Differentiation of Ca2+ pumps linked to plasma membrane and endoplasmic reticulum in the microsomal fraction from intestinal smooth muscle

ATP promotes ⁴⁵Ca uptake by the microsomal fraction from the longitudinal smooth muscle of guinea-pig ileum and this uptake is stimulated by oxalate. As the microsomal fraction is made up of various subcellular entities, we examined the localization of the Ca²⁺-transport activity by density gradient centrifugation, taking advantage of the selective effect of digitonin (at low concentration) on the density of plasmalemmal elements. When the ⁴⁵Ca-uptake activity was measured in the absence of oxalate, its behavior in subfractionation experiments closely paralleled that of the plasmalemmal marker 5′-nucleotidase. In contrast, the additional Ca²⁺-transport activity elicited by oxalate behaved like NADH-cytochrome $\text{c}$ reductase, a putative endoplasmic reticulum marker. The endoplasmic reticulum vesicles constituted only a small part of the membranes in the microsomal fraction, which explains that their Ca²⁺-storage capacity was not detectable in the absence of Ca²⁺-trapping agent. Low digitonin concentrations selectively increased the Ca²⁺ permeability of the plasmalemmal vesicles. The two Ca²⁺-transport activities were further differentiated by their distinct sensitivities to K⁺, vanadate and calmodulin. In this respect, the oxalte-insensitive and oxalate-stimulated Ca²⁺-transport systems resembled, respectively, the sarcolemmal and sarcoplasmic reticulum Ca²⁺ pumps in cardiac and skeletal muscle, in accordance with the subcellular locations established by density gradient centrifugation.     

Differentiation of Ca pumps linked to plasma membrane and endoplasmic reticulum in the microsomal fraction from intestinal smooth muscle

ATP promotes ⁴⁵Ca uptake by the microsomal fraction from the longitudinal smooth muscle of guinea-pig ileum and this uptake is stimulated by oxalate. As the microsomal fraction is made up of various subcellular entities, we examined the localization of the Ca²⁺-transport activity by density gradient centrifugation, taking advantage of the selective effect of digitonin (at low concentration) on the density of plasmalemmal elements. When the ⁴⁵Ca-uptake activity was measured in the absence of oxalate, its behavior in subfractionation experiments closely paralleled that of the plasmalemmal marker 5′-nucleotidase. In contrast, the additional Ca²⁺-transport activity elicited by oxalate behaved like NADH-cytochrome c reductase, a putative endoplasmic reticulum marker. The endoplasmic reticulum vesicles constituted only a small part of the membranes in the microsomal fraction, which explains that their Ca²⁺-storage capacity was not detectable in the absence of Ca²⁺-trapping agent. Low digitonin concentrations selectively increased the Ca²⁺ permeability of the plasmalemmal vesicles. The two Ca²⁺-transport activities were further differentiated by their distinct sensitivities to K⁺, vanadate and calmodulin. In this respect, the oxalte-insensitive and oxalate-stimulated Ca²⁺-transport systems resembled, respectively, the sarcolemmal and sarcoplasmic reticulum Ca²⁺ pumps in cardiac and skeletal muscle, in accordance with the subcellular locations established by density gradient centrifugation.     

Sodium/calcium exchange in smooth-muscle microsomal fractions.

The existence of Na+ -dependent Ca2+ transport was investigated in microsomal fractions from the longitudinal smooth muscle of the guinea-pig ileum and from the rat aorta, and its activity was compared with that of the plasmalemmal ATP-dependent Ca2+ pump previously identified in these preparations. The rate of Ca2+ release from plasmalemmal vesicles previously loaded with Ca2+ through the ATP-dependent Ca2+ pump was transiently faster in the presence of 150 mM-NaCl in the medium than in the presence of 150 mM-KCl or -LiCl or 300 mM-sucrose. Na+-loaded vesicles took up Ca2+ when an outwardly directed Na+ gradient was formed across the membrane. The Ca ionophore A23187 induced a rapid release of 85% of the sequestered Ca2+, whereas only 15% was displaced by La3+. Ca2+ accumulated by the Na+-induced Ca2+ transport was released by the addition of NaCl, but not KCl, to the medium. Ca2+ uptake in Na+-loaded vesicles was inhibited in the presence of increasing NaCl concentration in the medium. Half-maximum inhibition was observed with 28 mM-NaCl. Data fitted the Hill equation, with a Hill coefficient (h) of 1.9. Na+-induced Ca2+ uptake was a saturable function of Ca2+ concentration in the medium. Half-maximum activity was obtained with 18 microM-Ca2+ in intestinal-smooth-muscle microsomal fraction and with 50 microM-Ca2+ in aortic microsomal fraction. The results suggest that in these membrane preparations a transmembrane movement of Ca2+ can be driven by a Na+ gradient. However, the Na+-induced Ca2+ transport had a lower capacity, a lower affinity and a slower rate than the ATP-dependent Ca2+ pump.     

Kinetic properties of the Ca2+-accumulation system of a rat liver microsomal fraction.

1. By using Ca-EGTA buffers, the Km for Ca2+ uptake into rat liver heavy microsomes (microsomal fraction) was found to be 0.2 microM free Ca2+. 2. In the absence of oxalate, these vesicles accumulate about 20 nmol of Ca2+/mg of protein. Efflux of Ca2+ from the vesicles is much faster at pH 7.6 than at pH 6.8, but does not apparently show saturation kinetics or any stringent requirement for external ions. 3. The steady-state distribution of Ca2+ between the microsomes and the medium in the presence of ATP and the absence of oxalate is dependent on Ca2+ load. When the vesicles are loaded to 50% capacity, the external free Ca2+ concentration is 70 nM. 4. The affinity of heavy microsomes for Ca2+ is such that is seems likely that they has a dominant role in the determination of cytoplasmic free Ca2+ concentrations.     

Calcium transport in endoplasmic reticulum of the rat liver during lipid peroxidation

Some parameters of calcium transport in rat liver microsomes under conditions of lipoperoxidation activation modelled by antioxidant deficiency (AOD) were studied. This process was shown to be associated with a sharp stimulation of NADPH- and ascorbate-dependent lipid peroxidation in hepatocyte endoplasmic reticulum. The activation of lipid peroxidation was accompanied by disturbances in the kinetic properties of Ca2(+)-ATPase. This was paralleled with a considerable decrease of the ATP-dependent 45Ca-accumulation, increase in the passive permeability of microsomal vesicles for Ca2+ and Ca2+ elevation in the microsomal fraction. The AOD-induced diminution of the Ca2(+)-pump efficiency was slightly prevented by injections of rats with the antioxidants, alpha-tocopherol acetate and ionol which enable Ca2+ compartmentation correction in liver cytosol and membrane fractions.     

Inhibition by orthovanadate of ATP-dependent Ca2+ transport in microsomes isolated from rat liver

A technique employing sucrose-density centrifugation for the enrichment of rat liver microsomes and rat liver plasma membranes in separate subcellular fractions is described. The fractions are enriched in glucose 6-phosphatase and 5'-nucleotidase, respectively, and are free of cytochrome oxidase activity. Vanadate-sensitive Ca2+ transport activity (half-maximal inhibition at approximately 10 microM vanadate, corresponding to approximately 12 nmol/mg of protein) was detected in only that fraction enriched in microsomal membranes. Inhibition by vanadate of ATP-dependent Ca2+ transport is noncompetitive with respect to added Ca2+ but competitive with respect to added ATP. Because it inhibits ATP-dependent Ca2+ transport in rat liver microsomes but not in rat liver plasma membranes, vanadate becomes a useful tool to distinguish in vitro between these two transport systems.     

Carbachol partially inhibits the plasma-membrane Ca2+-pump in microsomes from pig stomach smooth muscle

A plasmalemmal enriched membrane fraction, prepared from pig stomach smooth-muscle, contains a calmodulin-stimulated (Ca2+ + Mg2+)-ATPase and presents an ATP-dependent 45Ca-uptake. If these smooth-muscle strips are preincubated with 10(-3) M-carbachol, this Ca2+ + Mg2+)-ATPase and the 45Ca-uptake are reduced by 21.4% and 13.5%, respectively, as compared to controls. This inhibitory effect of carbachol can be completely blocked by atropine. Carbachol does neither affect the passive permeability of the microsomes to 45Ca, nor the passive 45Ca-binding to the vesicles. Neither does it exert an effect on the proportion of closed inside-out plasma-membrane vesicles. Likewise, preincubation of rat myometrium with 90 nM-oxytocin induces a 20.4% inhibition of the ATP-dependent 45Ca-uptake, without having an effect on the passive 45Ca-binding, the permeability to 45Ca or the sideness of the vesicles. From these results, it is concluded that some agonists as carbachol and oxytocin induce a decrease in the activity of the plasmalemmal Ca2+-pump.     

Glucose 6-phosphate stimulation of MgATP-dependent Ca2+ uptake by rat kidney microsomes

(1) The features of MgATP-dependent Ca2+ accumulation under stimulation with glucose 6-phosphate were studied in rat kidney microsomes. (2) Ca2+ accumulated in the presence of MgATP alone does not exceed approx. 2 nmol/mg protein. (3) Glucose 6-phosphate markedly stimulates Ca2+ accumulation, up to steady-state levels approx. 15-fold higher than in its absence. (4) The hydrolysis of glucose 6-phosphate by glucose-6-phosphatase is essential for the stimulation, as shown by inhibiting the glucose 6-phosphate hydrolysis with adequate concentrations of vanadate. Inorganic phosphate is accumulated in microsomal vesicles during glucose 6-phosphate-stimulated Ca2+ uptake in equimolar amounts with respects to Ca2+. (5) Increasing concentrations of glucose 6-phosphate result in increasing stimulations of Ca2+ uptake, until a maximal Ca2(+)-loading capacity of approx. 27 nmol/mg microsomal protein is reached. It is suggested that the enlargement of the kidney microsomal Ca2+ pool induced by glucose 6-phosphate (an important metabolite in kidney) might play a role in the regulation of Ca2+ homeostasis in kidney tubular cells.     

Liver cytosolic non-dialysable factor(s) can counteract GTP-dependent Ca2+ release in rat liver microsomal fractions

Readdition to rat liver microsomes of dialysed liver post-microsomal supernatant resulted in an almost complete inhibition of the Ca2+-releasing effect of GTP. Such inhibition was heat-labile, and was associated with non-ultrafiltrable supernatant components with a molecular weight higher than 30,000 D. A preliminary fractionation of liver supernatant showed that the inhibitory effect is recovered in the 40-50% ammonium sulfate-precipitated proteins, with an approx. 10-fold enrichment. The active ammonium sulfate fraction did not modify the GTP-induced Ca2+ increase of passive Ca2+ efflux from microsomes, nor did it affect microsomal GTP hydrolysis, which is likely required for its Ca2+ releasing effect. The active ammonium sulfate fraction appears to markedly favour the translocation of GTP-released Ca2+ into a microsomal GTP-insensitive pool. Separation of liver microsomes in smooth and rough fractions revealed that such GTP-insensitive Ca2+ pool is almost completely associated with smooth microsomes.     

Localization of ATP-dependent calcium transport activity in mouse pancreatic microsomes

Electron-dense deposits representing calcium oxalate crystals which result from ATP-dependent calcium uptake have been localized within vesicles of of a heavy microsomal fraction prepared from mouse pancreatic acini. In the absence of either ATP or oxalate, no electron-dense deposits could be observed. By subfractionation of microsomes on discontinuous sucrose gradients, it could be shown that the highest energy-dependent calcium transport activity was associated with the rough endoplasmic reticulum. In rough microsomes, the 45Ca2+-uptake measured was 7 times greater than that of smooth microsomes in the presence of ATP and oxalate and about 3 times greater in he presence of ATP alone. When ribosomes were released from the rough endoplasmic reticulum vesicles by treatment with KCl in the presence of puromycin, the stripped microsomes showed a 40% increase in the specific 45Ca2+-uptake activity measured in he presence of ATP and oxalate and an increase of 80 to 90% in the presence of ATP alone. From these results it can be concluded that the calcium transport activity of microsomes prepared from mouse pancreatic acini is located predominantly in the rough endoplasmic reticulum membrane.     

Subcellular origin of the oxalate- or inorganic phosphate-stimulated Ca2+ transport by smooth muscle microsomes: revisitation of the old problem by a new approach using saponin

Saponin, a cell-skinning reagent which perforates the cell membrane via its specific interaction with plasmalemmal cholesterol, was used to identify the subcellular origin of ATP-dependent Ca2+ accumulation in the presence and absence of inorganic phosphate and oxalate by microsomal fractions isolated from rat vas deferens and dog aorta. The purified plasma membranes from rat gastric fundus muscle, which elicit the stimulation of ATP-dependent Ca2+ accumulation by inorganic phosphate but not by oxalate, were used as a control reference. Saponin at concentrations effective for skinning smooth muscle fibres (10-50 micrograms/ml) inhibited Ca2+ binding in the absence of ATP to a similar extent in all fractions, but the inhibition of ATP-dependent Ca2+ accumulation was more pronounced in dog aorta microsomes and rat gastric fundus muscle plasma membranes than in rat vas deferens microsomes. The resistance of phosphate- and oxalate-stimulated ATP-dependent Ca2+ accumulation to inhibition by saponin was much greater in rat vas deferens than in dog aorta microsomes. Our results suggest that phosphate- and oxalate-stimulated ATP-dependent Ca2+ accumulation also occurs in plasma membrane vesicles isolated from smooth muscle and is by no means an unique property of endoplasmic reticulum.     

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.     

Ca2+- and inositol-1,4,5-triphosphate induced release of Ca2+ in the microsomal fraction of the rat brain

Using the fluorescent probes, Quin 2 and chlortetracycline, a comparative study of the Ca2+ and inositol-1.4.5-triphosphate (IP3)-induced Ca2+ release from rabbit skeletal muscle sarcoplasmic reticulum (SR) terminal cisterns and rat brain microsomal vesicles was carried out. It was shown that Ca2+ release from rat brain microsomal vesicles is induced both by IP3 and Ca2+, whereas that in SR terminal cisterns is induced only by Ca2+. Data from chlorotetracycline fluorescence analysis revealed that CaCl2 (50 microM) causes the release of 15-20% and 40-50% of the total Ca2+ pool accumulated in rat brain microsomal vesicles and rabbit SR terminal cisterns, respectively. Using Quin 2, it was found that IP3 used at the optimal concentration (1.5 mM) caused the release of 0.4-0.6 nmol of Ca2+ per mg microsomal protein, which makes up to 10-15% of the total Ca2+ pool. IP3 does not induce Ca2+ release in SR. Preliminary release of Ca2+ from brain microsomes induced by IP3 diminishes the liberation of this cation induced by Ca2+. It is suggested that brain microsomes contain a Ca2+ pool which is exhausted under the action of the both effectors, Ca2+ and IP3.     

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.     

Mechanism of arachidonic acid-induced Ca2+ mobilization from rat liver microsomes

Recent evidence indicates that unesterified arachidonic acid functions as a mediator of intracellular Ca2+ mobilization by inducing Ca2+ release from the endoplasmic reticulum of pancreatic islet beta cells in a manner closely similar to that of inositol 1,4,5-trisphosphate. To test the generality and explore the mechanism of this phenomenon we have examined the effects of arachidonic acid on calcium accumulation and release by hepatocyte subcellular fractions enriched in endoplasmic reticulum (microsomes). At concentrations above 0.017 mumol/mg microsomal protein, arachidonate induced rapid (under 2 min) 45Ca2+ release from microsomes that had been preloaded with 45Ca2+. Arachidonate also suppressed microsomal 45Ca2+ accumulation when present during the loading period, as reflected by reduction both of 45Ca2+ accumulation at steady state and of the rate of uptake. Neither the cyclooxygenase inhibitor indomethacin nor the lipoxygenase/cyclooxygenase inhibitor BW755C suppressed arachidonate-induced 45Ca2+ release, indicating that this effect was not dependent upon oxygenation of the fatty acid to metabolites. The long-chain unsaturated fatty acids oleate and linoleate were less potent than arachidonate in inducing 45Ca2+ release, and the saturated fatty acid stearate did not exert this effect. Albumin prevented 45Ca2+ release by arachidonate, presumably by binding the fatty acid. As is the case for inositol 1,4,5-trisphosphate, the ability of arachidonate to induce 45Ca2+ release was dependent on the ambient free Ca2+ concentration. Arachidonate did not influence microsomal membrane permeability or Ca2+-ATPase activity and may exert its effects on microsomal Ca2+ handling by activation of a Ca2+ extrusion mechanism or by dissociating Ca2+ uptake from Ca2+-ATPase activity.     

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.     

Electron cytochemistry of oxalate-stimulated calcium uptake in microsomes from the smooth muscle of the pig stomach

Summary A microsomal fraction was isolated from the smooth muscle of the antrum of the pig stomach by differential centrifugation. Electron microscopy of the negatively stained material showed that this fraction is heterogeneous in composition. The microsomes accumulated calcium in the presence of ATP, magnesium and oxalate. The amount of calcium taken up per mg protein was in the same range as observed for other smooth muscle microsomal preparations. Although this amount is much smaller than that in the microsomal fraction of skeletal muscle, calcium oxalate crystals were formed in some vesicles, as occurs in the skeletal muscle fragmented sarcoplasmic reticulum. Through the presence of the calcium oxalate crystals, many of these vesicles acquired sufficient mass and density to allow them to be isolated by centrifugation. A purification of about 40 fold in terms of calcium content was reached.     

Parotid microsomal Ca2+ transport. Subcellular localization and characterization.

Rat parotid gland homogenates were fractionated into mitochondrial, heavy microsomal and light microsomal fractions by differential centrifugation. ATP-dependent 45Ca2+ uptake by the subcellular fractions paralleled the distribution of NADPH-cytochrome c reductase, an enzyme associated with the endoplasmic reticulum. The highest rate of Ca2+ uptake was found in the heavy microsomal fraction. Ca2+ uptake by this fraction was dependent on the presence of ATP and was sustained at a linear rate by 5 mM-oxalate. Inhibitors of mitochondrial Ca2+ transport had no effect on the rate of Ca2+ uptake. Na+ and K+ stimulated Ca2+ uptake. At optimal concentrations. Na+ stimulated Ca2+ uptake by 120% and K+ stimulated Ca2+ uptake by 260%. Decreasing the pH from 7.4 to 6.8 had little effect on Ca2+ uptake. The Km for Ca2+ uptake was 3.7 microM free Ca2+ and 0.19 mM-ATP. Vanadate inhibited Ca2+ uptake; 60 microM-vanadate inhibited the rate of Ca2+ accumulation by 50%. It is concluded that the ATP-dependent Ca2+ transport system is located on the endoplasmic reticulum and may play a role in maintaining intracellular levels of free Ca2+ within a narrow range of concentration.     

Characteristics of bile acid-mediated Ca2+ release from permeabilized liver cells and liver microsomes

Saponin-treated liver cells and a microsomal fraction were used to characterize the mechanism of the Ca2+ release induced by different bile acids. The saponin-treated cells accumulated 0.8-1 nmol/mg of protein of the medium Ca2+ in a nonmitochondrial, high affinity, and inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3)-sensitive Ca2+ pool. Three of five bile acids tested, lithocholate and the conjugates taurolithocholate and taurolithocholate sulfate, released 85% of the Ca2+ pool within 45-60 s and with ED50 from 16 to 28 microM. Ins(1,4,5)P3 released 80% from the same Ca2+ pool with an ED50 of 0.3 microM. The Ca2+-Mg2+-ATPase inhibitor vanadate (1 mM) had no effect on the Ca2+ released by the bile acids and Ins(1,4,5)P3. The Ins(1,4,5)P3-binding antibiotic neomycin (1 mM) and the receptor competitor heparin (16 micrograms/ml) abolished the releasing effect of Ins(1,4,5)P3 but had no effect on the bile acid-mediated Ca2+ release. The 45Ca2+ accumulated by the microsomal fraction (8 nmol of 45Ca2+/mg of protein) was released by the bile acids within 45-90 s and with an ED50 of 17 microM. In contrast, the bile acids had no effect on the Ca2+ permeability of other natural and artificial membranes. The resting 45Ca2+ influx of intact cells (0.45 nmol/mg of protein/min), the 45Ca2+ accumulated by mitochondria (2-13 nmol of 45Ca2+/mg of protein), and the 45Ca2+ trapped in sonicated phosphatidylcholine vesicles (5 mM 45Ca2+) were not altered by the different bile acids. These results suggest that the Ca2+ release initiated by lithocholate and its conjugates results from a direct action on the Ca2+ permeability of the Ins(1,4,5)P3-sensitive pool. It is not mediated by Ins(1,4,5)P3 or via activation of the Ins(1,4,5)P3 receptor, and it is specific for the membrane of the internal pool.     

Effects of the anti-calmodulin drugs calmidazolium and trifluoperazine on 45Ca transport in plasmalemmal vesicles from gastric smooth muscle

The anti-calmodulin drugs calmidazolium (CMZ) and trifluoperazine (TFP) were shown to have a number of effects on 45Ca transport by plasmalemmal vesicles from gastric smooth muscle. Although these compounds produced the expected dose-dependent inhibition of the plasmalemmal ATP-dependent Ca2+ transport system, they also evoked a Ca2+ release comparable to that observed in the presence of the Ca2+ ionophore, ionomycin. This increased transmembrane Ca2+ flux was so large that it accounted for much of the apparent decrease in 45Ca uptake produced by these agents. Thus, direct effects of CMZ and TFP on ATP-dependent 45Ca uptake could only be reliably assessed for brief (less than or equal to 30 seconds) drug exposures. The explanation for the observed effects of CMZ and TFP on membrane Ca2+ permeability is unclear. The increased transmembrane Ca2+ flux may reflect nonspecific effects on membrane permeability or it may reflect a specific interaction of the anticalmodulin drugs with a Ca2+ release channel or with the Ca2+ transport ATPase. In any case, these results suggest the need for caution in the design and interpretation of studies using both CMZ and TFP as anticalmodulin agents.