic techniques. During Ca2+ uptake, both the EP level and the tryptophan fluorescence gradually decreased following a time course similar to that of the Ca2+ accumulation. Upon inducing Ca2+ release by addition of either caffeine or ADP, there was a transient increase of the EP level (from 0.3-0.5 to 1-2 nmol/mg protein) preceding the release of Ca2+. Similarly, a transient increase of the tryptophan fluorescence prior to Ca2+ release produced by the application of a Ca2+ jump was also found. These results indicate that the Ca2+-ATPase enzyme undergoes a rapid conformational change in response to triggering of Ca2+ release.     

The effect of oxytocin and sigetin on Ca2+ transport in the fraction of plasma membranes of myometrial cells

Oxytocin and sigetin were studied for their effect on the active and passive transport of Ca2+ in the fraction of myometrium sarcolemma in women. Oxytocin (5.10(-7) M) introduced into the sarcolemma vesicles and sigetin (5.10(-3) M) added into the incubation medium inhibit Mg2+, ATP-dependent accumulation of Ca2+ in these structures. The both agents in the mentioned concentration do not affect the passive release of cation from vesicles. A conclusion is drawn that inhibition of the calcium pump of myometrium cell plasma membranes underlies the physiological action of oxytocin and sigetin as stimulators of the contractile activity of the myometrium.     

Histamine and a guanine nucleotide increase calcium permeability in pig aortic microsomal fractions.

ATP-dependent Ca2+ accumulation was measured in pig aortic microsomal fractions containing plasmalemma and endoplasmic reticulum. In vesicles sonicated with histamine, to allow access to internally located receptor sites, guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG), added to activate externally located guanine-nucleotide-transducing proteins, caused a concentration-dependent decrease in steady-state Ca2+ accumulation that was reversed by guanosine 5'-[beta-thio]diphosphate. In the presence of p[NH]ppG, sonication with histamine produced a concentration-dependent inhibition of Ca2+ accumulation that could be antagonized by the H1 antagonist mepyramine, but not by the H2 antagonist cimetidine. The inhibition of steady-state Ca2+ accumulation could have resulted from an inhibition of ATP-dependent Ca2+ uptake or a stimulation of Ca2+ release. We observed, however, that p[NH]ppG plus histamine stimulated, rather than inhibited, Ca2(+)-ATPase activity. We concluded that p[NH]ppG and histamine acted together to increase Ca2+ permeability. In support of this, p[NH]ppG accelerated efflux of Ca2+ from passively loaded vesicles sonicated with, but not without, histamine. The effect of p[NH]ppG was unlikely to be due to Ins(1,4,5)P3 (and hence release from endoplasmic-reticulum vesicles), since addition of Ins(1,4,5)P3 to vesicles sonicated with histamine did not alter steady-state Ca2+ accumulation. Our results therefore suggest that histamine and p[NH]ppG increased the permeability of the plasmalemma vesicles and may thus model the process of receptor-mediated Ca2+ entry into intact cells.     

Ca2+ transport studied with arsenazo III in Tetrahymena microsomes. Effects of calcium ionophore A23187 and trifluoperazine

Transport of Ca2+ in microsomal membrane vesicles of the Tetrahymena has been investigated using arsenazo III as a Ca2+ indicator. The microsomes previously shown to carry a Mg2+-dependent, Ca2+-stimulated ATPase (Muto, Y. and Nozawa, Y. (1984) Biochim. Biophys. Acta 777, 67-74) accumulated calcium upon addition of ATP and Ca2+ sequestered into microsomal vesicles was rapidly discharged by the Ca2+ ionophore A23187. Kinetic studies indicated that the apparent Km for free Ca2+ and ATP are 0.4 and 59 microM, respectively. The Vmax was about 40 nmol/mg protein per min at 37 degrees C. The calcium accumulated during ATP-dependent uptake was released after depletion of ATP in the incubation medium. Furthermore, addition of trifluoperazine which inhibited both (Ca2+ + Mg2+)-ATPase and ATP-dependent Ca2+ uptake rapidly released the calcium accumulated in the microsomal vesicles. These observations suggest that Tetrahymena microsome contains both abilities to take up and to release calcium and may act as a Ca2+-regulating site in this organism.     

4',6-Diamidino-2-phenylindole, a novel conformational probe of the sarcoplasmic reticulum Ca2+ pump, and its effect on Ca2+ release

Sarcoplasmic reticulum vesicles were noncovalently labeled at micromolar concentrations with the polycationic fluorescent reagent 4',6-diamidino-2-phenylindole (DAPI), and changes in the fluorescence intensity of the membrane-bound dye associated with functions of the Ca2+ pump and Ca2+ release were investigated. It was found that 1) DAPI fluorescence changed in the [Ca2+] range in which high affinity Ca2+ binding to the Ca2+-ATPase takes place. The time course of the Ca2+-induced changes of DAPI fluorescence was essentially the mirror image of that of tryptophan fluorescence. 2) The fluorescence intensity of bound DAPI decreased upon increase of the intravesicular [Ca2+] by either ATP-dependent Ca2+ accumulation or incubation with millimolar Ca2+ in the presence of a calcium ionophore. 3) Upon induction of Ca2+ release by adding caffeine after the completion of Ca2+ uptake, DAPI fluorescence showed transient changes. Two classes of binding sites of the sarcoplasmic reticulum membrane for DAPI were clearly distinguishable: a high affinity site (Ka = 3.0 X 10(5) M-1) with a capacity of about 1 mol/mol of Ca2+-ATPase (8.0 nmol/mg of protein) and low affinity sites with about 20-fold lower affinity and 10-fold larger capacity. The partially purified Ca2+-ATPase showed similar characteristics of high affinity DAPI binding, suggesting that DAPI bound to its high affinity site on the Ca2+-ATPase monitors the enzyme conformational changes coupled with the events described above. The high affinity binding of DAPI to the enzyme led to an increase of the initial rate of Ca2+ uptake and the inhibition of Ca2+ release induced by caffeine or ionic replacement. These results suggest that the Ca2+-ATPase is involved in some steps of the Ca2+ release mechanism.     

Activation of the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum by caffeine and related compounds

The caffeine-sensitive Ca2+ release pathway in skeletal muscle was identified and characterized by studying the release of 45Ca2+ from heavy sarcoplasmic reticulum (SR) vesicles and by incorporating the vesicles or the purified Ca2+ release channel protein complex into planar lipid bilayers. First-order rate constants for 45Ca2+ efflux of 1 s-1 were obtained in the presence of 1-10 microM free Ca2+ or 2 X 10(-9) M free Ca2+ plus 20 mM caffeine. Caffeine- and Ca2+-induced 45Ca2+ release were potentiated by ATP and Mg.ATP, and were both inhibited by Mg2+. Dimethylxanthines were similarly (3,9-dimethylxanthine) or more (1,7-, 1,3-, and 3,7-dimethylxanthine) effective than caffeine in increasing the 45Ca2+ efflux rate. 1,9-Dimethylxanthine and 1,3-dimethyluracil (which lacks the imidazole ring) did not appreciably stimulate 45Ca2+ efflux. Recordings of calcium ion currents through single channels showed that the Ca2+- and ATP-gated SR Ca2+ release channel is activated by addition of caffeine to the cis (cytoplasmic) and not the trans (lumenal) side of the channel in the bilayer. The single channel measurements further revealed that caffeine activated Ca2+ release by increasing the number and duration of open channel events without a change of unit conductance (107 pS in 50 mM Ca2+ trans). These results suggest that caffeine exerts its Ca2+ releasing effects in muscle by activating the high-conductance, ligand-gated Ca2+ release channel of sarcoplasmic reticulum.     

Calcium transport mechanisms in basolateral plasma membrane-enriched vesicles from rat parotid gland.

Ca2+ transport was studied by using basolateral plasma membrane vesicles from rat parotid gland prepared by a Percoll gradient centrifugation method. In these membrane vesicles, there were two Ca2+ transport systems; Na+/Ca2+ exchange and ATP-dependent Ca2+ transport. An outwardly directed Na+ gradient increased Ca2+ uptake. Ca2+ efflux from Ca2+-preloaded vesicles was stimulated by an inwardly directed Na+ gradient. However, Na+/Ca2+ exchange did not show any 'uphill' transport of Ca2+ against its own gradient. ATP-dependent Ca2+ transport exhibited 'uphill' transport. An inwardly directed Na+ gradient also decreased Ca2+ accumulation by ATP-dependent Ca2+ uptake. The inhibition of Ca2+ accumulation was proportional to the external Na+ level. Na+/Ca2+ exchange was inhibited by monensin, tetracaine and chlorpromazine, whereas ATP-dependent Ca2+ transport was inhibited by orthovanadate, tetracaine and chlorpromazine. Oligomycin had no effect on either system. These results suggest that in the parotid gland cellular free Ca2+ is extruded mainly by an ATP-dependent Ca2+ transport system, and Na+/Ca2+ exchange may modify the efficacy of that system.     

Calcium release from vesicles of heavy sarcoplasmic reticulum of rabbit skeletal muscles

The release of Ca2+ from vesicles of heavy sarcoplasmic reticulum after its accumulation due to hydrolysis of ATP, GTP, CTP, UTP or ITP has been studied using Antipyrylazo III, a metal-chromic Ca-indicator. All the studied substrates of the Ca-pump provide Ca2+ accumulation inside the heavy sarcoplasmic reticulum vesicles, the spontaneous Ca2+ outflux rate being different for different nucleoside triphosphates. It is only ATP that provides Ca-(caffeine)-induced Ca2+ release, however AMP, ADP, beta, gamma-methylene-ATP induce Ca2+ ejection in the presence of nonadenylic nucleotides. The ruthenium red (10(-7M) inhibits the induced ejection of Ca2+ from vesicles of the heavy sarcoplasmic reticulum, but does not prevent the spontaneous release of Ca2+ in the same concentrations. A conclusion is drawn that besides Ca-channels sensitive to Ca2+ and caffeine in the presence of ATP (or to AMP, ADP, beta, gamma-methylene-ATP in the presence of nonadenylic nucleotides) and possessing high sensitivity to the ruthenium red there is another pathway for Ca2+ in the heavy reticulum membranes along which its spontaneous release occurs after the substrate exhaustion. It is supposed that this release is provided by the presence of the Ca-ATPase protein.     

Evidence against parallel operation of sodium/calcium antiport and ATP-driven calcium transport in plasma membrane vesicles from kidney tubule cells

The present study aimed to clarify the existence of a Na+/Ca2+ antiport device in kidney tubular epithelial cells discussed in the literature to represent the predominant mechanistic device for Ca2+ reabsorption in the kidney. Inside-out oriented plasma membrane vesicles from tubular epithelial cells of guinea-pig kidney showed an ATP-driven Ca2+ transport machinery similar to that known to reside in the plasma membrane of numerous cell types. It was not affected by digitalis compounds which otherwise are well-documented inhibitors of Ca2+ reabsorption. The vesicle preparation contained high, digitalis-sensitive (Na+ + K+)-ATPase activities indicating its origin from the basolateral portion of plasma membrane. The operation of a Na+/Ca2+ antiport device was excluded by the findings that steep Ca2+ gradients formed by ATP-dependent Ca2+ accumulation in the vesicles were not discharged by extravesicular Na+, and did not drive 45Ca2+ uptake into the vesicles via a Ca2+-45Ca2+ exchange. The ATP-dependent Ca2+ uptake into the vesicles became increasingly depressed with time by extravesicular Na+. This was not due to an impairment of the Ca2+ pump itself, but caused by Na+/Ca2+ competition for binding sites on the intravesicular membrane surface shown to be important for high Ca2+ accumulation in the vesicles. Earlier observations on Na+-induced release of Ca2+ from vesicles pre-equilibrated with Ca2+, seemingly favoring the existence of a Na+/Ca2+ antiporter in the basolateral plasma membrane, were likewise explained by the occurrence of Na+/Ca2+ competition for binding sites. The weight of our findings disfavors the transcellular pathway of Ca2+ reabsorption through tubule epithelium essentially depending on the operation of a Na+/Ca2+ antiport device.     

Fluorescence conformational probe study of calcium release from sarcoplasmic reticulum

Sarcoplasmic reticulum isolated from rabbit skeletal muscle was labeled with a limited (0.625 nmol/mg sarcoplasmic reticulum protein) amount of the fluorescent thiol reagent N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide (DACM). The fluorescence intensity of the membrane-attached DACM decreased concurrently with (Ca2+ and caffeine)-induced Ca2+ release, depolarization-induced Ca2+ release and Ca2+-dependent dependent passive efflux of Ca2+. The decreased DACM fluorescence level initiated by a Ca2+ jump was subsequently reversed under passive efflux conditions when there was no ATP-dependent Ca2+ uptake, suggesting spontaneous closing of the channels. Therefore, the higher fluorescence level corresponds to a larger population of closed channels, whereas the lower level represents a larger population of opened channels. Under conditions when the Ca2+ release-coupled fluorescence change was maximal, a stoichiometric incorporation of DACM took place only into a 32-kDa protein. Furthermore, reconstituted vesicles, in which purified DACM-labeled 32-kDa protein was incorporated into unlabeled sarcoplasmic reticulum vesicles, were capable of both (Ca2+ and caffeine)-induced Ca2+ release and the release-coupled DACM fluorescence change. These results suggest that the 32-kDa protein is a constituent of the Ca2+ release channel or a protein which is in close contact with the channel.     

ATP-dependent transport of Ca2+ in myocardium sarcolemma vesicles and its activation by phorbol esters

Highly purified pig myocardium sarcolemma vesicles possess the Ca2+,Mg2+-ATPase activity (4.1 mumol Pi/mg protein/hour) and induce the ATP-dependent accumulation of 45Ca2+ (6.0 nmol/mg protein/min). This reaction is not stimulated by oxalate; Ca2+ are released from the vesicles by saponin and Na+ treatment, which suggests that Ca2+ transport against the concentration gradient is induced by myocardium sarcolemma vesicles and not by sarcoplasmic reticulum fragments. The phorbol ester possessing a biological activity of a growth-promoting factor and activating membrane-bound protein kinase C stimulates the Ca2+,Mg2+-ATPase activity and the ATP-dependent accumulation of Ca2+, whereas its counterpart devoid of biological activity does not influence Ca2+ transport. Polymixin B, a specific inhibitor of protein kinase C, prevents the activating effect of phorbol esters on Ca2+ accumulation inside the vesicles. It is suggested that the ATP-dependent transport of Ca2+ in myocardium sarcolemma is controlled by Ca2+-phospholipid-dependent phosphorylation catalyzed by protein kinase C.     

Bromo-eudistomin D, a novel inducer of calcium release from fragmented sarcoplasmic reticulum that causes contractions of skinned muscle fibers

Bromo-eudistomin D induced a contraction of the chemically skinned fibers from skeletal muscle at concentrations of 10 microM or more. This contractile response to bromo-eudistomin D was completely blocked by 10 mM procaine. The extravascular Ca2+ concentrations of the heavy fractions of the fragmented sarcoplasmic reticulum (HSR) were measured directly by a Ca2+ electrode to examine the effect of bromo-eudistomin D on the sarcoplasmic reticulum. After the HSR was loaded with Ca2+ by the ATP-dependent Ca2+ pump, the addition of 10 microM bromo-eudistomin D caused Ca2+ release that was followed by spontaneous Ca2+ reuptake. In the presence of 2 microM ruthenium red or 4 mM MgCl2, no Ca2+ release was induced by 20 microM bromo-eudistomin D. The rate of 45Ca2+ efflux from HSR, which had been passively preloaded with 45Ca2+, was accelerated 7 times by 10 microM bromo-eudistomin D. The concentration of bromo-eudistomin D for half-maximum effect on the apparent efflux rate was 1.5 microM, while that of caffeine was 0.6 mM. The bromo-eudistomin D-evoked efflux of 45Ca2+ was abolished by 2 microM ruthenium red or 0.5 mM MgCl2. Bromo-eudistomin D was found to be 400 times more potent than caffeine in its Ca2+-releasing action but was similar in its action in other respects. These results indicate that bromo-eudistomin D may induce Ca2+ release from the sarcoplasmic reticulum through physiologically relevant Ca2+ channels.     

Enhanced Ca2+-induced calcium release by isolated sarcoplasmic reticulum vesicles from malignant hyperthermia susceptible pig muscle

To further define the possible involvement of sarcoplasmic reticulum calcium accumulation and release in the skeletal muscle disorder malignant hyperthermia (MH), we have examined various properties of sarcoplasmic reticulum fractions isolated from normal and MH-susceptible pig muscle. A sarcoplasmic reticulum preparation enriched in vesicles derived from the terminal cisternae, was further fractionated on discontinuous sucrose density gradients (Meissner, G. (1984) J. Biol. Chem. 259, 2365-2374). The resultant MH-susceptible and normal sarcoplasmic reticulum fractions, designated F0-F4, did not differ in yield, cholesterol and phospholipid content, or nitrendipine binding capacity. Calcium accumulation (0.27 mumol Ca/mg per min at 22 degrees C), Ca2+-ATPase activity (0.98 mumol Pi/mg per min at 22 degrees C), and calsequestrin content were also similar for MH-susceptible and normal sarcoplasmic reticulum fraction F3. To examine sarcoplasmic reticulum calcium release, fraction F3 vesicles were passively loaded with 45Ca (approx. 40 nmol Ca/mg), and rapidly diluted into a medium of defined Ca2+ concentration. Upon dilution into 1 microM Ca2+, the extent of Ca2+-dependent calcium release measured after 5 s was significantly greater for MH-susceptible than for normal sarcoplasmic reticulum, 65.9 +/- 2.8% vs. 47.7 +/- 3.9% of the loaded calcium, respectively. The C1/2 for Ca2+ stimulation of this calcium release (5 s value) from MH-susceptible sarcoplasmic reticulum also appeared to be shifted towards a higher Ca2+-sensitivity when compared to normal sarcoplasmic reticulum. Dantrolene had no effect on calcium release from fraction F3, however, halothane (0.1-0.5 mM) increased the extent of calcium release (5 s) similarly in both MH-susceptible and normal sarcoplasmic reticulum. Furthermore, Mg2+ was less effective at inhibiting, while ATP and caffeine were more effective in stimulating, this Ca2+-dependent release of calcium from MH-susceptible, when compared to normal sarcoplasmic reticulum. Our results demonstrate that while sarcoplasmic reticulum calcium-accumulation appears unaffected in MH, aspect(s) of the sarcoplasmic reticulum Ca2+-induced calcium release mechanism are altered. Although the role of the Ca2+-induced calcium release mechanism of sarcoplasmic reticulum in situ is not yet clear, our results suggest that an abnormality in the regulation of sarcoplasmic reticulum calcium release may play an important role in the MH syndrome.     

Effect of oxytocin on the calcium pump in myometrium sarcolemma

Oxytocin (10(-7) M) administered inside the myometrium sarcolemma vesicles closed outward by the cytoplasmic side is shown to inhibit Mg2+, ATP-dependent Ca2+ accumulation in these structures having no effect on the passive release of cation out of them. According to these results and to the data available in literature on the inhibitory action of the peptide hormone on Mg2+, Ca2+-ATPase of myometrium sarcolemma a conclusion is drawn that oxytocin inhibits the Ca pump activity in plasma membranes of the myometrium cells.     

Ca2+ currents and acid secretion in the isolated parietal cell involved in response to gastrin, compound 48/80, and ethylenediamine tetraacetic acid

In intact guinea pig parietal cells, gastrin or compound 48/80 caused an initial increase in cytosolic Ca2+ concentration and subsequent acid secretion, owing to release of intracellulary stored Ca2+ besides the Ca2+ entry from the extracellular space. However, the maximum gastrin-induced Ca2+ entry into the cell was delayed by 60 min, a time which coincided with sustained acid secretion (by gastrin) that was dependent on medium Ca2+. On the other hand, there are two ATP-dependent Ca2+-removal systems detected in either plasmalemma or smooth surfaced membrane besides that of mitochondria. The plasmalemmal Ca2+-removal system was dependent on calmodulin. Smooth surfaced membrane vesicles caused an ATP-dependent Ca2+ uptake that was almost similar to that taken by saponin-permiabilized cell. In this system (permeable cell), myo-inositol 1,4,5-triphosphate (InsP3) caused the release of ATP-accumulated Ca2+ into the cytosol, suggesting an ATP-dependent and InsP3-sensitive Ca2+ pool(s) is in or near the smooth surfaced membranes. The ATP-dependent Ca2+ uptake by vesicles was markedly enhanced by the stimulation of cells with gastrin, compound 48/80, or EDTA. The increase of this Ca2+ uptake in stimulated cells by plasmalemmal vesicles exceeded that by smooth surfaced ones. The increase of the Ca2+ uptake by plasmalemmal vesicles was abolished by the cease of intracellular Ca2+ release without Ca2+ entry. In addition, gastrin or compound 48/80 evoked an early Ca2+ efflux across the plasma membrane owing to a pump that was independent of medium Ca2+ in intact cells. These results suggest that in the first acid secretion by gastrin or others, the Ca2+ released, which may be derived from an ATP-dependent and InsP3-sensitive Ca2+ pool, is mainly pumped out by the plasmalemmal Ca2+-removal system rather than the intracellular Ca2+-removal system; whereas the sustained acid secretion by gastrin required medium Ca2+ and in this phase, Ca2+ efflux across the plasma membrane became lower, suggesting that an ATP-dependent Ca2+ pool may be replenished by Ca2+ entering from the extracellular space.     

Changes in ryanodine receptor-mediated calcium release during skeletal muscle differentiation.

We have observed a disparity between the actions of caffeine and ryanodine, two agents known to affect the same site of intracellular calcium (Ca2+) release in muscle. The site of intracellular Ca2+ release, the ryanodine receptor (RyR), is established as the route of Ca2+ movement from the sarcoplasmic reticulum (SR) to the cytosol during excitation-contraction coupling. We measured Ca2+ release fluorimetrically in both saponin-permeabilized and intact L6 cells, in response to known modulators (i.e., caffeine and ryanodine), during differentiation in vitro. The undifferentiated L6 cells showed little response to caffeine. However, a substantial caffeine-induced calcium release (caffCR) was evident by Day 3 of differentiation, and was nearly maximal by Day 7 of differentiation. By contrast, ryanodine failed to stimulate Ca2+ release until Day 4, lagging behind the caffeine response. Ryanodine-stimulated Ca2+ release was also maximal by Day 7. Higher concentrations of ryanodine, known to inhibit Ca2+ release, only began to affect caffCR at Day 4, indicating that cells were insensitive to both ryanodine stimulation and ryanodine inhibition prior to this time. Most of the results could be obtained both in permeabilized and intact cells. Using intact cells, we measured the time course of K+ -dependent (i.e., depolarization-induced) Ca2+ release. This time course matched caffeine and not ryanodine-induced Ca2+ release suggesting the action of caffeine was not due to Ca2+ release unrelated to excitation-contraction coupling. These findings suggest that ryanodine binding sites on the RyR may not be functional at early stages of muscle development, that ryanodine sensitivity is a poor indicator of Ca2+ flux through the RyR, or that other proteins are involved in Ca2+ release under certain circumstances.     

Calcium regulation by lens plasma membrane vesicles

The role of the plasma membrane in the regulation of lens fiber cell cytosolic Ca2+ concentration has been examined using a vesicular preparation derived from calf lenses. Calcium accumulation by these vesicles was ATP dependent, and was releasable by the ionophore A23187, indicating that calcium was transported into a vesicular space. Calcium accumulation was stimulated by Ca2+ (K1/2 = 0.08 microM Ca2+) potassium (maximally at 50 mM K+), and cAMP-dependent protein kinase; it was inhibited by both vanadate (IC50 = 5 microM) and the calmodulin inhibitor R24571 (IC50 = 5 microM), indicating that this pump was plasma-membrane derived and likely calmodulin dependent. Valinomycin, in the presence of K+, stimulated calcium uptake, suggesting that the calcium pump either countertransports K+, or is regulated in an electrogenic fashion. Inhibition of calcium uptake by selenite and p-chloromercuribenzoate demonstrates the presence of an essential -SH group(s) in this enzyme. Calcium release from calcium-filled lens vesicles was enhanced by Na+, demonstrating that these vesicles also contain a Na:Ca exchange carrier. p-Chloromercuribenzoate and p-chloromercuribenzoate sulfonic acid also promoted calcium release from calcium-filled vesicles, suggesting that this release, like calcium uptake, is in part mediated by a cysteine-containing protein. We conclude that lens fiber cell cytosolic Ca2+ concentration could be regulated by a number of plasma membrane processes. The sensitivity of both calcium uptake and release to -SH reagents has implications in lens cataract formation, where oxidation of lens proteins has been proposed to account for the elevated cytosolic Ca2+ in this condition.     

Novel ATP-dependent calcium transport component from rat liver plasma membranes. The transporter and the previously reported (Ca2+-Mg2+)-ATPase are different proteins

An ATP-dependent calcium transport component from rat liver plasma membranes was solubilized by cholate and reconstituted into egg lecithin vesicles by a cholate dialysis procedure. The uptake of Ca2+ into the reconstituted vesicles was ATP-dependent and the trapped Ca2+ could be released by A23187. Nucleotides, including ADP, UTP, GTP, CTP, GDP, AMP, and adenyl-5'-yl beta, gamma-imidophosphate, and p-nitrophenylphosphate did not substitute for ATP. The concentration of ATP required for half-maximal stimulation of Ca2+ uptake into the reconstituted vesicles was 6.2 microM. Magnesium was required for calcium uptake. Inhibitors of mitochondrial calcium-sequestering activities, i.e. oligomycin, sodium azide, ruthenium red, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and valinomycin did not affect the uptake of Ca2+ into the vesicles. In addition, strophanthidin and p-chloromercuribenzoate did not affect the transport. Calcium transport, however, was inhibited by vanadate in a concentration-dependent fashion with a K0.5 of 10 microM. A calcium-stimulated, vanadate-inhibitable phosphoprotein was demonstrated in the reconstituted vesicles with an apparent molecular weight of 118,000 +/- 1,300. These properties of Ca2+ transport by vesicles reconstituted from liver plasma membranes suggest that this ATP-dependent Ca2+ transport component is different from the high affinity (Ca2+-Mg2+)-ATPase found in the same membrane preparation (Lotersztajn, S., Hanoune, J. and Pecker, F. (1981) J. Biol. Chem. 256, 11209-11215; Lin, S.-H., and Fain, J.N. (1984) J. Biol. Chem. 259, 3016-3020). When the entire reconstituted vesicle population was treated with ATP and 45Ca in a buffer containing oxalate, the vesicles with Ca2+ transport activity could be separated from other vesicles by centrifugation in a density gradient and the ATP-dependent Ca2+ transport component was purified approximately 9-fold. This indicates that transport-specific fractionation may be used to isolate the ATP-dependent Ca2+ transport component from liver plasma membrane.     

Depolarization promotes caffeine induced [3H]-noradrenaline release in calcium-free solution from peripheral sympathetic nerves

The transmitter releasing action of caffeine was studied in the absence of extracellular Ca2+ from the peripheral sympathetic nerves of the rabbit main pulmonary artery. Caffeine (10 mM) increased the release of [3H]-noradrenaline moderately, but not significantly in Ca2(+)-free (+1 mM EGTA) Krebs solution. When peripheral nerve endings/varicosities were depolarized by elevating extracellular K+ to 47.2 mM and 70.8 mM in Ca2(+)-free solution, the transmitter releasing effect of 10 mM caffeine became significant. Ca2+ removal itself transiently increased the [3H]-noradrenaline outflow. In the individual experiments the amount of the caffeine evoked transmitter release at 47.2 mM and 70.8 mM K(+)-depolarization was inversely correlated to the release evoked by Ca2(+)-removal. Our results suggest that caffeine-sensitive calcium stores are present in peripheral nerve terminals of rabbit pulmonary artery, and part of the caffeine sensitive calcium stores may discharge during Ca2(+)-removal from the extracellular solution.