Comparison of Ca uptake characteristics of microsomal fractions isolated from unfertilized and fertilized sea urchin eggs

The microsomal fraction isolated from sea urchin H. pulcherrimus eggs has the ability to actively accumulate Ca²⁺ in the presence of ATP. The Ca²⁺ uptake was sustained by addition of oxalate and was apparently insensitive to sodium azide. The sequestered microsomal Ca was readily released by the divalent cation ionophore A23187. The microsomal fraction obtained from fertilized eggs accumulated Ca²⁺ about five times more quickly than did that from unfertilized eggs. The increased Ca²⁺ uptake by microsomal fraction obtained from fertilized eggs was due to an increase in the maximum velocity of Ca²⁺ uptake and there was no difference in K_m for calcium between the two fractions.     

Calcium pump activity of the renal plasma membrane and renal microsomes

ATP-dependent Ca²⁺ uptake distinct from that of the mitochondria is found in both plasma membrane and microsomal membranes of rat kidney. Activity attributed to these fractions is enhanced by ammonium oxalate and is apparently insensitive to NaN₃. In contrast, rat kidney mitochondrial Ca²⁺ uptake is blocked by NaN₃. The pH of optimal activity is significantly higher for the mitochondrial fraction. Microsomal membrane Ca²⁺ uptake differs from that of the plasma membrane. Microsomal membranes are four times as active as the plasma membrane at high (5 mM) ATP levels. Apparent K_m values for Mg²⁺-ATP differ in the two preparations with a higher affinity for Mg²⁺-ATP found in the plasma membrane Ca²⁺ uptake activity of the plasma membrane preparation is readily inhibited by Na⁺. Sucrose gradient density fractionation indicates that the observed microsomal membrane Ca²⁺ pump activity is associated with membrane vesicles derived from the endoplasmic reticulum. Ca²⁺ pump activity of both plasma membrane and microsomal fraction is depressed din the adrenalectomized rat. This activity is not restored by a single natriuretic dose of aldosterone.     

Effects of ruthenium red on Ca2+ uptake and ATPase of sarcoplasmic reticulum of rabbit skeletal muscle

Ruthenium red, a powerful inhibitor of Ca²⁺ transport by mitochondria, does not inhibit the active Ca²⁺ uptake by sarcoplasmic reticulum isolated from rabbit skeletal muscle promoted by 5 mM ATP-Mg in the presence or absence of potassium oxalate. Although concentrations of ruthenium red up to 100 μM do not affect the active uptake of Ca²⁺, 25 μM of the inorganic dye inhibit the passive binding of Ca²⁺ by about 50%. This inhibitory effect is observed in sarcoplasmic reticulum even after its lipid fraction is extracted with acetone.Although active Ca²⁺ uptake by sarcoplasmic reticulum is not inhibited by ruthenium red, in the absence of oxalate it inhibits significantly the Ca²⁺-dependent ATPase activity but not the Mg²⁺-ATPase. However, if potassium oxalate is present, the Ca²⁺-stimulated ATPase is not sensitive to the dye. It is not clear how oxalate functions to protect the Ca²⁺-ATPase against the inhibitor effect of ruthenium red.The high sensitivity to ruthenium red of the Ca²⁺ transport mechanism in mitochondria as compared to the Ca²⁺ transport in sarcoplasmic reticulum may be useful in determining the extent to which each organelle functions in the cell to regulate intracellular free Ca²⁺.     

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.     

ATP-dependent strontium uptake by basolateral membrane vesicles from rat renal cortex in the absence or presence of calcium

ATP-dependent Sr²⁺ transport was examined in vitro using basolateral membrane (BLM) vesicles isolated from rat renal cortex to clarify the discrimination mechanisms between strontium (Sr) and calcium (Ca) in renal tubules during reabsorption. ATP-dependent Sr²⁺ uptake and Ca²⁺ uptake were observed in renal BLM vesicles and were inhibited by vanadate. Hill plots indicate similar kinetic behavior for Ca²⁺ and Sr²⁺ uptake. The apparentK _m andV _max of ATP-dependent Sr²⁺ uptake were both higher than those for Ca²⁺ uptake. ATP-dependent Sr²⁺ uptake by BLM vesicles diminished in the presence of 0.1 μM Ca²⁺ and was more markedly inhibited by 1 μM Ca²⁺. Hill plots of Sr²⁺ uptake data with and without 0.1 μM Ca²⁺ showed that the cooperative behavior of Sr²⁺ uptake was not changed by Ca²⁺. In the presence of 0.1 μM Ca²⁺, the affinity of the transport system for Sr²⁺ and the velocity of Sr²⁺ uptake in the BLM were both decreased. However, the rate of Ca²⁺ uptake was not diminished by Sr²⁺ concentrations of <1.6 μM. These results suggest that Ca²⁺ is preferentially transported in the renal cortex BLM when Ca²⁺ and Sr²⁺ are present at the same time.     

Influence of Ca<Superscript>2+</Superscript> ions on metabolism of active oxygen species in wheat calli cocultured with the bunt pathogen <Emphasis Type="Italic">Tilletia caries</Emphasis>

The effect of Ca²⁺ on morphophysiological parameters of wheat calli (Triticum aestivum L.) infected by the bunt pathogen Tilletia caries, in particular on the level of active oxygen species, activity of oxalate oxidase, peroxidase, and catalase is investigated. The concentration of O²⁻, H₂O₂, and activity of oxidoreductases (oxalate oxidase, peroxidase, and catalase) depended on the content of Ca²⁺ in the culture medium of calli. The increase of the concentration of Ca²⁺ ions in the culture medium led to forming of calli with high structure, induction of activity of oxalate oxidase and of some isoperoxidase, and to accumulation of active oxygen species. These changes contributed to inhibition of development of the fungus. So this dependence confirm the role of calcium as the intermediant in biochemical reactions related to the formation of the protective response of plant cells to biotic stress.     

ATP-dependent calcium transport in rat parotid basolateral membrane vesicles is modulated by membrane potential

Summary The ATP-dependent Ca²⁺ transport activity (T. Takuma, B.L. Kuyatt and B.J. Baum,Biochem. J. 227:239–245, 1985) exhibited by inverted basolateral membrane vesicles isolated from rat parotid gland was further characterized. The activity was dependent on Mg²⁺. Phosphate (5mm), but not oxalate (5mm), increased maximum Ca²⁺ accumulation by 50%. Half-maximal Ca²⁺ transport was achieved at 70nm Ca²⁺ in EGTA-buffered medium while maximal activity required >1 m Ca²⁺ (V _max=54 nmol/mg protein/min). Optimal rates of Ca²⁺ transport were obtained in the presence of KCl, while in a KCl-free medium (mannitol or sucrose) 40% of the total activity was achieved, which could not be stimulated by FCCP. The initial rate of Ca²⁺ transport could be significantly altered by preimposed membrane potentials generated by K⁺ gradients in the presence of valinomycin. Compared to the transport rate in the absence of membrane potential, a negative (interior) potential stimulated uptake by 30%, while a positive (interior) potential inhibited uptake. Initial rates of Ca²⁺ uptake could also be altered by imposing pH gradients, in the absence of KCl. When compared to the initial rate of Ca²⁺ transport in the absence of a pH gradient, pH _i =7.5/pH _o =7.5; the activity was 60% higher in the presence of an outwardly directed pH gradient, pH _i =7.5/pH _o =8.5; while it was 80% lower when an inwardly directed pH gradient was imposed, pH _i =7.5/pH _o =6.2. The data show that the ATP-dependent Ca²⁺ transport in BLMV can be modulated by the membrane potential, suggesting therefore that there is a transfer of charge into the vesicle during Ca²⁺ uptake, which could be compensated by other ion movements.     

Phosphate and calcium uptake in beech (Fagus sylvatica) in the presence of aluminium and natural fulvic acids

In the present study we examine the effects of Al on the uptake of Ca²⁺ and H₂PO^-₄ in beech (Fagus sylvatica L.) grown in inorganic nutrient solutions and nutrient solutions supplied with natural fulvic acids (FA). All the solutions used were chemically well characterized. The uptake of Al by roots of intact plants exposed to solutions containing 0, 0.15 or 0.3 mM AlCl₃ for 24 h, was significantly less if FA (300 mg l⁻¹) were also present in the solutions. The Ca²⁺(⁴⁵Ca²⁺) uptake was less affected by Al in solutions supplied with FA than in solutions without FA. There was a strong negative correlation between the Al and Ca²⁺ uptake (r²=0.98). When the Al and Ca²⁺ (⁴⁵Ca²⁺) uptake were plotted as a function of the Al³⁺ activity (or concentration of inorganic mononuclear Al), almost the same response curves were obtained for the -FA and +FA treatments. We conclude that FA-complexed Al was not available for root uptake and therefore could not affect the Ca²⁺ uptake. The competitive effect of Al on the Ca²⁺ uptake was also shown in a 5-week cultivation experiment, where the Ca concentration in shoots decreased at an AlCl₃ concentration of 0.3 mM. The effect of Al on H₂PO⁻₄ uptake was more complex. The P content in roots and shoots was not significantly affected, compared with the control, by cultivation for 5 weeks in a solution supplied with 0.3 mM AlCl₃, despite a reduction of the H₂PO⁻₄ concentration in the nutrient solution to about one-tenth. At this concentration Al obviously had a positive effect on H₂PO⁻₄ uptake. The presence of FA decreased ³²P-phosphate uptake by more than 60% during 24 h, and the addition of 0.15 or 0.3 mM AlCl₃ to these solutions did not alter the uptake of ³²P-phosphate.     

Demarcation of Ca2+ transport processes in guinea pig stomach smooth muscle

Summary Microsomal fractions were isolated from gastric antrum and fundus smooth muscle of guinea pigs. Ca²⁺ uptake into and Ca²⁺ release from the membrane vesicles were studied by a rapid filtration method, and Ca²⁺ transport properties of the different regions of the stomach were compared. ATP-dependent Ca²⁺ uptake was similar in microsomes isolated from both regions. This uptake was increased by oxalate and was not affected by NaN₃. Oxalate affected Ca²⁺ permeability of both antrum and fundus microsome vesicles similarly. Fundus microsome vesicles preincubated in 100mm NaCl and then diluted to 1/20 concentration with Na⁺-free medium had significantly higher ATP-independent Ca²⁺ uptake than vesicles preincubated in 100mm KCl and treated the same way. This was not true for antrum vesicles. Monensin abolished Na⁺-dependent Ca²⁺ uptake, and NaCl enhanced Ca²⁺ efflux from fundus microsome vesicles. The halflife values of Ca²⁺ loss from fundus vesicles in the presence of NaCl were significantly smaller than those in the presence of KCl. The release of Ca²⁺ from the vesicles within the first 3 min was accelerated by NaCl to three times that by KCl. However, NaCl had ro effect on Ca²⁺ release from antrum microsome vesicles.Results suggest two distinct mechanisms of stomach membrane Ca²⁺ transport: (1) ATP-dependent Ca²⁺ uptake and (2) Na⁺–Ca²⁺ exchange; the latter in the fundus only.     

Serotonin Regulation of Serotonin Uptake in RN46A Cells

1. Aim: The role of the serotonin transporter (SERT) is to remove serotonin (5-HT) from the synaptic space. In vitro studies have shown that 5-HT uptake via SERT is influenced by the availability of its substrate, 5-HT. We used RN46A cells, a line that expresses SERT, to investigate 5-HT regulation of 5-HT uptake and the intracellular signaling pathways involved. RN46A cells also express mRNAs for 5-HT receptors (5-HT_1A, 5-HT_1B, 5-HT_2A, and 5-HT_2C) and as cAMP and intracellular Ca²⁺ are modulated by different 5-HT receptors, we studied both pathways.2. Methods: 5-HT uptake was determined as imipramine-inhibitable uptake of [³H]5-HT, intracellular cAMP was measured by RIA and intracellular Ca²⁺ changes were determined using the ratiometric method of intracellular Ca²⁺ imaging.3. Results: For uptake experiments, cells were kept for 30 min either with or without 1 μM 5-HT in the medium before measuring uptake. Removal of 5-HT for 30 min significantly decreased [³H]5-HT uptake. The absence of 5-HT for 15 min failed to induce any changes in intracellular cAMP levels. Removal of 5-HT from the medium did not change intracellular Ca²⁺ levels either; however, adding 1 μM 5-HT after 5 min in 5-HT-free conditions rapidly increased intracellular Ca²⁺ levels in 50% of the cells. The remaining cells showed no changes in the intracellular Ca²⁺ levels.4. Conclusions: We have shown that in RN46A cells, that endogenously express SERT and mRNAs for several 5-HT receptors, changes in 5-HT levels influence 5-HT uptake rate as well as induce changes in intracellular Ca²⁺ levels. This suggests that 5-HT may utilize intracellular Ca²⁺ to regulate 5-HT uptake.     

Interaction of caffeine-, IP3- and vanadate-sensitive Ca2+ pools in acinar cells of the exocrine pancreas

Summary Previous studies have shown the existence of functionally distinguishable inositol 1,4,5-trisphosphate- (IP₃) sensitive and IP₃-insensitive nonmitochondrial intracellular Ca²⁺ pools in acinar cells of the exocrine pancreas. For further characterization of Ca²⁺ pools, endoplasmic reticulum (ER) membrane vesicles were separated by Percoll gradient centrifugation which allowed us to distinguish five discrete fractions designatedP ₁ toP ₅ from the top to the bottom of the gradient. Measuring Ca²⁺ uptake and Ca²⁺ release with a Ca²⁺ electrode, we could differentiate three nonmitochondrial intracellular Ca²⁺ pools; (i) an IP₃-sensitive Ca²⁺ pool (IsCaP), vanadate- and caffeine-insensitive, (ii) a caffeine-sensitive Ca²⁺ pool (CasCaP), vanadate- and IP₃-insensitive, and (iii) a vanadate-sensitive Ca²⁺ pool (VasCaP), neither IP₃- nor caffeine-sensitive, into which Ca²⁺ uptake is mediated via a Ca²⁺ ATPase sensitive to vanadate at 10^–4 mol/liter. A fourth Ca²⁺ pool is neither IP₃- nor caffeine- or vanadate-sensitive. Percoll fractionP ₁ contained essentially the IsCaP, CasCaP and VasCaP and was mainly used for studies on Ca²⁺ uptake and Ca²⁺ release.When membrane vesicles were incubated in the presence of caffeine (2×10^–2 mol/liter), Ca²⁺ uptake up to the steady state [Ca²⁺] did not appear to be altered as compared to the control Ca²⁺ uptake. However, in control vesicles spontaneous Ca²⁺ release occurred after the steady state had been reached, whereas cfffeine-pretreated vesicles did not spontaneously release Ca²⁺. Addition of IP₃ at steady state [Ca²⁺] induced similar Ca²⁺ release followed by Ca²⁺ reuptake in both caffeine-pretreated and control vesicles. However, when caffeine was acutely added at steady state, Ca²⁺ was released from all Ca²⁺ pools including the IsCaP. Following Ca²⁺ reuptake after IP₃ had been added, a second addition of IP₃ to control vesicles induced further but smaller Ca²⁺ release, and a third addition resulted in a steady Ca²⁺ efflux by which all Ca²⁺ that had been taken up was released. This steady Ca²⁺ release started at a Ca²⁺ concentration between 5.5–8 ×10^–7 mol/liter and could also be induced by the IP₃ analogue inositol 1,4,5-trisphosphorothioate (IPS₃) or by addition of Ca²⁺ itself. Ruthenium red (10^–5 mol/liter) inhibited both caffeine-induced as well as Ca²⁺-induced but not IP₃-induced Ca²⁺ release. Heparin (100 g/m) inhibited IP₃-but not caffeine-induced Ca²⁺ release. The data indicate the presence of at least three separate Ca²⁺ pools in pancreatic acinar cells: the IsCaP, CasCaP and VasCaP. During Ca²⁺ uptake these Ca²⁺ pools appear to be separate. However, when steady state is reached, we assume that these Ca²⁺ pools come into contact and total Ca²⁺ release from all three pools can occur. The mechanism of this contact of Ca²⁺ pools is not clear but seems to be different from that induced by GTP in the presence of polyethylene glycol, which probably involves fusion of membranes.     

The effect of glucagon on the kinetics of hepatic mitochondrial calcium uptake

Summary Previous work by this and other laboratories has shown that glucagon administration stimulates calcium uptake by subsequently isolated hepatic mitochondria. This stimulation of hepatic mitochondrial Ca²⁺ uptake byin vivo administration of glucagon was further characterized in the present report. Maximal stimulation of mitochondrial Ca²⁺ accumulation was achieved between 6–10 min after the intravenous injection of glucagon into intact rats. Under control conditions, Ca²⁺ uptake was inhibited by the presence of Mg²⁺ in the incubation medium. Glucagon treatment, however, appeared to obliterate the observed inhibition by Mg²⁺ of mitochondrial Ca²⁺ uptake. Kinetic experiments revealed the usual sigmoidicity associated with initial velocity curves for mitochondrial calcium uptake. Glucagon treatment did not alter this sigmoidal relationship. Glucagon treatment significantly increased the V_max for Ca²⁺ uptake from 292±22 to 377±34 nmoles Ca²⁺ /min per mg protein (n=8) but did not affect the K_0.5, (6.5–8.6 μM). Since the major kinetic change in mitochondrial Ca²⁺ uptake evoked by glucagon is an increase in V_max, the enhancement mechanism is likely to be an increase either in the number of active transport sites available to Ca²⁺ or in the rate of Ca²⁺ carrier movement across the mitochondrial membranes.     

Characterization of calcium uptake into rough endoplamic reticulum of rat pancreas

Summary ATP-dependent Ca²⁺ uptake into isolated pancreatic acinar cells with permeabilized plasma membranes, as well as into isolated endoplasmic reticulum prepared from these cells, was measured using a Ca²⁺-specific electrode and⁴⁵Ca²⁺. Endoplasmic reticulum was purified on an isopycnic Percoll gradient and characterized by marker enzyme distribution. When compared to the total homogenate, the typical marker for the rough endoplasmic reticulum RNA was enriched threefold and the typical marker for the plasma membrane Na⁺,K⁺(Mg²⁺)ATPase was decreased 20-fold. When different fractions of the Percoll gradient were compared,⁴⁵Ca²⁺ uptake correlated with the RNA content and not with the Na⁺,K⁺(Mg²⁺)ATPase activity. The characteristics of nonmitochondrial Ca²⁺ uptake into leaky isolated cells and⁴⁵Ca²⁺ uptake into isolated endoplasmic reticulum were very similar: Calcium uptake was maximal at 0.3 and 0.2 mmol/liter free Mg²⁺, at 1 and 1 mmol/liter ATP, at pH 6.0 and 6.5, and free Ca²⁺ concentration of 2 and 2 mol/liter, respectively. Calcium uptake decreased at higher free Ca²⁺ concentration.⁴⁵Ca²⁺ uptake was dependent on monovalent cations (Rb⁺>K⁺>Na⁺>Li⁺>choline⁺) and different anions (Cl^–>Br^–>SO ₄ ^2– >NO ₃ ^– >I^–>cyclamate^–>SCN^–) in both preparations. Twenty mmol/liter oxalate enhanced⁴⁵Ca²⁺ uptake in permeabilized cells 10-fold and in vesicles of endoplasmic reticulum, fivefold. Calcium oxalate precipitates in the endoplasmic reticulum of both preparations could be demonstrated by electron microscopy. The nonmitochondrial Ca²⁺ pool in permeabilized cells characterized in this study has been previously shown to regulate the cytosolic free Ca²⁺ concentration to 0.4 mol/liter. Our results provide firm evidence that the endoplasmic reticulum plays an important role in the regulation of the cytosolic free Ca²⁺ concentration in pancreatic acinar cells.     

Relationship Between Ca2+ Uptake and Catecholamine Secretion in Primary Dissociated Cultures of Adrenal Medulla

Abstract: Carbachol or elevated K⁺ stimulated ⁴⁵Ca²⁺ uptake into chromaffin cells two- to fourfold. The uptake was stimulated by cholinergic drugs with nicotinic activity, but not by those with only muscarinic activity. Ca²⁺ uptake and catecholamine secretion induced by the mixed nicotinic-muscarinic agonist carbachol were inhibited by the nicotinic antagonist mecamylamine, but not by the muscarinic antagonist atropine. Significant Ca²⁺ uptake occurred within 15 s of stimulation by carbachol or elevated K⁺ at a time before catecholamine secretion was readily detected. At later times the time course of secretion induced by carbachol or elevated K⁺ was similar to that of Ca²⁺ uptake. There was a close correlation between Ca²⁺ uptake and catecholamine secretion at various concentrations of Ca²⁺. The concentration dependencies for inhibition of both processes by Mg²⁺ or Cd²⁺ were similar. Ca²⁺ uptake saturated with increasing Ca²⁺ concentrations, with an apparent K_m for both carbachol-induced and elevated K⁺-induced Ca²⁺ uptake of approximately 2 mM. The Ca²⁺ dependency, however, was different for the two stimuli. The studies provide strong support for the notion that Ca²⁺ entry and a presumed increase in cytosolic Ca²⁺ concentration respectively initiates and maintains secretion. They also provide evidence for the existence of saturable, intracellular, Ca²⁺- dependent processes associated with catecholamine secretion. Ca²⁺ entry may, in addition, enhance nicotinic receptor desensitization and may cause inactivation of voltage-sensitive Ca²⁺ channels.     

Phosphatidylinositol 3,5‐bisphosphate regulates Ca2+ transport during yeast vacuolar fusion through the Ca2+ ATPase Pmc1

The transport of Ca²⁺ across membranes precedes the fusion and fission of various lipid bilayers. Yeast vacuoles under hyperosmotic stress become fragmented through fission events that requires the release of Ca²⁺ stores through the TRP channel Yvc1. This requires the phosphorylation of phosphatidylinositol‐3‐phosphate (PI3P) by the PI3P‐5‐kinase Fab1 to produce transient PI(3,5)P₂ pools. Ca²⁺ is also released during vacuole fusion upon trans‐SNARE complex assembly, however, its role remains unclear. The effect of PI(3,5)P₂ on Ca²⁺ flux during fusion was independent of Yvc1. Here, we show that while low levels of PI(3,5)P₂ were required for Ca²⁺ uptake into the vacuole, increased concentrations abolished Ca²⁺ efflux. This was as shown by the addition of exogenous dioctanoyl PI(3,5)P₂ or increased endogenous production of by the hyperactive fab1^T2250A mutant. In contrast, the lack of PI(3,5)P₂ on vacuoles from the kinase dead fab1^EEE mutant showed delayed and decreased Ca²⁺ uptake. The effects of PI(3,5)P₂ were linked to the Ca²⁺ pump Pmc1, as its deletion rendered vacuoles resistant to the effects of excess PI(3,5)P₂. Experiments with Verapamil inhibited Ca²⁺ uptake when added at the start of the assay, while adding it after Ca²⁺ had been taken up resulted in the rapid expulsion of Ca²⁺. Vacuoles lacking both Pmc1 and the H⁺/Ca²⁺ exchanger Vcx1 lacked the ability to take up Ca²⁺ and instead expelled it upon the addition of ATP. Together these data suggest that a balance of efflux and uptake compete during the fusion pathway and that the levels of PI(3,5)P₂ can modulate which path predominates.     

Calcium regulation in the freshwater-adapted mummichog

In light of recent findings of an unusual pattern of ionoregulation (high Na⁺ uptake and negligible Cl^- uptake) in the freshwater-adapted mummichog Fundulus heteroclitus, the pattern of Ca²⁺ regulation was examined. Under control conditions (water Ca²⁺= 200μEq l^-1), unidirectional Ca²⁺ influx was 11 ± 4 nEq g^-1 h^-1. Acute variation of external Ca²⁺ levels revealed a saturable Ca²⁺ uptake system with a relatively high affinity (K_m= 125 ± 36 μEq 1^-1) and a transport capacity (J_max= 31 ± 4 nEq g^-1 h^-1) comparable to those of other teleosts. Lanthanum (equimolar to [Ca²⁺]) significantly blocked Ca²⁺ uptake by 67% whereas magnesium had no effect. Chronic low Ca²⁺ exposure (50 μEq 1^-1) stimulated Ca²⁺ uptake almost three-fold above control values, whereas chronic high Ca²⁺ exposure (20000 μEq 1^-1) had no effect. Lanthanum and chronic low Ca²⁺ treatments disturbed the normally positive Ca²⁺ and Na⁺ balances of the animals whereas acid-base balance and ammonia excretion were undisturbed. The results indicate that Ca²⁺ regulation by the mummichog conforms to the model for freshwater Ca²⁺ transport whereby chloride cells on the gills take up Ca²⁺ actively from the water. However, the absence of extra-intestinal Cl^- uptake and the recent demonstration of significant Ca²⁺ uptake by opercular epithelia raise questions about the relative roles of branchial and opercular epithelial chloride cells in freshwater F. heteroclitus.     

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.     

ATP-driven Ca2+ transport in sealed plasma membrane vesicles prepared by aqueous two-phase partitioning from leaves of Commelina communis

Sealed plasma membrane vesicles were obtained in high purity from leaves of Commelina communis L. by aqueous two-phase partitioning. Based on the analysis of a range of markers, the preparations (U₃+U₃′ phases) were shown to be devoid of tonoplast, Golgi and thylakoid membranes, and showed only trace mitochondrial contamination. One-third of the vesicles were oriented inside out and exhibited ATP-driven ⁴⁵Ca²⁺ transport [? 15 pkat (mg protein)⁻¹]. Ca²⁺ uptake into the vesicles had a pH optimum of 7.2 and apparent K_m values for Ca²⁺ of 4.4 μM and for Mg-ATP of 300 μM. Ca²⁺ uptake, K⁺, Mg²⁺-ATPase (EC 3.6.1.3) activity as well as glucan synthase II (EC 2.4.1.34) activity were all maximal at the same equilibrium density (1.17 g cm⁻³) on continuous sucrose density gradients. The protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP) did not inhibit the ATP-dependent Ca²⁺ transport into the vesicles, excluding a Ca²⁺/H⁺ exchange driven by a proton gradient. ATP-dependent Ca²⁺ uptake was inhibited by erythrosin B (I₅₀= 0.1 μM), ruthenium red (I₅₀= 30 μM), La³⁺ (I₅₀= 10 μM) and vanadate (I₅₀= 500 μM), but not by azide, cyanide and oligomycin. The calmodulin antagonists, trifluoperazine (I₅₀= 70 μM) and W-7 (I₅₀= 100 μM) were also inhibitory, However, this inhibition was not overcome by calmodulin. Trifluoperazine and W-7, on the other hand, stimulated Ca²⁺ efflux from the vesicles rather than inhibit Ca²⁺ uptake. Our results demonstrate the presence of a Ca²⁺-ATPase in the plasma membrane of C. communis. In the intact cell, the enzyme would pump Ca²⁺ out of the cell. Its high affinity for Ca²⁺ makes it a likely component involved in adjusting low cytoplasmic Ca²⁺ levels. No indications for a secondary active Ca²⁺/H⁺ transport mechanism in the plasma membrane of C. communis were obtained. Both, the nucleotide specificity and the sensitivity towards vanadate. distinguish the Ca²⁺-ATPase from the H⁺-translocating K⁺. Mg²⁺-ATPase in C. communis plasma membranes.     

State of translocated Ca2+ by sarcoplasmic reticulum inferred from kinetic analysis of calcium oxalate precipitation

Uptake of Ca²⁺ by sarcoplasmic reticulum in the presence of oxalate displays biphasic kinetics. An initial phase of normal uptake is followed by a second phase coincident with precipitation of calcium oxalate inside the vesicles. The precipitation rate induced by actively transported Ca²⁺ is depressed by increasing the added Ca²⁺ concentration. This correlates linearly with the reciprocal of precipitation rate. Therefore, a maximal limit rate could be extrapolated at zero Ca²⁺ ($\text{V}{}_0$). The rate of precipitation, also a function of added amount protein, gives a linear correlation in a double reciprocal plot. Thus, it was possible to estimate the maximal precipitation rate occurring at infinite protein concentration ($\text{V}{}^{\mathrm{\infty }}$). With the combined extrapolated values a maximal expected precipitation rate could be calculated ($\text{V}{}_0{}^{\mathrm{\infty }}$). Kinetics of calcium oxalate precipitation was studied in the absence of calcium uptake and empirical equations relating the rate of precipitation with the added Ca²⁺ were established. Entering $\text{V}{}_0{}^{\mathrm{\infty }}$ in the equations, an internal free Ca²⁺ concentration of approx. 2.5 mM was estimated. Additionally, it is shown that the ionophore X-537A does not supress the Ca²⁺ uptake, if added during the oxalate-dependent phase, albeit the uptake proceeds at a slower rate after the release of approx. 70 nmol Ca²⁺/mg protein. This amount presumably equals the internal free Ca²⁺ not sequestered by oxalate, producing a maximal concentration approx. 14 mM. Taking into account low affinity binding of internal binding sites and the transmembrane Ca²⁺ gradients built up during the uptake of Ca²⁺, values of free Ca²⁺ ranging from 3 to 6 mM, approaching those estimated by the precipitation analysis, could be estimated.     

Effects of Monovalent and Divalent Cations on Ca2+ Fluxes Across Chromaffin Secretory Membrane Vesicles

Abstract: Bovine chromaffin secretory vesicle ghosts loaded with Na⁺ were found to take up Ca²⁺ when incubated in K⁺ media or in sucrose media containing micromolar concentrations of free Ca²⁺. Li⁺- or choline⁺loaded ghosts did not take up Ca²⁺. The Ca²⁺ accumulated by Na⁺-loaded ghosts could be released by the Ca²⁺ ionophore A23187, but not by EGTA. Ca²⁺ uptake was inhibited by external Sr²⁺, Na ⁺, Li ⁺, or choline ⁺. All the ⁴⁵Ca²⁺ accumulated by Na⁺-dependent Ca²⁺ uptake could be released by external Na ⁺, indicating that both Ca²⁺ influx and efflux occur in a Na⁺-dependent manner. Na ⁺ -dependent Ca²⁺ uptake and release were only slightly inhibited by Mg²⁺. In the presence of the Na⁺ ionophore Monensin the Ca²⁺ uptake by Na ⁺-loaded ghosts was reduced. Ca²⁺ sequestered by the Na⁺-dependent mechanism could also be released by external Ca²⁺ or Sr²⁺ but not by Mg²⁺, indicating the presence of a Ca²⁺/Ca²⁺ exchange activity in secretory membrane vesicles. This Ca²⁺/Ca²⁺ exchange system is inhibited by Mg²⁺, but not by Sr²⁺. The Na ⁺ -dependent Ca²⁺ uptake system in the presence of Mg²⁺ is a saturable process with an apparent K_m of 0.28 μM and a V_max= 14.5 nmol min^?1 mg protein^?1. Ruthenium red inhibited neither the Na⁺/Ca²⁺ nor the Ca²⁺/Ca²⁺ exchange, even at high concentrations.