Facilitated transport of calcium by cells and subcellular membranes of Bacillus subtilis and Escherichia coli.

The level of calcium in growing cells is lower than that in the growth medium. Non-energy-dependent uptake of 45-Ca by log-phase cells of Bacillus subtilis occurs under two conditions: at 0 C or in the presence of m-chlorophenyl carbonylcyanide hydrazone. Similar uptake, but quantitatively less, occurs with Escherichia coli cells under the same conditions. Membrane vesicles prepared from B. subtilis or E. coli accumulate 45-Ca by a process that does not depend on added energy sources and is not inhibited by the respiratory poison cyanide. The properties of calcium transport in all cases is consistent with carrier-mediated, facilitated transport with specificity Ca-2+ greater than Sr-2+ greater than Mn-2+ greater than Mg-2+. Upon transfer of cells from 0 C to 20 C, pre-accumulated 45-Ca is released. Heat-killed cells do not accumulate 45-Ca and calcium is released by cells upon addition of toluene (under conditions that do not cause visible lysis). These results suggest that the facilitated uptake of calcium may be utilizing a transport system that normally is responsible for the energy-dependent excretion of calcium from the cells.     

Energy-dependent calcium transport in endoplasmic reticulum of adipocytes.

The endoplasmic reticulum from isolated rat adipocytes has the ability to actively accumulate calcium. The calcium uptake was characterized using the 20,000 X g supernatant (S1 fraction) of total cellular homogenate. Endoplasmic reticulum vesicles isolated from the S1 fraction as a 160,000 X g microsomal pellet prior to testing demonstrated little ability to accumulate calcium. The calcium uptake in the S1 fraction was localized to the endoplasmic reticulum vesicles by morphologic appearance, by the use of selective inhibitors of calcium uptake, and by high speed sedimentation of the accumulated calcium. The uptake was MgATP- and temperature-dependent and was sustained by the oxalate used as the intravesicular trapping agent. Uptake was linear with time for at least 30 min at all calcium concentrations tested (3 to 100 muM) and exhibited a pH optimum of approximately 7.0. The sulfhydryl inhibitor p-chloromercuribenzene sulfonate produced a dose-dependent inhibition of calcium uptake with total inhibition at 0.07 mumol/mg protein. Ruthenium red and sodium azide inhibited less than 5% of the uptake at concentrations (5 muM and 10 mM, respectively) which completely blocked calcium uptake by mitochondria isolated from the same cells. The Km for calcium uptake was 10 muM total calcium which corresponded to approximately 3.6 muM ionized calcium in the assay system. The maximum velocity of the uptake was 5.0 nmol (mg of microsomal protein)-1 (min)-1 at 24 degrees under the assay conditions used and exhibited a Q10 of 1.8. The uptake activity of the endoplasmic reticulum vesicles in the S1 fraction exhibited a marked time- and temperature-dependent lability which might account in part for the lack of uptake in the isolated microsomal fraction. This energy-dependent calcium uptake system would appear to be of physiologic importance to the regulation of intracellular calcium.     

Effect of cell density on energy-dependent calcium uptake by Balb/c 3T3 membranes is independent of protein synthesis and attachment to substratum.

Membranes isolated from subconfluent cultures of Balb/c 3T3 cells have low energy-dependent calcium uptake activity. Replating confluent cells at low density results in a prompt fall of energy-dependent calcium uptake by membrane fractions. The level to which uptake activity falls is a function of the density at which the cells are plated (Moore and Pastan, '77b). To determine if regulation of energy-dependent uptake of calcium by membrane fractions is dependent upon attachment to a substrate and to further characterize conditions that regulate the process, we examined calcium uptake activity of membranes isolated from cells in suspension. With cells in suspension energy-dependent calcium uptake activity of isolated membranes falls promptly if cells are diluted to a low density (less than 10(5) cells/ml) and is a function of cell density. When cells in suspension at low cell densities are concentrated to high cell densities (greater than 2 x 10(6) cells/ml), calcium uptake activity of the isolated membrane fraction is increased as a function of cell density. These changes of membrane calcium uptake activity occur promptly and do not require protein synthesis.     

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.     

Calcium transport in isolated bone cells. II. Calcium transport studies

Calcium transport was studied in bone cells isolated from fetal rat calvaria. ⁴⁵Ca uptake experiments revealed an active component of calcium exchange. Calcium uptake was inhibited by iodoacetamide, DNP, CCCP and oligomycin and appeared to be dependent on medium phosphate concentration. Initial influx values exhibited saturation kinetics from 0.6 mM to 1.5 mM extracellular calcium. Efflux of ⁴⁵Ca from loaded cells increased in the presence of iodoacetamide, DNP and CCCP. Incubation of the cells af 4° C inhibited both influx and efflux of calcium. Parathyroid hormone had no consistent effect on calcium uptake although characteristic increases in cyclic AMP levels were seen with the hormone. Calcitonin appeared to cause a transient increase in calcium uptake.     

Calcium channels are present in the apical plasma membranes of the hepatopancreatic B-cells of<Emphasis Type="Italic"> Marsupenaeus japonicus</Emphasis>

This study demonstrates the existence of calcium channels in the apical membranes of the hepatopancreatic blister (B) cells of Marsupenaeus japonicus. Using brush-border membrane vesicles we demonstrated that the channel-mediated calcium passive flux was saturable and was stimulated by a transmembrane electrical potential difference and inhibited by barium. We raised a monoclonal antibody (Mab 24A4) against the calcium channel, which allowed us to inhibit the channel-mediated calcium uptake. By immunocytochemistry, using Mab 24A4, we demonstrated that these channels are located at the apical membrane of hepatopancreatic B cells. Finally, by measuring the calcium uptake in R- and B-enriched cell suspensions, we showed that only the plasma membrane of the B cells expresses a channel-mediated calcium uptake inhibited by barium, verapamil and the monoclonal antibody 24A4. The plasma membrane of R cells did not show calcium channels.Abbreviations ELISA enzyme-linked immunosorbent assay - BBMV brush-border membrane vesiclesCommunicated by G. Heldmaier     

Characterization of an active transport system for calcium in inverted membrane vesicles of Escherichia coli.

The energy-dependent uptake of calcium by inverted membrane vesicles of Escherichia coli was investigated. Methods for preparation and storage of the vesicles were devised to allow for the maximal activity and stability of the calcium transport system. The pH and temperature optima for the reaction were observed to occur at pH 8.0 AND 30 DEGREES, RESPECTIVELY. The eft was found that the extent of the reaction depended on the presence of phosphate or oxalate. Phosphate was found to enter the vesicles at a rate slower than that of calcium. A Ca2+:Pi ratio of approximately 1.5 was found, suggesting formation of Ca3(PO4)2. Monovalent cations stimulated calcium uptake, with the order of effectiveness being K+ is greater than Na+ is greater than Li+ is greater than NH4+. Inhibition was found with certain divalent cations, but these also inhibited the electron transport chain. Of the divalent cations examined only Mg2+ and Sr2+ inhibited calcium transport without a corresponding inhibition of respiration. Calcium transport exhibited biphasic Kinetics, with a low affinity system and a high affinity system. The low affinity system showed a Km of 0.34 mM and a Vmax of 85 nmol/min/mg of protein. The kinetic constants of the high affinity system were 4.5 muM and 2 nmol/min/mg of protein. The energy for calcium transport could be derived from the electron transport chain by oxidation of NADH, D-lactate, and succinate, in order of their effectiveness. Respiration-driven calcium transport was inhibited by inhibitors of the electron transport chain and by uncouplers of oxidative phosphorylation. ATP could also be used to supply enerty for calcium transport. The ATP-driven reaction was inhibited by inhibitors of the Mg2+ATPase and by an antiserum prepared against that protein, demonstrating that that enzyme is involved in the utilization of ATP for active transport in inverted vesicles.     

Increased Calcium Absorption in Nephrolithiasis Explained by Uptake Studies in Ileal Brush Border Membrane Vesicles

We previously showed that recurrent calcium renal stone formers have enhanced urinary excretions of calcium and oxalate resulting from malabsorption of citrate. In the present investigation, the mechanism of the citrate-induced increased calcium uptake was studied using guinea pig ileal brush border membrane vesicles. In this model, calcium is absorbed in a concentration dependent, single mechanism uptake with a K_m of 275 ± 30 umol/liter (SD) and a V_max of 4.0 ± 0.5 nmol/min · mg protein. Under conditions of maximal calcium uptake, both citrate and phosphate inhibited calcium absorption into brush border membrane vesicles (BBMVs). In contrast, when phosphate and citrate were added together, calcium absorption normalized. Citrate inhibition of calcium absorption appeared to be due to free citrate ions, and phosphate ions overcame this inhibition. Phosphate inhibition was mostly due to decreased concentrations of ionized calcium and partly to precipitation of insoluble calcium phosphate. These studies confirm that the effects of citrate in humans in enhancing calcium absorption occur in the lumen of the gut and are not related to further biochemical conversions of citrate by the gut cells, to effects of citrate on calcium-related hormones, or to the renal handling of calcium. Also, the effects of citrate on increasing calcium absorption should be increased or attenuated in patients who malabsorb citrate, and this explains the increased urinary calcium and oxalate excretions reported for recurrent calcium stone formers.     

Different Subunit Location of the Inhibition and Transport Sites in the Mitochondrial Calcium Uniporter

The mitochondrial calcium uniporter behaves as a cooperative mechanism, where the velocity is dependent on [Ca2+]ex. Transport kinetics follows a sigmoidal behavior with a Hill coefficient near 2.0, indicating the binding of at least two calcium molecules. Calcium transport in mitochondria is dependent on a negative inner membrane potential and is inhibited by policationic ruthenium compounds. In this study, calcium uptake activity was reconstituted into cytochrome oxidase vesicles by incorporating solubilized mitochondrial proteins. Calcium accumulation plotted against increasing Ca2+ concentrations followed a sigmoidal behavior with a Hill coefficient of 1.53. The uptake was sensitive to ruthenium policationic inhibitors, e.g. ruthenium red and Ru360. After mitochondrial proteins were separated by preparative isoelectrofocusing and incorporated into cytochrome oxidase vesicles, two peaks of calcium uptake activity were recovered. One of the activities was inhibited by Ru360, while the second activity was insensitive to Ru360 and was associated with proteins focused at very acidic isoelectric points. By using a thiol-group crosslinker and radiolabeled Ru360, we proposed a scheme of partial dissociation of the uniporter inhibitor-binding subunit under acidic conditions.     

Calcium sequestration activity in rat liver microsomes. Evidence for a cooperation of calcium transport with glucose-6-phosphatase

Mechanisms regulating the energy-dependent calcium sequestering activity of liver microsomes were studied. The possibility for a physiologic mechanism capable of entrapping the transported Ca2+ was investigated. It was found that the addition of glucose 6-phosphate to the incubation system for MgATP-dependent microsomal calcium transport results in a marked stimulation of Ca2+ uptake. The uptake at 30 min is about 50% of that obtained with oxalate when the incubation is carried out at pH 6.8, which is the pH optimum for oxalate-stimulated calcium uptake. However, at physiological pH values (7.2-7.4), the glucose 6-phosphate-stimulated calcium uptake is maximal and equals that obtained with oxalate at pH 6.8. The Vmax of the glucose 6-phosphate-stimulated transport is 22.3 nmol of calcium/mg protein per min. The apparent Km for calcium calculated from total calcium concentrations is 31.9 microM. After the incubation of the system for MgATP-dependent microsomal calcium transport in the presence of glucose 6-phosphate, inorganic phosphorus and calcium are found in equal concentrations, on a molar base, in the recovered microsomal fraction. In the system for the glucose 6-phosphate-stimulated calcium uptake, glucose 6-phosphate is actively hydrolyzed by the glucose-6-phosphatase activity of liver microsomes. The latter activity is not influenced by concomitant calcium uptake. Calcium uptake is maximal when the concentration of glucose 6-phosphate in the system is 1-3 mM, which is much lower than that necessary to saturate glucose-6-phosphatase. These results are interpreted in the light of a possible cooperative activity between the energy-dependent calcium pump of liver microsomes and the glucose-6-phosphatase multicomponent system. The physiological implications of such a cooperation are discussed.     

Calcium Uptake and Associated Adenosine Triphosphatase Activity of Isolated Platelet Membranes

A platelet subcellular fraction, sedimenting between 14,000 and 40,000 g and consisting primarily of membrane vesicles, accumulates up to 200–400 nmoles calcium/mg protein in the presence of ATP and oxalate. Steady-state levels of calcium accumulation are attained in 40–60 min. Calcium uptake requires adenosine triphosphate (ATP), is enhanced by oxalate, and is accompanied by the release of inorganic phosphate. Calcium accumulation and phosphate release require magnesium and are inhibited by Salyrgan (10 µM) and adenosine diphosphate (ADP) (1 mM), but not by ouabain (0.1 mM). The ATPase activity is stimulated by low concentrations of calcium (5–10 µM) and is inhibited by 2 mM EGTA. Electron microscopic histochemistry using lead nitrate to precipitate released phosphate results in lead precipitates localized primarily at the inner surface of membrane vesicles. These results provide evidence for a membrane ATPase that is stimulated by low concentrations of calcium and may be involved in the transport of calcium across the membrane. It is postulated that the observed calcium uptake activity is an in vitro manifestation of a calcium extrusion pump in the intact platelet.     

Comparative study of the effect of aliphatic alcohols on energy-dependent accumulation of Ca2+ in intracellular membranes of smooth muscle cells

The effects of ethanol and other aliphatic alcohols on energy-dependent Ca2+ transport in endoplasmic reticulum and mitochondria were studied in digitonin-treated myometrium cells. The Ca2+ uptake in mitochondria increased (on 15-20%) with increasing methanol, ethanol and propanol concentrations in medium, whereas further rise of concentration inhibited this process. Treatments of myometrial cells with short-chain alcohols caused an inhibition of calcium uptake in endoplasmic reticulum. Butanol inhibited both calcium uptake in mitochondria and endoplasmic reticulum. Ca2+ accumulation in intracellular pools is inhibited by aliphatic alcohols in the following order of potency: butanol > propanol > ethanol > methanol. It is concluded that modifying effect of aliphatic alcohols on energy dependent calcium accumulation in intracellular membrane structures is defined as on origin of Ca(2+)-transporting system and (or) properties of these membrane structures so on properties of alcohols.     

Calcium Transport by Frankia sp. Strain EAN1pec

When incubated at 25°C, N₂-grown cells of Frankia strain EAN1pec actively accumulated calcium, while NH₄Cl-grown cells did not accumulate calcium. When incubated at 0°C, both N₂-grown and NH₄Cl-grown cells did not actively accumulate calcium. Inhibitors of respiration inhibited calcium accumulation by N₂-grown cells at 25°C. Isolated vesicles also accumulated calcium in an energy- and temperature-dependent manner. Two lines of evidence show that Frankia strain EAN1pec has an active calcium extrusion mechanism. First, NH₄Cl-grown cells incubated under deenergizing conditions accumulated calcium. Second, calcium efflux from calcium-loaded cells required an energy source and was blocked by inhibitors. The results of this study indicate that Frankia strain EAN1pec has two systems for calcium transport: a calcium extrusion system and a developmentally regulated calcium uptake system. Received: 1 December 1997 / Accepted: 9 January 1998     

Calcium Transport in Sealed Vesicles from Red Beet (Beta vulgaris L.) Storage Tissue : II. Characterization of Ca Uptake into Plasma Membrane Vesicles

Calcium uptake was examined in sealed plasma membrane vesicles isolated from red beet (Beta vulgaris L.) storage tissue using (45)Ca(2+). Uptake of (45)Ca(2+) by the vesicles was ATP-dependent and radiotracer accumulated by the vesicles could be released by the addition of the calcium ionophore A23187. The uptake was stimulated by gramicidin D but slightly inhibited by carbonylcyanide m-chlorophenylhydrazone. Although the latter result might suggest some degree of indirect coupling of (45)Ca(2+) uptake to ATP utilization via deltamuH(+), no evidence for a secondary H(+)/Ca(2+) antiport in this vesicle system could be found. Following the imposition of an acid-interior pH gradient, proton efflux from the vesicle was not enhanced by the addition of Ca(2+) and an imposed pH gradient could not drive (45)Ca(2+) uptake. Optimal uptake of (45)Ca(2+) occurred broadly between pH 7.0 and 7.5 and the transport was inhibited by orthovanadate, N,N'-dicyclohexylcarbodiimide, and diethylstilbestrol but insensitive to nitrate and azide. The dependence of (45)Ca(2+) uptake on both calcium and Mg:ATP concentration demonstrated saturation kinetics with K(m) values of 6 micromolar and 0.37 millimolar, respectively. While ATP was the preferred substrate for driving (45)Ca(2+) uptake, GTP could drive transport at about 50% of the level observed for ATP. The results of this study demonstrate the presence of a unique primary calcium transport system associated with the plasma membrane which could drive calcium efflux from the plant cell.     

Active Transport of Manganese in Isolated Membranes of Escherichia coli

Accumulation of manganese was measured in subcellular membrane vesicles isolated from Escherichia coli. Accumulation of (54)Mn by vesicles in 0.5 m sucrose is stimulated by glucose and d-lactate and is inhibited by metabolic poisons such as dinitrophenol, m-chlorophenyl carbonylcyanide hydrazone, valinomycin, and nigericin. Manganese uptake by vesicles requires 10 mm calcium, which is not required for uptake of manganese by intact cells. The calcium requirement is specific and cannot be replaced by magnesium, sodium, or potassium. Strontium can replace calcium but is somewhat less effective than calcium. The uptake of manganese is via a manganese-specific system which shows saturation kinetics with manganese with a K(m) of 8 x 10(-6)m and a V(max) of 4 nmoles per min per g (wet weight) at 25 C. Magnesium and calcium do not compete for uptake. The accumulated manganese can be released from the vesicles by lipid active agents such as toluene, and can be exchanged for external manganese.     

Calcium transport systems in the LLC-PK1 renal epithelial established cell line

ATP-dependent calcium uptake was measured in membrane vesicles prepared from the renal epithelial LLC-PK1 established cell line. The relative contribution of the nonmitochondrial versus the mitochondrial calcium uptake is larger in LLC-PK1 cell homogenates than in homogenates from renal cortex. Two types of calcium pump, characterized by the formation of calcium-dependent phosphointermediates of 135 kDa and 115 kDa, were found in membrane fractions from LLC-PK1 cells. The 135 kDa calcium pump was also detected by 125I-labelled calmodulin overlay. Although the subcellular localization in LLC-PK1 cell membranes could not be unambiguously determined, it is conceivable that the 135 kDa and the 115 kDa molecules represent the plasma membrane calcium pump and the endoplasmic reticulum calcium pump respectively, in agreement with what was found for renal cortex preparations. Extravesicular sodium partially inhibits ATP-driven calcium uptake in a plasma-membrane-enriched fraction of the LLC-PK1 cells. The effect is potentiated by a vesicle inside-negative membrane potential. Although the effect is less pronounced than in renal cortex basal-lateral membranes, this observation suggests that an Na+-Ca2+ exchange mechanism is also present in LLC-PK1 cells. ATP-dependent calcium uptake in nonmitochondrial intracellular stores was investigated, using saponin-permeabilized cells. Permeabilized LLC-PK1 cells lowered the free calcium concentration in the medium to less than 0.4 microM. More than 60% of the accumulated calcium can be released by addition of inositol 1,4,5-trisphosphate. Our data indicate that the LLC-PK1 cell line can be successfully used as model system for the study of renal calcium handling.     

Regulation of intracellular calcium in chick embryo fibroblast: calcium uptake by the microsomal fraction.

The total membrane fraction of a chick embryo fibroblast (CEF) homogenate accumulates calcium in an energy-dependent manner. This activity can be dissociated into azide-sensitive and azide-insensitive components. The azide-sensitive component of calcium uptake is believed to represent mitochondrial calcium uptake. The azide-insensitive component of calcium uptake is enhanced by the presence of a calcium trapping agent such as oxalate, and cannot utilize, ADP, inorganic phosphate and a Krebs cycle substrate to support uptake. The distribution of the azide-insensitive calcium uptake in subcellular fractions suggests that this uptake occurs in other than mitochondrial membranes. The membranes most likely to contribute to the azide-insensitive component of calcium uptake are the endoplasmic reticulum and plasma membrane. A microsomal preparation from CEF cells is essentially devoid of the azide-sensitive calcium uptake activity. This microsomal activity is similar in characteristics to the sarcoplasmic reticulum of skeletal muscle. However the specific activity of CEF microsomal calcium uptake system is much less than that found in the skeletal muscle system. The transport of calcium by these membranes provide a mechanism for the regulation of cytosol calcium levels and may play a role in the control of movement and growth of cultured cells.     

Calcium transport by bull spermatozoa plasma membranes

Plasma membrane isolated from frozen ejaculated bull spermatozoa were found to contain calcium transport activity. Thin-section electronmicrography of these membranes revealed relatively homogeneous vesicular membranes with sizes ranging from 2000 to 6000 A in diameter. Membrane vesicles that were exposed to oxalate as a calcium-trapping agent accumulated Ca2+ in the presence of Mg2+ and ATP. One microM of the calcium-ionophore A23187, added initially, completely inhibited net Ca2+ uptake and, if added later, caused the release of Ca2+ accumulated previously. An Arrhenius plot for the rate of Ca2+ uptake revealed a break at 32--33 degrees C, and Ea of 4.4 kcal/mol above the break and 32.2 kcal/mol below. The Ca+ uptake was inhibited by low concentrations of quercetin, which is known to be an inhibitor of (Ca2+ + Mg2+)-ATPase in many systems.     

Calcium and glucose uptake in rat small intestinal brush-border membrane vesicles. Modulation by exogenous hypercortisolism and 1,25-dihydroxyvitamin D-3

The effect of exogenous hypercortisolism and 1,25-dihydroxyvitamin D-3 on small-intestinal calcium and glucose transport in the rat was studied at the level of brush-border membrane vesicles generated from isolated villous cells by a freeze-thaw procedure. At 5 X 10(-5) M extravesicular calcium, initial uptake rates in vesicles prepared from triamcinolone-treated adult rats were decreased by 30% after 5 days. Since calcium ionophore A23187 virtually abolished the difference in calcium uptake, triamcinolone appeared to affect calcium channel density or activity rather than intravesicular binding capacity. Kinetic analysis showed that a decrease in Vmax of a saturable calcium transport system could entirely account for the diminished rate of vesicular calcium uptake. Calcium transport rates could be partially restored by in vivo administration of 1,25-dihydroxyvitamin D-3 at a dosage which did not affect vesicular calcium uptake in control animals. Conversely, sodium-driven glucose accumulation in brush-border vesicles from triamcinolone-treated rats was stimulated by 50-70% after 36 h and appeared insensitive to vitamin D. A specific triamcinolone action on the glucose carrier itself rather than on the driving force of the sodium gradient was indicated by (i) a similar stimulation of glucose transport under equilibrium exchange conditions and (ii) an opposite effect of triamcinolone on sodium-driven alanine transport. The triamcinolone-induced changes in calcium and glucose uptake were not accompanied by a gross alteration of membrane integrity in vitro or by major alterations in vesicular protein composition, intravesicular glucose space and sucrase or alkaline phosphatase activity. The modification of vesicular transport properties is discussed in relation to the vitamin D-antagonized inhibition of intestinal calcium uptake and the stimulation of glucose absorption in response to supraphysiologic amounts of glucocorticoids observed in intact epithelium.     

Calcium oxalate and calcium phosphate capacities of cardiac sarcoplasmic reticulum

Both oxalate-supported and phosphate-supported calcium uptake by canine cardiac sarcoplasmic reticulum initially increase linearly with time but fall to a steady-state level within 20 min. The departure from linearity could be due to a decrease in influx or to an increase in efflux of calcium. Because Ca2+-ATPase activity is linear, a decrease in the influx of calcium is an unlikely cause of the non-linear calcium uptake curves. A possible cause of an increase in calcium efflux is rupture of the vesicles. This hypothesis was tested by investigating the amount of calcium which could be released upon addition of 5 mM EGTA. The amount of rapidly releasable calcium was zero until a threshold calcium uptake of about 4-6 mumol calcium oxalate or calcium phosphate per mg was reached. After that point the rapidly releasable calcium continued to increase with calcium oxalate to reach more than 23 mumol/mg, but stayed constant at about 0.7 mumol/mg for calcium phosphate. The rapidly releasable calcium was attributed to calcium oxalate or calcium phosphate crystals externalized by vesicle rupture. The differences in the amounts of rapidly releasable calcium were attributed to different kinetics of calcium phosphate and calcium oxalate dissolution. Addition of ryanodine caused a marked increase in the threshold for rapidly releasable calcium oxalate. Transmission electron micrographs showed that vesicles can become filled with calcium oxalate crystals, but the vesicles were heterogeneous with respect to their size and their sensitivity to ryanodine. These observations support the hypothesis that calcium oxalate and calcium phosphate capacities are limited by vesicle rupture and that ryanodine increases the capacity by closing a calcium channel in a subpopulation of vesicles that otherwise would not accumulate calcium.