Cytosolic calcium dependent proteinase of human erythrocytes: Formation of an enzyme-natural inhibitor complex induced by Ca2+ ions

A calcium dependent soluble neutral proteinase has been purified to homogeneity from human erythrocytes. The proteinase is composed of two different polypeptide chains of approximate molecular weight of 80 k and 30 k daltons. Maximum activity is expressed at 50 μM Ca²⁺. The enzyme is regulated by reversible binding to a natural inhibitor, also present in the cytosolic compartment. The formation of the enzyme-inhibitor complex is dependent on high Ca²⁺ concentrations and is reversed by chelating agents. The proteinase is inhibited by leupeptin, chymostatin, antipain and free hemin and has a marked specificity for native or denatured human globin chains.     

Evidence for an endogenous protein inhibitor of sarcoplasmic reticulum calcium pump in heart muscle

A cytosolic protein fraction, termed CPF-I, derived by (NH₄)₂ SO₄ fractionation of rabbit heart cytosol caused marked inhibition (up to 95%) of ATP-dependent Ca²⁺ uptake by cardiac sarcoplasmic reticulum. The inhibitory effect of CPF-I was concentration-dependent (50% inhibition with ～ 80–100 μg CPF-I) and heat labile. The inhibitor reduced the velocity of Ca²⁺ uptake without altering the apparent affinity of the transport system for Ca²⁺. Concomitant with the inhibition of Ca²⁺ uptake, Ca²⁺-sensitive ATP hydrolysis was also inhibited by CPF-I. The inhibitor did not cause release of Ca²⁺ from Ca²⁺-preloaded membrane vesicles. The inhibitor activity of CPF-I could be adsorbed to a DEAE cellulose column and could be eluted with a linear gradient of KCl. These results demonstrate the presence of a soluble protein inhibitor of sarcoplasmic reticulum calcium pump in cardiac muscle and raises the intriguing possibility of its participation in the regulation of calcium pump $\text{in}\text{vivo}$.     

Effects of calcium on a parathyroid hormone-sensitive adenylate cyclase inhibitor

Inhibition of parathyroid hormone (PTH)-sensitive adenylate cyclase by {Nle-8, Nle-18, Tyr-34} bPTH-(3–34) amide was studied in thyroparathyroid-ectomized dogs. The inhibitory effect was shown to be markedly enhanced by the addition of calcium ions into the $\text{in}\text{,}\text{vitro}$ assay system. At 0.1 mM Ca²⁺, complete inhibition by the antagonist was obtained. Chelation of exogenous Ca²⁺ by EGTA eliminated the Ca²⁺-induced inhibition. Both the basal and hormone-stimulated activities were decreased in the presence of 0.1 mM Ca²⁺, whereas the addition of EGTA increased both activities. Our results suggest that Ca²⁺ modulates canine renal PTH-sensitive adenylate cyclase and its inhibition by substituted bPTH-(3–34).     

Effects of ambient calcium concentration on morphological form of callus-like cells in Saccharina japonica (Phaeophyceae) sporophyte

Explants obtained from young sporophytes of Saccharina japonica were cultured in an artificial medium with different concentrations of Ca²⁺ (0–20?mM). The culture with 10?mM Ca²⁺ promoted the formation of unpigmented filamentous callus-like cells in the cortical layer. In contrast, explants cultured with 5?mM Ca²⁺ formed pigmented round callus-like cells in the epidermis at a high percentage. The thallus regeneration rate of explants in 5?mM Ca²⁺ was ten times higher than those of explants cultured in 10?mM Ca²⁺. Ambient Ca²⁺ concentrations also influenced the production of radical oxygen species (ROS) in explants. Explants cultured in 10?mM Ca²⁺ produced higher ROS than did those cultured in 5?mM. The ROS production was histologically observed mainly in the plasma membrane of callus-like cells using 2′, 7′-dichlorodihydrofluorescein diacetate. Moreover, a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, diphenyleneiodonium chloride, inhibited the ROS production with propagation of filamentous callus-like cells. These results suggest that Ca²⁺ concentration in medium influences the development of callus-like cells and thallus regeneration by affecting NADPH oxidase activity and ROS production in the plasma membrane of the callus-like cells. Therefore, the morphological form of callus-like cells and the development of thallus will be controlled by Ca²⁺ concentration in the medium.     

Strontium induces murine keratinocyte differentiation in vitro in the presence of serum and calcium

Primary mouse keratinocytes in culture are induced to terminally differentiate by increasing extracellular Ca²⁺ concentrations (Ca_O) from 0.05 mM to ≥ 0.1 mM. The addition of Sr²⁺ (≥ 2.5 mM) to medium containing 0.05 mM Ca²⁺ induces focal stratification and terminal differentiation, which are similar to that found after increasing the Ca_O to 0.12 mM. Sr²⁺ in 0.05 mM Ca²⁺ medium induces the expression of the differentiation-specific keratins, keratin 1 (K1), keratin 10 (K10), and the granular cell marker, filaggrin, as determined by both immunoblotting and immunofluorescence. Sr²⁺ induces the expression of those differentiation markers in a dose dependent manner, with an optimal concentration of 5 mM. In the absence of Ca²⁺ in the medium, the Sr²⁺ effects are reduced, and Sr²⁺ is ineffective when both Ca²⁺ and serum are deleted from the medium. Sr²⁺ treatment increases the ratio of fluorescence intensity of the intracellular Ca²⁺ sensitive probe, fura-2, indicating an associated rise in the level of intracellular free Ca²⁺ and/or Sr²⁺. At doses sufficient to induce differentiation, Sr²⁺ also increases the level of inositol phosphates in primary keratinocytes within 30 min. The uptake curves of ⁸⁵Sr²⁺ by primary keratinocytes are similar to those of ⁴⁵Ca²⁺. At low concentrations, the initial uptake of both ⁴⁵Ca²⁺ and ⁸⁵Sr²⁺ reaches a plateau within 1 hr; at higher concentrations, the uptake of both ⁴⁵Ca²⁺ and ⁸⁵Sr²⁺ increases continuously for 12 hr. In keratinocytes pre-equilibrated with ⁴⁵Ca²⁺ in 0.05 mM Ca²⁺ medium, Sr²⁺ causes an increase of ⁴⁵Ca²⁺ uptake, which is dependent on the presence of serum. These results suggest that Sr²⁺ utilizes the same signalling pathway as Ca²⁺ to induce keratinocyte terminal differentiation and that Ca²⁺ may be required to exert these effects. © 1993 Wiley-Liss, Inc.     

Inhibition of exogenous NADH oxidation in plant mitochondria by chlorotetracycline in the presence of calcium ions

Chlorotetracycline inhibits the uncoupled oxidation of exogenous NADH by Jerusalem artichoke (Helianthus tuberosus L.) mitochondria extensively (over 80%) and rapidly (inhibition complete in 10 s) in the presence of added Ca²⁺. Half-maximal inhibition is observed at 15 μM chlorotetracycline in the presence of 2 mM Ca²⁺. The oxidation of succinate is only affected marginally by chlorotetracycline plus Ca²⁺. The inhibition of NADH oxidation and the fluorescence of CTC are well correlated. Mn²⁺ is the only other cation which shows an (increased) inhibition in the presence of chlorotetracycline. The inhibition by Ca²⁺ and chlorotetracycline disappears at acid pH, and the pH optimum in their presence is 6.4. The inhibition caused by other lipid-soluble Ca²⁺-chelators is not reversible or is enhanced by the addition of excess Ca²⁺. In contrast, inhibition caused by relatively water-soluble chelators is completely reversed by added Ca²⁺. It is suggested that a neutral 1:2 complex is formed between Ca²⁺ and chlorotetracycline which can substitute for Ca²⁺ bound at sites in the lipophilic phase of the inner mitochondrial membrane, which are essential for the activity of the external NADH dehydrogenase.     

The uptake of calcium by isolated chromaffin granules of the adrenal medulla

Bovine chromaffin secretory granules were purified by isopycnic Metrizamide gradient centrifugation and their Ca²⁺ sequestration pathways were characterized. The rate of Ca²⁺ sequestration at 37°C was first order, with a maximal uptake of 26.9 ±0.46 (mean ± S.D., n = 3) nmol Ca²⁺/mg protein and a first order rate constant (k) of 0.046 ± 0.002 min^–1. At 4°C the rate of uptake was substantially attenuated, with only 2.47 ± 0.2 (mean ± S.D, n = 3) nmol Ca²⁺/mg protein sequestered in 60 min. Ca²⁺ sequestration was 93% inhibited by 180 mM NaCl [I_50% of 78.7 ± 9.3 mM NaCl (mean ± S.D., n = 11)] but only slightly inhibited by KCl or MgCl₂. Ca ²⁺ sequestration was not stimulated by incubation with MgATP but was inhibited by 57% after incubation with 30 M monensin. Ca ²⁺ sequestration was dependent on extravesicular Ca ²⁺ with half-maximal sequestration at pCa²⁺ 6.81 ± 0.028 (mean ± S.D., n = 3). Sequestered Ca²⁺ could be exchanged with external ⁴⁵Ca²⁺, the exchange rate was first order (k of 0.042 ± 0.004: mean ± S.D., n = 3) and saturated at 27.7 ± 1.1 nmol Ca²⁺/mg (mean ± S.D., n = 3). The Ca²⁺/Ca²⁺ exchange system was totally inhibited by NaCl or KCl but only slightly by MgCl₂. About 75% of sequestered ⁴⁵Ca²⁺ could be released by incubation with NaCl, but only 8% was released by incubation with KCI. Half-maximal release of sequestered ⁴⁵Ca²⁺ required 69.3 ± 12.2 mM NaCl (mean ± S.D., n = 3). The Na⁺-induced release of sequestered ⁴⁵Ca²⁺ was rapid, t_0.5 of 2.80 ± 0.63 min (mean ± S.D., n = 3) and inhibited at 4°C. The concurrent incubation of chromaffin granules with ⁴⁵Ca²⁺ and either annexin proteins V or VI resulted in attenuated uptake of ⁴⁵Ca²⁺. These results suggest that Ca²⁺ uptake in adrenal chromaffin granules is regulated by Na⁺ and Ca²⁺ gradients and also possibly by annexins V and VI.Abbreviations EGTA ethylene glycol bis (-aminoethyl ether)-N,-N,N,N-tetraacetic acid - SDS Sodium dodecyl sulphate - PAGE Polyacrylamide gel electrophoresis - BSA bovine serum albumin - AI Annexin I - AIIt Annexin II tetramer - AIII Annexin III - AIV Annexin IV - AV Annexin V - AVI Annexin VI - k first order rate constant - AT total extent of Ca²⁺ uptake (nmol) - BufferA 300 mM sucrose, 10 mM potassium phosphate (pH 7.0), 5 mM EGTA - Buffer B 300 mM sucrose, 10 mM potassium phosphate (pH 7.0) and 1 mM EGTA - Buffer C 300 mM sucrose, 10 mM potassium phosphate (pH 7.0) - Buffer D 300 mM sucrose, 10 mM potassium phosphate (pH 7.0), 0.5 mM EGTA and 0.65 MM CaCl₂ - Buffer E 300 mM sucrose, 10 mM potassium phosphate (pH 7.0), 0.25 mM EGTA and 0.325 mM CaCl₂     

Endogenous inhibitor of calcium activated neutral proteinase from human placenta: Purification and possible mechanism of proteinase regulation

An endogenous inhibitor of calcium activated neutral proteinase has been purified from human placenta. The procedure included chromatography on DEAE cellulose, Ultrogel AcA 22 and milli calcium activated neutral proteinase-sepharose in succession. Endogenous calcium activated neutral proteinase inhibitor was a tetramer with identical subunits of molecular weight 68 kDa. It was specific for milli calcium activated neutral proteinase (Calpain II) which is inhibited by the formation of an inactive enzyme-inhibitor complex and not by sequestering Ca₂₊ from the medium. Although micro calcium activated neutral proteinase (Calpain I) was not inhibited by endogenous calcium activated neutral proteinase inhibitor, it was protected from autolysis in the presence of the inhibitor. The placental endogenous calcium activated neutral proteinase inhibitor thus regulates Ca₂₊ activated proteolysis by ensuring micro calcium activated neutral proteinase activity, while inhibiting milli calcium activated neutral proteinase.     

Mercuric chloride induces increases in both cytoplasmic and nuclear free calcium ions through a protein phosphorylation-linked mechanism

The mechanism of the lymphocyte stimulatory action of sulfhydryl group-reactive mercuric ions was studied with respect to its potential ability to induce a protein tyrosine phosphorylation-linked signal for mobilization of free Ca²⁺ into cytoplasm and nucleus of the cell. Exposure of human leukamic T cell line (Jurkat) cells to high (1 mM) and low (0.01 mM) concentrations of HgCl₂ induced tyrosine phosphorylation of multiple proteins in a concentration-dependent manner. Confocal microscopy directly visualized the time course localization of Ca²⁺ inside the cells after exposure to HgCl₂. The onset and level of Ca²⁺ mobilization following HgCl₂ exposure were in parallel to those of protein tyrosine phosphorylation. Interestingly, by either concentration of HgCl₂, Ca²⁺ was mobilized in both cytoplasm and nucleus almost simultaneously, and the level of Ca²⁺ mobilization in the nucleus was more than that in the cytoplasm. All the HgCl₂-mediated Ca²⁺ mobilization was prevented by addition of protein kinase inhibitor staurosporin prior to HgCl₂. These results suggest that heavy metal stress triggers a protein tyrosine phosphorylation-linked signal that leads to a nuclear event-dominant Ca²⁺ mobilization.     

Alleviation of cadmium toxicity on maize seedlings by calcium

The rate of germination, radicle and plumule length, fresh and dry mass of maize seedlings were increased as Ca²⁺ was added to the nutrient solution, which contained different levels of Cd²⁺, especially at low concentration of Ca²⁺ (5 mM) and high concentrations of Cd²⁺ (1.4 and 1.8 mM). The biosynthesis of pigments, respiration rate and content of soluble saccharides in endosperm were reduced sharply as the concentration of Cd²⁺ in the medium increased. This effects was alleviated by Ca²⁺ addition. Cd²⁺ content in seedlings was increased as the Cd²⁺ concentration in medium was increased and decreased sharply as Ca²⁺ was present in the culture medium. The study suggests liming of soil with CaCO₃ to improve the yield of many crops.     

Specificity of the requirements for magnesium and calcium in the growth and metabolism of chick embryo fibroblasts

The rate of entry of chick embryo fibroblasts (CEF) into the S-period of the cell cycle is reduced by lowering the external supply of Mg²⁺ below 0.2 mM. This slowdown, which is measured by the rate of incorporation of ³H thymidine into DNA, can be largely reversed by doubling or tripling the concentration of Ca²⁺ in the medium, normally 1.7 mM. The Ca²⁺-induced stimulation is shown not to depend on contaminating traces of Mg²⁺ in the added Ca²⁺. The increase in cell number in the Ca²⁺-stimulated cultures is delayed, possibly due to cell detachment. The effect of Ca²⁺ on thymidine incorporation can be simulated almost quantitatively by Sr²⁺. Ba²⁺ does not produce the effect, nor do any of the other cations tested. As little as 0.2 mM Mg²⁺ produces a full stimulation of thymidine incorporation in the absence of added Ca²⁺, and no substitute was found that is effective in the same concentration range. In short term experiments, i.e., 16 hours, even 5.0 mM Ca²⁺ cannot stimulate thymidine incorporation to the extent achieved with 0.2 mM Mg²⁺. Large amounts of Ca²⁺ or Sr²⁺ can accelerate the uptake of 2-dGlc in Mg²⁺-deprived cultures, but they are much less efficient than Mg²⁺ in this regard also. It is suggested that Mg²⁺ is the direct intracellular effector in controlling the diverse reactions of the coordinate response, and that Ca²⁺ and Sr²⁺ act indirectly by making Mg²⁺ available to participate in these reactions.     

Stromal free calcium concentration and light-mediated activation of chloroplast fructose-1,6-bisphosphatase

Light-mediated activation of fructose-1,6-bisphosphatase (EC 3.1.3.11) in intact spinach chloroplasts (Spinacia oleracea L.) is enhanced in the presence of 10⁻⁵ molar external free Ca²⁺. The most pronounced effect is observed during the first minutes of illumination. Ruthenium red, an inhibitor of light-induced Ca²⁺ influx, inhibits this Ca²⁺ stimulated activation. In isolated stromal preparations, the activation of fructose-1,6-bisphosphatase is already enhanced by 2 minutes of exposure to elevated Ca²⁺ concentrations in the presence of physiological concentrations of Mg²⁺ and fructose-1,6-bisphosphate. Maximal activation of the enzyme is achieved between 0.34 and 0.51 millimolar Ca²⁺. The Ca²⁺ mediated activation decreases with increasing fructose-1,6-bisphosphate concentration and with increasing pH. The data are consistent with the proposal that the illumination of chloroplasts leads to a transient increase of free stromal Ca²⁺. In dark-kept chloroplasts the steady-state concentration of free stromal Ca²⁺ is 2.4 to 6.3 micromolar as determined by null point titration. These observations support our previous proposal that light-induced Ca²⁺ influx into chloroplasts does not only influence the cytosolic concentration of free Ca²⁺ but also regulates enzymatic processes inside the chloroplast.     

Mechanisms of low Na+-induced increase in intracellular calcium in KCl-depolarized rat cardiomyocytes

Although low Na⁺ is known to increase the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in cardiac muscle, the exact mechanisms of low Na⁺-induced increases in [Ca²⁺]_i are not completely defined. To gain information in this regard, we examined the effects of low Na⁺ (35 mM) on freshly isolated cardiomyocytes from rat heart in the absence and presence of different interventions. The [Ca²⁺]_i in cardiomyocytes was measured fluorometrically with Fura-2 AM. Following a 10 min incubation, the low Na⁺-induced increase in [Ca²⁺]_i was only observed in cardiomyocytes depolarized with 30 mM KCl, but not in quiescent cardiomyocytes. In contrast, low Na⁺ did not alter the ATP-induced increase in [Ca²⁺]_i in the cardiomyocytes. This increase in [Ca²⁺]_i due to low Na⁺ and elevated KCl was dependent on the extracellular concentration of Ca²⁺ (0.25–2.0 mM). The L-type Ca²⁺-channel blockers, verapamil and diltiazem, at low concentrations (1 M) depressed the low Na⁺, KCl-induced increase in [Ca²⁺]_i without significantly affecting the response to low Na⁺ alone. The low Na⁺, high KCl-induced increase in [Ca²⁺]_i was attenuated by treatments of cardiomyocytes with high concentrations of both verapamil (5 and 10 M), and diltiazem (5 and 10 M) as well as with amiloride (5–20 M), nickel (1.25–5.0 mM), cyclopiazonic acid (25 and 50 M) and thapsigargin (10 and 20 M). On the other hand, this response was augmented by ouabain (1 and 2 mM) and unaltered by 5-(N-methyl-N-isobutyl) amiloride (5 and 10 M). These data suggest that in addition to the sarcolemmal Na⁺–Ca²⁺ exchanger, both sarcolemmal Na⁺–K⁺ATPase, as well as the sarcoplasmic reticulum Ca²⁺-pump play prominent roles in the low Na⁺-induced increase in [Ca²⁺]_i. (Mol Cell Biochem 263: 151–162, 2004)     

Subunit A of calmodulin-dependent protein phosphatase requires Mn2+ for activity

Bovine brain calmodulin-dependent protein phosphatase comprises a catalytic subunit A (Mr 60,000) and a regulatory subunit B (Mr 19,000). The native enzyme was active with Ca²⁺ or Mn²⁺. Upon resolution into its subunits in 6 M urea and 15 mM EDTA, subunit A was active with Mn²⁺; Co²⁺ and Ni²⁺ partially substituted for Mn²⁺, but Ca²⁺, Mg²⁺ and Zn²⁺ were ineffective. The stimulating effect of Mn²⁺ was not easily reversed by EGTA. Like the native phosphatase, subunit A was markedly stimulated by calmodulin or by controlled trypsinization. Unlike the native enzyme, however, trypsinized subunit A still required Mn²⁺ for activity. These findings provide evidence that the catalytic subunit of phosphatase may be a metallo (possibly Mn²⁺) enzyme.     

Extracellular calcium promotes the migration of breast cancer cells through the activation of the calcium sensing receptor

Breast cancer is the most frequent form of cancer in women, with the highest incidence of metastasis to the bone. The reason for the preferential destination to the bone is believed to be due to chemoattractant factors released during bone resorption, which act on the cancer cells facilitating their metastasis. One of the factors released during osteolysis that may mediate breast cancer bone localization is Ca²⁺. Here, we show that extracellular Ca²⁺ (Ca²⁺_o) acting via the calcium-sensing receptor (CaSR), greatly promotes the migration of bone-preferring breast cancer cells. In Boyden Chamber and Scratch Wound migration assays, an increase in breast cancer cell migration was observed at 2.5 mM and 5 mM Ca²⁺_o compared to basal levels for three of the four breast cancer cell lines tested. However, a significantly greater migratory response was observed for the highly bone metastatic MDA-MB-231 cells, compared to the MCF7 and T47D, which have a lower metastatic potential in vivo. The BT474 cells, which do not metastasize to the bone, did not respond to elevated concentrations of Ca²⁺_o in the migration assays. Inhibition of either ERK1/2 MAPK or phospholipase Cβ (PLCβ) led to an abolition of the Ca²⁺_o-induced migration, implicating these pathways in the migratory response. Knockdown of the CaSR by siRNA resulted in an inhibition of the Ca²⁺_o-induced migration, demonstrating the involvement of this receptor in the effect. These results suggest that the activation of the CaSR by elevated Ca²⁺_o concentrations, such as those found near resorbing bone, produces an especially strong chemoattractant effect on bone metastatic breast cancer cells toward the Ca²⁺-rich environment.     

The effect of calcium ionophore A23187 on the ATP level of human erythrocytes

Incubation of red cells at 37° with the ionophore A23187 results in a loss of ATP that is dependent on the concentrations of A23187 and Ca²⁺ in the medium. ATP hydrolysis is greatest at micromolar concentrations of Ca²⁺ and decreases as Ca²⁺ in the medium is raised to millimolar levels. The ATP depletion is due to stimulation of calcium ATPase by A23187-mediated Ca²⁺ influx into the cell. The biphasic nature of Ca²⁺-stimulated ATP depletion in whole cells reflects the activity of Ca²⁺-ATPase in membrane preparations at varying Ca²⁺ concentrations. The ionophore can be removed by washing the cells with plasma or bovine serum albumin-containing medium and the ATP levels restored to normal by reincubating with 5 mM adenosine for 1 hr.     

Isolation and purification of calcium and magnesium dependent endonuclease from rat liver nuclei

An endonuclease associated with rat liver chromatin was extracted with 0.6 M NaCl and purified by ammonium sulfate fractionation and Sephadex G-100 gel filtration. The enzyme produces single strand scissions on native DNA at neutral pH in the presence of 1 mM CaCl₂ and 5 mM MgCl₂. Alkali-denatured DNA was not nicked by the enzyme. Omission of Ca²⁺ reduced the enzyme activity to about one seventh. Without Ca²⁺, however, Mn²⁺ was more effective than Mg²⁺. The molecular weight of the enzyme is about 27,000.     

The effect of 2,4-dinitrophenol on the absorption and translocation of calcium by pumpkin plants

The study deals with the absorption and translocation of Ca²⁺ by án intact plant of pumpkinCucurbita pepo L. and with the effect of various concentrations of 2,4-dinitrophenol (DNP) on these processes. The absorption of Ca²⁺ was reduced by the application of this inhibitor, but not completely stopped. The translocation of Ca²⁺ into shoots was affected more expressively: it was almost completely inhibited by higher DNP concentrations. The uptake of Rb⁺ is aubstantially more influeneed than that of Ca²⁺: higher inhibitor concentrations caused its complete cessation.     

Stabilization of calcium uptake in rat rod outer segments by taurine and ATP

Summary. Calcium ion (Ca²⁺) uptake was measured in rod outer segments (ROS) isolated from rat retina in the presence of varying concentrations of CaCl₂ in the incubation buffer (1.0–2.5 mM). It is known that taurine increases Ca²⁺ uptake in rat ROS in the presence of ATP and at low concentrations of CaCl₂ (Lombardini, 1985a); taurine produces no significant effects when CaCl₂ concentrations are increased to 1.0 and 2.5 mM. With the removal of both taurine and ATP, Ca²⁺ uptake in rat ROS increased significantly in the presence of 2.5 mM CaCl₂. Taurine treatment in the absence of ATP was effective in decreasing Ca²⁺ uptake at the higher levels of CaCl₂ (2.0 and 2.5 mM). Similar effects were observed with ATP treatment. The data suggest that taurine and ATP, alone or in combination, limit the capacity of the rat ROS to take up Ca²⁺ to the extent that a stable uptake level is achieved under conditions of increasing extracellular Ca²⁺, indicating a protective role for both agents against calcium toxicity. Received January 25, 2000/Accepted January 31, 2000