The effect of Na+ and K+ on the thermal denaturation of Na+ and + K+-dependent ATPase.

To increase our understanding of the physical nature of the Na+ and K+ forms of the Na+ + K+-dependent ATPase, thermal-denaturation studies were conducted in different types of ionic media. Thermal-denaturation measurements were performed by measuring the regeneration of ATPase activity after slow pulse exposure to elevated temperatures. Two types of experiments were performed. First, the dependence of the thermal-denaturation rate on Na+ and K+ concentrations was examined. It was found that both cations stabilized the pump protein. Also, K+ was a more effective stabilizer of the native state than was Na+. Secondly, a set of thermodynamic parameters was obtained by measuring the temperature-dependence of the thermal-denaturation rate under three ionic conditions: 60 mM-K+, 150 mM-Na+ and no Na+ or K+. It was found that ion-mediated stabilization of the pump protein was accompanied by substantial increases in activation enthalpy and entropy, the net effect being a less-pronounced increase in activation free energy.     

铜、铅、镉、锌、汞和银离子复合污染对水螅的急性毒性效应

以水螅(Hydrasp)为例,通过单因子静态急性毒性试验方法和等毒性溶液法,分别研究Hg2 、Cu2 、Cd2 、Ag 、Zn2 和Pb2 对其单一和复合毒性效应。单一实验结果表明,它们对水螅毒性大小顺序为Hg2 >Cu2 >Cd2 >Ag >Zn2 >Pb2 。复合毒性实验表明,Zn2 与Cu2 、Hg2 、Pb2 、Ag ;Pb2 与Cu2 ;Hg2 与Ag ;Pb2 与Ag 这些组合对水螅联合急性毒性总体上表现出拮抗作用,Cd2 与Cu2 、Hg2 、Pb2 、Ag 组合总体上则是协同作用,Zn2 与Cd2 、Pb2与Hg2 、Cu2 与Hg2 ,Ag 在不同的浓度水平组合下明显表现出不同的毒性效应。     

Macroscopic Ca2+ -Na+ and K+ currents in single heart and aortic cells

The whole-cell voltage clamp technique was used to study the slow inward currents and K+ outward currents in single heart cells of embryonic chick and in rabbit aortic cells. In single heart cells of 3-day-old chick embryo three types of slow inward Na+ currents were found. The kinetics and the pharmacology of the slow INa were different from those of the slow ICa in older embryos. Two types of slow inward currents were found in aortic single cells of rabbit; angiotensin II increased the sustained type and d-cAMP and d-cGMP decreased the slow transient component. Two types of outward K+ currents were found in both aortic and heart cells. Single channel analysis demonstrated the presence of a high single K+ channel conductance in aortic cells. In cardiac and vascular smooth muscles, slow inward currents do share some pharmacological properties, although the regulation of these channels by cyclic nucleotides and several drugs seems to be different.     

Different cation sensitivities and binding site domains of Na+-Ca2+-K+ and Na+-Ca2+ exchangers

We examined inhibitory effects of external multivalent cations Ni(2+), Co(2+), Cd(2+), La(3+), Mg(2+), and Mn(2+) on reverse-mode exchange of the K(+)-dependent Na(+)/Ca(2+) exchanger NCKX2 and the K(+)-independent exchanger NCX1 expressed in CCL-39 cells by measuring the rate of Ca(2+) uptake with radioisotope tracer and electrophysiological techniques. The apparent affinities for block of Ca(2+) uptake by multivalent cations was higher in NCKX2 than NCX1, and the rank order of inhibitory potencies among these cations was different. Additional experiments also showed that external Li(+) stimulated reverse-mode exchange by NCX1, but not NCKX2 in the presence of 5 mM K(+). Thus, both exchangers exhibited differential sensitivities to not only K(+) but also many other external cations. We attempted to locate the putative binding sites within the alpha motifs for multivalent cations by site-directed mutagenesis experiments. The cation affinities of NCKX2 were altered by mutations of amino acid residues in the alpha-1 motif, but not by mutations in the alpha-2 motif. These results contrast with those for NCX1 where mutations in both alpha-1 and alpha-2 motifs have been shown previously to affect cation affinities. Susceptibility tests with sulfhydryl alkylating agents suggested that the alpha-1 and alpha-2 motifs are situated extracellularly and intracellularly, respectively, in both exchangers. A topological model is proposed in which the extracellular-facing alpha-1 motif forms an external cation binding site that includes key residues N203, G207C, and I209 in NCKX2, while both alpha-1 and alpha-2 motifs together form the binding sites in NCX1.     

Macroscopic Ca2+ -Na+ and K+ currents in single heart and aortic cells

Summary The whole-cell voltage clamp technique was used to study the slow inward currents and K⁺ outward currents in single heart cells of embryonic chick and in rabbit aortic cells. In single heart cells of 3-day-old chick embryo three types of slow inward Na⁺ currents were found. The kinetics and the pharmacology of the slow I_Na, were different from those of the slow Ica in older embryos. Two types of slow inward currents were found in aortic single cells of rabbit; angiotensin 11 increased the sustained type and d-cAMP and d-cGMP decreased the slow transient component. Two types of outward K⁺ currents were found in both aortic and heart cells. Single channel analysis demonstrated the presence of a high single K⁺ channel conductance in aortic cells. In cardiac and vascular smooth muscles, slow inward currents do share some pharmacological properties, although the regulation of these channels by cyclic nucleotides and several drugs seems to be different.     

Activity coefficients of the peptide and the electrolyte in ternary systems water+glycylglycine+NaCl, +NaBr, +KCl and +KBr at 298.2 K

The activity coefficients of glycylglycine in four aqueous electrolyte solutions (+NaCl, +NaBr, +KCl and +KBr) were obtained at 298.2 K. The mean ionic activity coefficient of the electrolyte in aqueous solutions containing the peptide was determined from measurements of the potential differences of a cation and an anion ion-selective-electrode, each vs. a double junction reference electrode. The results show that the nature of the anion has a major effect on the activity coefficients of glycylglycine. Comparison of activity coefficient data for glycylglycine with literature data for glycine, both in aqueous NaCl solutions, indicates that the effect of the electrolyte is larger for the peptide than for the amino acid. For the peptide, in all cases, the effect of the electrolyte is more important at low molalities of the electrolyte. The Wilson equation was used to correlate the activity coefficient data obtained. The correlation results were satisfactory for the region of concentrated electrolyte.     

Effects of Ca2+ and Mg2+ on Elongation and H+ Secretion of Vigna radiata Hypocotyl Sections

The effects exerted by divalent cations Ca²⁺ and Mg²⁺ on spontaneous,IAA- and H⁺-induced elongation were investigated with excisedmung bean hypocotyl segments. Low concentrations of calcium(<1 mM) were sufficient to inhibit spontaneous and IAA-inducedelongation of abraded segments without any lag time whereaseffects of magnesium were not so evident. The initial growthrate could be restored, at least partly, with EDTA or acidicsolutions. Both calcium and magnesium enhanced H⁺ excretionbut Mg²⁺ was a more potent activator. Divalent cations alsoinduced simultaneously an inhibition of growth and a stimulationof proton release. Such contrasting effects are discussed withrespect to the functions of the cuticle. (Received May 1, 1984; Accepted September 10, 1984)     

Homooligomeric and heterooligomeric associations between K+-Cl- cotransporter isoforms and between K+-Cl- and Na+-K+-Cl- cotransporters

Little is known regarding the quaternary structure of cation-Cl- cotransporters (CCCs) except that the Na+-dependent CCCs can exist as homooligomeric units. Given that each of the CCCs exhibits unique functional properties and that several of these carriers coexist in various cell types, it would be of interest to determine whether the four K+-Cl- cotransporter (KCC) isoforms and their splice variants can also assemble into such units and, more importantly, whether they can form heterooligomers by interacting with each other or with the secretory Na+-K+-Cl- cotransporter (NKCC1). In the present work, we have addressed these questions by conducting two groups of analyses: 1) yeast two-hybrid and pull-down assays in which CCC-derived protein segments were used as both bait and prey and 2) coimmunoprecipitation and functional studies of intact CCCs coexpressed in Xenopus laevis oocytes. Through a combination of such analyses, we have found that KCC2 and KCC4 could adopt various oligomeric states (in the form of KCC2-KCC2, KCC4-KCC4, KCC2-KCC4, and even KCC4-NKCC1 complexes), that their carboxyl termini were probably involved in carrier assembly, and that the KCC4-NKCC1 oligomers, more specifically, could deploy unique functional features. Through additional coimmunoprecipitation studies, we have also found that KCC1 and KCC3 had the potential of assembling into various types of CCC-CCC oligomers as well, although the interactions uncovered were not characterized as extensively, and the protein segments involved were not identified in yeast two-hybrid assays. Taken together, these findings could change our views on how CCCs operate or are regulated in animal cells by suggesting, in particular, that cation-Cl- cotransport achieves higher levels of functional diversity than foreseen.     

Characterization of Foxp3+CD4+CD25+ and IL-10-secreting CD4+CD25+ T cells during cure of colitis

CD4+CD25+ regulatory T cells can prevent and resolve intestinal inflammation in the murine T cell transfer model of colitis. Using Foxp3 as a marker of regulatory T cell activity, we now provide a comprehensive analysis of the in vivo distribution of Foxp3+CD4+CD25+ cells in wild-type mice, and during cure of experimental colitis. In both cases, Foxp3+CD4+CD25+ cells were found to accumulate in the colon and secondary lymphoid organs. Importantly, Foxp3+ cells were present at increased density in colon samples from patients with ulcerative colitis or Crohn's disease, suggesting similarities in the behavior of murine and human regulatory cells under inflammatory conditions. Cure of murine colitis was dependent on the presence of IL-10, and IL-10-producing CD4+CD25+ T cells were enriched within the colon during cure of colitis and also under steady state conditions. Our data indicate that although CD4+CD25+ T cells expressing Foxp3 are present within both lymphoid organs and the colon, subsets of IL-10-producing CD4+CD25+ T cells are present mainly within the intestinal lamina propria suggesting compartmentalization of the regulatory T cell response at effector sites.     

Dependence of Na+-Ca2+ exchange and Ca2+-Ca2+ exchange on monovalent cations

Two mechanisms of passive Ca2+ transport, Na+-Ca2+ exchange and Ca2+-Ca2+ exchange, were studied using highly-purified dog heart sarcolemmal vesicles. About 80% of the Ca2+ accumulated by Na+-Ca2+ exchange or Ca2+-Ca2+ exchange could be released as free Ca2+, while up to 20% was probably bound. Na+-Ca2+ exchange was simultaneous, coupled countertransport of Na+ and Ca2+. The movement of anions during Na+-Ca2+ exchange did not limit the initial rate of Na+-Ca2+ exchange. Na+-Ca2+ exchange was electrogenic, with a reversal potential of about -105 mV. The apparent flux ratio of Na+-Ca2+ exchange was 4 Na+:1 Ca2+. Coupled cation countertransport by the Na+-Ca2+ exchange mechanism required a monovalent cation gradient with the following sequence of ion activation: Na+ much greater than Li+ greater than Cs+ greater than K+ greater than Rb+. In contrast to Na+-Ca2+ exchange, Ca2+-Ca2+ exchange did not require a monovalent cation gradient, but required the presence of Ca2+ plus a monovalent cation on both sides of the vesicle membrane. The sequence of ion activation of Ca2+-Ca2+ exchange was: K+ much greater than Rb+ greater than Na+ greater than Li+ greater than Cs+. Na+ inhibited Ca2+-Ca2+ exchange when Ca2+-Ca2+ exchange was supported by another monovalent cation. Both Na+-Ca2+ exchange and Ca2+-Ca2+ exchange were inhibited, but with different sensitivities, by external MgCl2, quinidine, or verapamil.     

Lyt phenotype of cytotoxic T cells: shift from Lyt-1+2+3+ to a mixed population of Lyt-1+2+3+ and Lyt-1-2+3+ during in vitro culture

The Lyt phenotype of cytotoxic T cells generated in the primary H-2 response was investigated kinetically. The cytotoxicity generated in the early stage of culture was abolished by treatment with alpha Lyt-1,2,3, and complement (C), whereas that generated in the late stage was only partially eliminated by alpha Lyt-1, but was abolished by alpha Lyt-2, 3, and C. This suggested late expansion of the Lyt-1-2+3+ population. Lack of Lyt-1 antigen was confirmed with cells that were depleted of Lyt-1+ from primary culture and then stimulated in the secondary response by elimination of cytotoxicity and by direct Lyt typing. Results indicated that the response of proliferative and cytotoxic T cells of the Lyt-1+2+3+ phenotype in the early stage of culture was followed by activation of Lyt-1-2+3+ T cells. Cytotoxic T cells in the late stage were shown to be a mixture of Lyt-1+2+3+ and Lyt-1-2+3+ cells. This was confirmed with cytotoxic T cells from secondary culture and uncloned long-term T cell lines.     

Comparison of lipid effects on K+-Mg2+ activated p-nitrophenyl phosphatase and Na+-K+-Mg2+ activated adenosine triphosphatase of membrane

Abstract— A comparison was made between K⁺-Mg²⁺ activated p-nitrophenyl phosphatase and Na⁺-K⁺-Mg²⁺ activated adenosine triphosphatase with a solubilized enzyme preparation from a membrane fraction of cerebral cortex. The NPPase showed activity even in the absence of phospholipid, whereas the ATPase required the lipid for its activity. More varied types of phospholipids were effective in activating the NPPase than the ATPase, and with each phospholipid the extent and the pattern of the NPPase activation differed from that of the ATPase. By deoxycholate treatment the pH optimum of the NPPase was shifted independently from the pH optimum shift of the ATPase. The specific activity ratio of the NPPase to the ATPase was not constant during purification. These two enzymes were, however, not separable with ammonium sulphate fractionation, and their thermo-lability was identical regardless of the presence of phospholipid. The results suggested two possibilities: (1) the NPPase is a separate enzyme entity from the ATPase; (2) although the NPPase is a part of the ATPase system, the mechanism of action of lipids on the former part differs from that on the rest of the system.     

Na+ K+ pump and passive K+ transport in large and small red cell populations of anemic high and low K+ sheep

Reticulocytes, isolated by centrifugal elutriation from massively bled sheep and identified by cytometric techniques, were analyzed with respect to their cation transport properties. In sheep with genetically high K+ (HK) or low K+ (LK) red cells, two reticulocyte types were distinguished by conventional or fluorescence-staining techniques 5-6 days after hemorrhage: Large reticulocytes as part of a newly formed macrocytic (M) erythrocyte population, and small reticulocytes present among the adult red cell population (volume population III of normal sheep blood, Valet et al., 1978). Although cellular reticulin disappeared within a few days, the M-cell population persisted throughout weeks in the peripheral circulation permitting a transport study of in vivo maturation. At all times, M cells of LK sheep had lower K+ and higher Na+ contents than M cells of HK sheep. Regardless of the sheep genotypes, M cells apparently reduced their volume during their first days in circulation; however, throughout the observation period, they did not attain that characteristic for adult red cells. Both ouabain-sensitive K+ pump and ouabain-insensitive K+ leak fluxes were elevated in M cells of both HK and LK sheep. The increased K+ pump flux was mainly due to higher K+ pump turnover rather than to the modestly increased number of pumps as measured by [3H]ouabain binding. In contrast, small reticulocytes enriched from separated volume population III cells by a Percoll-density gradient exhibited transport parameters close to their prospective mature HK or LK red cells. The data support the concept that the M cells derived from emergency reticulocytes while the small reticulocytes represented precursors of normal red cell maturation. The Na+ and K+ composition found in M cells of HK and LK sheep, respectively, suggest development of the LK steady state at or prior to the reticulocyte state, a finding consistent with that of Lee and Kirk (1982) on low K+ dog red cells.     

Ca2+-binding of S-100 protein in the presence of K+ and Mg2+

Ca2+-binding of S-100 protein was studied using a Ca2+ electrode at pH 6.80. In the presence of 0.1 M KCl and 10 mM MgCl2 (ionic strength 0.13), Ca2+-binding to S-100 protein occurred in three steps with positive cooperativity. The numbers of bound Ca2+ ions in the three steps were 2, 2, and 4. The Ca2+-binding constants were 6.9 x 10(3) M-1, 2.9 x 10(3) M-1, and 3.7 x 10(2) M-1, respectively. The Ca2+-binding constants of the first and second steps obtained in the presence of 33.3 mM MgCl2 or 0.1 M KCl (ionic strength 0.10) were 1.4 times larger than those described above. This suggests that Mg2+ does not inhibit Ca2+-binding of S-100 protein. The increase of KCl concentration from 0.1 to 0.2 M caused a decrease of the Ca2+-binding constants to ca. 50%.     

Variable coupling of active NA+ + K+ transport in Ehrlich ascites tumor cells: Regulation by external NA+ and K+

The effects of altered external sodium and potassium concentrations on steady state, active Na⁺ + K⁺ transport in Ehrlich ascites tumor cells have been investigated. Membrane permeability to Na⁺ and K⁺, intracellular [Na⁺] and [K⁺], and membrane potential were measured. Active cation fluxes were calculated as equal and membrane potential were measured. Active cation fluxes were calculated as equal and opposite to the net, diffusional leak fluxes. Elevation of external K⁺ (6–60 Mm)by equivalent replacement of Na⁺ (154–91 mM) inhibits both active Na⁺ and K⁺ fluxes, but not proportionally. This results in a decrease of the coupling ratio (r_p = -J_k^p/J) as external K⁺ is increased. Elevation of external K⁺ (3–68 mM) at constant Na⁺ (92mM) inbibits J, but is without effect on J. The coupling ratio declines from 1.01 ± 0.14 to 0.07 ± 0.05, a 14-fold alteration. Reduction of external Na⁺ (154–25 mM) at constant K⁺ (6mM) depresses J, but is without effect on J. The coupling ratio increases from 0.63 ± 0.04 at 154 mM Na⁺ to 4.5 ± 2.04 at 25 mM Na⁺. The results of this investigation are consistent with the independent regulation of active cation fluxes by the transported species. Kinetic analysis of the data indicates that elevation of external sodium stimulates active sodium efflux by interacting at “modifier sites” at the outer cell surface. Similarly, external potassium inhibits active potassium influx by interaction at separate modifier sites.     

Role of Thy-1+ and Ia+ cells in ovarian function

Cryostat sections of anovulatory ovaries from persistent estrous rats following a single postnatal dose of testosterone and from persistent diestrous rats following long-term postnatal estradiol treatment were investigated. The indirect immunoperoxidase technique was used to localize ovarian Thy-1 and Ia glycoproteins, as well as several other cell surface markers, and the results were compared with those obtained in normal ovaries of cycling females. Folliculogenesis in persistent estrous rats proceeded up to cystic antral follicles and was associated with the occurrence of Thy-1+ stromal cells under follicular basal lamina. In contrast to normal ovaries, Thy-1+ material did not invade the basal layers of granulosa cells. There was also no association of Ia+ cells with follicular basal lamina, but Ia+ cells were usually found associated with some thecal vessels. In persistent diestrous rats folliculogenesis was significantly retarded in both advanced antrum formation and thecal development. Thy-1+ cells were usually present in theca. No Thy-1+ material was found among basal layers of granulosa cells and the depletion of thecal Ia+ cells was almost complete. We suggest that normal follicular development may be dependent on the correct effects of Thy-1+ and Ia+ cells in addition to appropriate gonadotropin and steroid stimulation. On the other hand, anovulatory syndromes following postnatal androgen or estrogen treatment might be induced by temporary direct ovarian effects disturbing the establishment of the normal relationship between follicular structures and the immune system.     

Na+ and K+ ion imbalances in Alzheimer's disease

Alzheimer's disease (AD) is associated with impaired glutamate clearance and depressed Na(+)/K(+) ATPase levels in AD brain that might lead to a cellular ion imbalance. To test this hypothesis, [Na(+)] and [K(+)] were analyzed in postmortem brain samples of 12 normal and 16 AD individuals, and in cerebrospinal fluid (CSF) from AD patients and matched controls. Statistically significant increases in [Na(+)] in frontal (25%) and parietal cortex (20%) and in cerebellar [K(+)] (15%) were observed in AD samples compared to controls. CSF from AD patients and matched controls exhibited no differences, suggesting that tissue ion imbalances reflected changes in the intracellular compartment. Differences in cation concentrations between normal and AD brain samples were modeled by a 2-fold increase in intracellular [Na(+)] and an 8-15% increase in intracellular [K(+)]. Since amyloid beta peptide (Aβ) is an important contributor to AD brain pathology, we assessed how Aβ affects ion homeostasis in primary murine astrocytes, the most abundant cells in brain tissue. We demonstrate that treatment of astrocytes with the Aβ 25-35 peptide increases intracellular levels of Na(+) (~2-3-fold) and K(+) (~1.5-fold), which were associated with reduced levels of Na(+)/K(+) ATPase and the Na(+)-dependent glutamate transporters, GLAST and GLT-1. Similar increases in astrocytic Na(+) and K(+) levels were also caused by Aβ 1-40, but not by Aβ 1-42 treatment. Our study suggests a previously unrecognized impairment in AD brain cell ion homeostasis that might be triggered by Aβ and could significantly affect electrophysiological activity of brain cells, contributing to the pathophysiology of AD.     

Kinetics of voltage- and Ca2+ activation and Ba2+ blockade of a large-conductance K+ channel fromNecturus enterocytes

Summary Potassium channels in membranes of isolatedNecturus enterocytes were studied using the patch-clamp technique. The most frequent channel observed had a conductance of 170 pS and reversal potential of 0 mV in symmetrical potassium-rich solutions. Channels were highly K⁺ selective. Channel activity was modulated by membrane potential and cytosolic Ca²⁺ concentration. Channel openings occurred in characteristic bursts separated by long closures. During bursts openings were interrupted by brief closures. Two gating modes controlled channel opening. The primary gate's sensitivity to intracellular Ca²⁺ concentration and membrane potential crucially determined long duration closures and bursting. In comparison, the second gate determining brief closures was largely insensitive to voltage and intracellular Ca²⁺ concentration. The channel was reversibly blocked by cytosolic barium exposure in a voltage-sensitive manner. Blockade reduced open-state probability without altering single-channel conductance and could be described, at relatively high Ca²⁺ concentration, by a three-state model where Ba²⁺ interacted with the open channel with a dissociation constant of about 10^–4 m at 0 mV.     

Cardiac sarcolemmal Na+-Ca2+ exchange and Na+-K+ ATPase activities and gene expression in alloxan-induced diabetes in rats

To determine the sequence of alterations in cardiac sarcolemmal (SL) Na⁺-Ca²⁺ exchange, Na⁺-K⁺ ATPase and Ca²⁺-transport activities during the development of diabetes, rats were made diabetic by an intravenous injection of 65 mg/kg alloxan. SL membranes were prepared from control and experimental hearts 1-12 weeks after induction of diabetes. A separate group of 4 week diabetic animals were injected with insulin (3 U/day) for an additional 4 weeks. Both Na⁺-K⁺ ATPase and Ca²⁺-stimulated ATPase activities were depressed as early as 10 days after alloxan administration; Mg²⁺ ATPase activity was not depressed throughout the experimental periods. Both Na⁺-Ca²⁺ exchange and ATP-dependent Ca²⁺-uptake activities were depressed in diabetic hearts 2 weeks after diabetes induction. These defects in SL Na⁺-K⁺ ATPase and Ca-transport activities were normalized upon treatment of diabetic animals with insulin. Northern blot analysis was employed to compare the relative mRNA abundances of _--subunit of Na⁺-K⁺ ATPase and Na⁺-Ca²⁺ exchanger in diabetic ventricular tissue vs. control samples. At 6 weeks after alloxan administration, a significant depression of the Na⁺-K⁺ ATPase _-- subunit mRNA was noted in diabetic heart. A significant increase in the Na⁺-Ca²⁺ exchanger mRNA abundance was observed at 3 weeks which returned to control by 5 weeks. The results from the alloxan-rat model of diabetes support the view that SL membrane abnormalities in Na⁺-K⁺ ATPase, Na⁺Ca²⁺ exchange and Ca²⁺-pump activities may lead to the occurrence of intracellular Ca²⁺ overload during the development of diabetic cardiomyopathy but these defects may not be the consequence of depressed expression of genes specific for those SL proteins.     

Dynamic buffering of mitochondrial Ca2+ during Ca2+ uptake and Na+-induced Ca2+ release

In cardiac mitochondria, matrix free Ca²⁺ ([Ca²⁺]_m) is primarily regulated by Ca²⁺ uptake and release via the Ca²⁺ uniporter (CU) and Na⁺/Ca²⁺ exchanger (NCE) as well as by Ca²⁺ buffering. Although experimental and computational studies on the CU and NCE dynamics exist, it is not well understood how matrix Ca²⁺ buffering affects these dynamics under various Ca²⁺ uptake and release conditions, and whether this influences the stoichiometry of the NCE. To elucidate the role of matrix Ca²⁺ buffering on the uptake and release of Ca²⁺, we monitored Ca²⁺ dynamics in isolated mitochondria by measuring both the extra-matrix free [Ca²⁺] ([Ca²⁺]_e) and [Ca²⁺]_m. A detailed protocol was developed and freshly isolated mitochondria from guinea pig hearts were exposed to five different [CaCl₂] followed by ruthenium red and six different [NaCl]. By using the fluorescent probe indo-1, [Ca²⁺]_e and [Ca²⁺]_m were spectrofluorometrically quantified, and the stoichiometry of the NCE was determined. In addition, we measured NADH, membrane potential, matrix volume and matrix pH to monitor Ca²⁺-induced changes in mitochondrial bioenergetics. Our [Ca²⁺]_e and [Ca²⁺]_m measurements demonstrate that Ca²⁺ uptake and release do not show reciprocal Ca²⁺ dynamics in the extra-matrix and matrix compartments. This salient finding is likely caused by a dynamic Ca²⁺ buffering system in the matrix compartment. The Na⁺- induced Ca²⁺ release demonstrates an electrogenic exchange via the NCE by excluding an electroneutral exchange. Mitochondrial bioenergetics were only transiently affected by Ca²⁺ uptake in the presence of large amounts of CaCl₂, but not by Na⁺- induced Ca²⁺ release.