Matrix free Mg(2+) and the regulation of mitochondrial volume.

Mitochondria must maintain volume homeostasis in order to carry out oxidative phosphorylation. It has been postulated that the concentration of free Mg(2+) ([Mg(2+)]) serves as the sensor of matrix volume and regulates a K(+)-extruding K(+)/H(+) antiport (K. D. Garlid. J. Biol. Chem. 255: 11273-11279, 1980). To test this hypothesis, the fluorescent probe furaptra was used to monitor [Mg(2+)] and free Ca(2+) concentration ([Ca(2+)]) in the matrix of isolated beef heart mitochondria, and K(+)/H(+) antiport activity was measured by passive swelling in potassium acetate. Concentrations that result in 50% inhibition of maximum activity of 92 microM matrix [Mg(2+)] and 2.2 microM [Ca(2+)] were determined for the K(+)/H(+) antiport. Untreated mitochondria average 670 microM matrix [Mg(2+)], a value that would permit <1% of maximum K(+)/H(+) antiport activity. Hypotonic swelling results in large decreases in matrix [Mg(2+)], but swelling due to accumulation of acetate salts does not alter [Mg(2+)]. Swelling in phosphate salts decreases matrix [Mg(2+)], but not to levels that permit appreciable antiport activity. We conclude that 1) it is unlikely that matrix [Mg(2+)] serves as the mitochondrial volume sensor, 2) if K(+)/H(+) antiport functions as a volume control transporter, it is probably regulated by factors other than [Mg(2+)], and 3) alternative mechanisms for mitochondrial volume control should be considered.

On the relationship between matrix free Mg2+ concentration and total Mg2+ in heart mitochondria.

The matrix free magnesium ion concentration, [Mg2+]m, estimated using the fluorescent probe furaptra, averaged 0.67 mM in 15 preparations of beef heart mitochondria containing an average of 21 nmol total Mg2+ per mg protein. [Mg2+]m was compared with total Mg2+ during respiration-dependent uptake and efflux of Mg2+ and during osmotic swelling. In the absence of external Pi these mitochondria contain about 32 nmol/mg non-diffusible Mg-binding sites with an apparent Kd of 0.34 mM. [Mg2+]m depends on both the size of the total Mg2+ pool and the ability of matrix anions to provide Mg-ligands. Pi interacts strongly with Mg2+ to decrease [Mg2+]m and, in the absence of external Mg2+, promotes respiration-dependent Mg2+ efflux and a decrease in [Mg2+]m to very low levels. The uptake of Pi by respiring mitochondria converts delta pH to membrane potential (delta psi) and provides additional Mg-binding sites. This permits large accumulations of Mg2+ and Pi with little change in [Mg2+]m. Nigericin also converts delta pH to delta psi in respiring mitochondria and induces a large and rapid increase in both total Mg2+ and [Mg2+]m. Mersalyl increases the permeability of the mitochondrial membrane to cations and this also induces a marked increase in both total Mg2+ and [Mg2+]m. These results suggest that mitochondria take up Mg2+ by electrophoretic flux through membrane leak pathways, rather than via a specific Mg2+ transporter. Mitochondria swollen by respiration dependent uptake of potassium phosphate show decreased [Mg2+]m, whereas those swollen to the same extent in potassium acetate do not. This suggests that [Mg2+]m is well-buffered during osmotic volume changes unless there is also a change in ligand availability.

On the use of fluorescent probes to estimate free Mg2+ in the matrix of heart mitochondria.

The fluorescent probe furaptra shows increases and decreases in the concentration of free magnesium ion, [Mg2+], in the mitochondrial matrix with changes in total Mg2+ and ligand availability. The factors involved in the calibration of these fluorescence changes in terms of absolute [Mg2+] have been investigated. The affinity of furaptra for Mg2+ is highly dependent on both temperature and ionic strength. The Kd for Mg-furaptra in solution in 100 mM KCl was found to be 2.1 +/- 0.1 mM at 25 degrees C. The use of this Kd to calculate matrix [Mg2+] is more reliable than in situ Kd measurements because ionophores, such as BrA23187 and ionomycin, do not equilibrate external Mg2+ with the matrix in an acceptable way. Furaptra is present at high concentrations (up to 500 microM) in the matrix when introduced by hydrolysis of the acetoxymethyl ester. However, absorbance spectra of aqueous solutions show no evidence of dimerization of the probe or other changes in properties at these concentrations. Fluorescence intensity at 340 nmex is strongly attenuated for matrix-sequestered furaptra, mag-fura-5, and mag-indo-1. This appears to result in part from preferential binding of the Mg-probe to mitochondrial proteins. The fluorescence of uncomplexed furaptra at 375-380 nmex seems unaffected by protein binding, however, and changes in intensity in this region of the spectrum can be used in conjunction with the Kd found in aqueous solution to estimate matrix [Mg2+]. The presence of secondary equilibria, such as protein binding, and possible changes in ionic strength may undermine exact quantitation by this method. However, values for matrix [Mg2+] obtained in this way (0.5 to 0.7 mM) correspond well to estimates by other available methods and each of these methods suffers from comparable uncertainties.

Cultured AIDS-related Kaposi's sarcoma (AIDS-KS) cells demonstrate impaired bioenergetic adaptation to oxidant challenge: implication for oxidant stress in AIDS-KS pathogenesis.

Despite its recognition as the most prevalent HIV associated cancer, speculation still abounds regarding the pathogenesis of AIDS-related Kaposi's sarcoma (AIDS-KS). However, it has been established that both cytokines, e.g. IL-6, and HIV-associated products, e.g., Tat, are integral in AIDS-KS cellular proliferation. Further, both experimental and clinical evidence is accumulating to link reactive oxygen intermediates (ROI) with both cytokine induction (primarily via nuclear factor-kappa B[NF-kappa B] dependent routes) as well as the subsequent cytokine, tumor necrosis factor alpha (TNF alpha) stimulation of HIV replication. Features of AIDS-KS patients, such as retention of phagocytes, presence of sustained immunostimulation, and a frequent history of KS lesions arising at traumatized sites, make oxidant stress a viable clinical factor in AIDS-KS development. Time course nucleotide profile analyses show that AIDS-KS cells have an inherent, statistically significant, biochemical deficit, even prior to oxidant stress, due to 1) a more glycolytic bioenergetic profile, resulting in lower levels of high energy phosphates (impairing capacity for glutathione [GSH] synthesis and DNA repair); 2) lower levels of NADPH (compromising the activities of GSSG reductase and peroxidase function of catalase); and 3) reduced levels of GSH (impeding both GSH peroxidase and GSH-S-transferases). Following exposure to physiologically relevant levels of H2O2, only the human microvascular endothelial cells (a putative AIDS-KS progenitor cell) responded with bioenergetic adaptations that reflected co-ordination of energy generating and cytoprotective pathways, e.g., retention of the cellular energy charge, increased NAD+, and an accentuation of the ATP, NADPH, and total adenine nucleotide differences relative to AIDS-KS cells. Also, some of the AIDS-KS strains retained intracellular GSSG subsequent to oxidant challenge, inviting the formation of deleterious protein mixed disulfides. While the results of our study address some AIDS-KS issues, they also raise an etiological question, i.e., Does the inability to tolerate oxidant stress arise in conjunction with AIDS-KS neoplastic development, or is it pre-existing in the population at risk? Regardless, use of antioxidant therapy (low risk/ potentially high benefit) in both the "at risk" population as well as in those individuals with active disease may prove a useful preventative and/or treatment modality.

Correlation between AIDS-related Kaposi sarcoma histological grade and in vitro behavior: reduced exogenous growth factor requirements for isolates from high grade lesions.

Kaposi sarcoma, the most common AIDS-associated malignancy, affects 10-30% of all AIDS patients. To date, research into the biological characteristics of AIDS-related Kaposi sarcoma (AIDS-KS) derived cell lines has been based on cultures established from skin explants or pleural effusions/peritoneal fluids. We have established several AIDS-KS lines from biopsy confirmed oral mucosal and epidermal AIDS-KS lesions and have found a correlation between AIDS-KS lesional grade and in vitro cellular growth characteristics. In comparison to epidermal AIDS-KS lesions, mucosal AIDS-KS lesions frequently possessed both a more advanced histologic grade and demonstrated a greater capacity to proliferate in minimal medium. We report the ability of AIDS-KS isolates from high grade lesions to sustain proliferation (greater than 60 population doubling levels) in medium not supplemented with endothelial cell growth supplement and/or cytokine rich conditioned medium. These findings indicate that AIDS-KS cells isolated from high grade lesions have reduced requirements for exogenously provided growth supplements, and suggest that increased autologous cytokine production accompanies AIDS-KS lesional progression.

Comparison of constitutive cytokine release in high and low histologic grade AIDS-related Kaposi's sarcoma cell strains and in sera from HIV+/KS+ and HIV+/KS- patients.

Kaposi's sarcoma (KS) is both an AIDS-defining disease and the most common HIV-associated malignancy. A cytokine-mediated pathogenesis for AIDS-KS is implicated because AIDS-KS-derived cell strains both respond to and express a variety of cytokines. We have reported the establishment of several (n = 18) AIDS-KS cell strains and determined that reduced exogenous growth factors are necessary to sustain proliferation in isolates from high histologic grade KS lesions. This current investigation explored the possibility that there are histologic grade-associated differences in either the qualitative and/or quantitative constitutive release of AIDS-KS growth stimulatory cytokines. Our findings showed that the incorporation of HTLV-II cytokine-rich conditioned media induced both qualitative and significant quantitative cytokine release, suggesting that exogenous growth promoters stimulate constitutive cytokine release. ELISA of our AIDS-KS cell strains demonstrated constitutive release of IL-6 (seven of seven), FGF-2 (five of seven), GM-CSF (three of seven), and IL-1 beta (one of seven). None of our AIDS-KS cell strains constitutively released detectable levels of Onco-M, IL-4, PDGF, TNF-alpha, or TNF-beta. In addition, we report that the method of cytokine result quantitation significantly affects reported cytokine levels. We determined that there was no significant histologic grade-dependent difference in the constitutive release of soluble cytokines by in vitro grown cultures of AIDS-KS cells. The presence of HIV influenced the sera cytokine profiles by elevating IL-6 and decreasing PDGF concentrations of HIV+ individuals relative to HIV- healthy controls. However, the presence of KS was not associated with unique serum cytokine profiles, because no differences were noted in comparisons of HIV+/KS+ versus HIV+/KS- individuals. Our findings suggest that the local environment is key in modulating AIDS-KS cytokine expression and that KS growth-promoting factors function at the local or paracrine, not the systemic, level. In conclusion, our previous results demonstrated a histologic grade-associated difference in the in vitro growth capacity of AIDS-KS cells; with high histologic grade isolates displaying a marked growth advantage during culture in minimally supplemented media. Findings from this current study reveal that although the potential for a constitutive growth loop exists in the high-grade isolates, it is not reflected in the free levels of soluble cytokines secreted into the culture medium.

The sodium-calcium antiport of heart mitochondria is not electroneutral.

Heart mitochondria contain a nNa+/Ca2+ antiport that participates in the regulation of matrix [Ca2+]. Based largely on a single study (Brand, M. D. (1985) Biochem. J. 229, 161-166), there has been a consensus that this antiport promotes the electroneutral exchange of two Na+ for one Ca2+. However, a recent study in our laboratory (Baysal, K., Jung, D. W., Gunter, K. K., Gunter, T. P., and Brierley, G. P. (1994) Am. J. Physiol. 266, C800-C808) has shown that the Na(+)-dependent efflux of Ca2+ from heart mitochondria has more energy available to it than can be supplied by a passive 2Na+/Ca2+ exchange. We have therefore re-examined Brand's protocols using fluorescent probes to monitor matrix pH and free [Ca2+]. Respiring heart mitochondria, suspended in KCl and treated with ruthenium red to block Ca2+ influx, extrude Ca2+ and establish a large [Ca2+]out:[Ca2+]matrix gradient. The extrusion of Ca2+ under these conditions is Na(+)-dependent and diltiazem-sensitive and can be attributed to the nNa+/Ca2+ antiport. Addition of nigericin increases the membrane potential (delta psi) and decreases delta pH to 0.1 or less, but has virtually no effect on the magnitude of the [Ca2+] gradient. Under these conditions a gradient maintained by electroneutral 2Na+/Ca2+ antiport should be abolished because the mitochondrial Na+/H+ antiport keeps the [Na+] gradient equivalent to the [H+] gradient. The [Ca2+] gradient is abolished, however, when an uncoupler is added to dissipate delta psi or when the exogenous electroneutral antiport BrA23187 is added. In addition, [Ca2+] influx via the nNa+/Ca2+ antiport in nonrespiring mitochondria is enhanced when delta psi is abolished. These results are consistent with Ca2+ extrusion by an electrophoretic antiport that can respond to delta psi but not with an electroneutral antiport.

Human microvascular endothelial cell-extracellular matrix interaction in cellular growth state determination.

We introduce two methods, both of which are based on cellular-extracellular matrix interaction, which will facilitate the study of human microvascular endothelial cells. One method describes the means to obtain a G1 population baseline in human microvascular endothelial cells. Because of the contribution of the extracellular matrix in endothelial cell growth, synchronization in G1 was possible only after the incorporation of angiostatic levels of heparin and hydrocortisone into the extracellular matrix. In the second method, we demonstrate that selective perturbation of human microvascular endothelial cell-extracellular matrix interactions results in the induction of a transitional growth state, between proliferative and differentiated growth states, in human microvascular endothelial cells. In the functional, microtubule formation assays, transitional growth state endothelial cells display rates that are indermediate between those obtained from differentiated and proliferative endothelial cells. Our results demonstrate the importance of the human microvascular endothelial cell-extracellular matrix interaction in the determination of cellular growth state. Our findings also imply that responsiveness of microvascular endothelial cells to their cellular-extracellular matrix environs is highest during the differentiated growth state.

Cultured AIDS-related Kaposi's sarcoma cells retain a proliferative bioenergetic profile but demonstrate reduced cytoprotective capabilities.

Features of AIDS-related Kaposi's sarcoma (AIDS-KS), such as the multifocal presentation at mucosal and epidermal sites subjected to trauma, suggest that AIDS-KS is initially a reactive hyperplasia that subsequently progresses to a neoplasia. It is recognized that there is an association between sustained inflammatory states and the subsequent development of neoplasia (e.g., ulcerative colitis/colonic adenocarcinoma). Furthermore, patients who develop AIDS-KS experience both a constant immune stimulation due to sustained high levels of virus-induced cytokines and, because of a sparing effect on their phagocytic cells, retention of the phagocytic inflammatory response. A component of phagocytic activation is the initiation of the oxidative burst, resulting in the generation of reactive oxygen species (ROS), which can be mutagenic to host cells if released beyond the phagolysosome and not inactivated. Our results demonstrate that cultured AIDS-KS cells possess decreased cytoprotective capabilities. Relative to either dermal fibroblasts, or human microvascular endothelial cells (HMECs), AIDS-KS cells contained significantly lower levels of glutathione, a tripeptide integral in both cytoprotection and maintenance of cellular thiol status. While HMECs increased catalase activity during culture in the cytokine-rich KS milieu (control medium supplemented with conditioned medium from MOT, an HTLV II-infected cell line), AIDS-KS cells demonstrated reduced catalase function under these conditions. Furthermore, HMEC cultures showed an inherent biochemical responsiveness, by increasing catalase activity following exposure to exogenous H2O2. In contrast, the catalase activity of AIDS-KS cells decreased following H2O2 challenge. Our results show that an inherent deficiency in cellular cytoprotection is present in AIDS-KS cells and suggest that oxidant stress may function in the development and progression of AIDS-KS.

Cation transport systems in mitochondria: Na+ and K+ uniports and exchangers.

It is now well established that mitochondria contain three antiporters that transport monovalent cations. A latent, allosterically regulated K+/H+ antiport appears to serve as a cation-extruding device that helps maintain mitochondrial volume homeostasis. An apparently unregulated Na+/H+ antiport keeps matrix [Na+] low and the Na(+)-gradient equal to the H(+)-gradient. A Na+/Ca2+ antiport provides a Ca(2+)-extruding mechanism that permits the mitochondrion to regulate matrix [Ca2+] by balancing Ca2+ efflux against influx on the Ca(2+)-uniport. All three antiports have well-defined physiological roles and their molecular properties and regulatory features are now being determined. Mitochondria also contain monovalent cation uniports, such as the recently described ATP- and glibenclamide-sensitive K+ channel and ruthenium red-sensitive uniports for Na+ and K+. A physiological role of such uniports has not been established and their properties are just beginning to be defined.

Magnesium transport by mitochondria.

The pathways for the uptake and extrusion of Mg2+ by mitochondria are now well defined, the present evidence suggests that uptake occurs by nonspecific diffusive pathways in response to elevated membrane potential. There is disagreement as to some of the properties of Mg2+ efflux from mitochondria, but the reaction resembles K+ efflux in many ways and may occur in exchange for H+. Matrix free magnesium ion concentration, [Mg2+], can be measured using fluorescent probes and is set very close to cytosol [Mg2+] by a balance between influx and efflux and by the availability of ligands, such as Pi. There are indications that matrix [Mg2+] may be under hormonal control and that it contributes to the regulation of mitochondrial metabolism and transport reactions.

Magnesium ion modulates the sensitivity of the mitochondrial permeability transition pore to cyclosporin A and ADP.

Regulation of the mitochondrial permeability transition pore has been investigated following the release of matrix solutes which normally participate in pore regulation. Under these conditions, neither cyclosporin A nor ADP induces pore closure, as judged by restoration of delta psi, unless Mg2+ is also added. Mg2+ alone is ineffective. In liver mitochondria, the Mg2+ effect is expressed over a 0 to 0.5 mM concentration range with higher concentrations inhibiting repolarization. In heart mitochondria, the inhibitory action of high Mg2+ is not seen and it can be shown that the Mg2+ effect on repolarization increases progressively up to a concentration of 5 mM. In liver mitochondria, when the pore is closed by maximally effective concentrations of Mg2+ plus cyclosporin A or Mg2+ plus ADP, reopening occurs upon the addition of carboxyatractyloside. The latter compound, however, fails to reopen the pore when Mg2+, cyclosporin A, and ADP are present simultaneously. In heart mitochondria, where higher Mg2+ concentrations can be employed, Mg2+ plus cyclosporin A or Mg2+ plus ADP produces pore closure in a carboxyatractyloside insensitive manner. Titration experiments support the adenine nucleotide translocase as the site at which carboxyatractyloside acts to regulate the pore. However, the action of ADP appears to involve a translocase-independent site. In intact mitochondria the action of carboxyatractyloside on pore opening is counteracted by oligomycin, apparently through inhibition of the F1F0 ATP synthase, with a consequent increase in the matrix space ADP/ATP ratio. It is concluded that the permeability transition pore induced by Ca2+ plus P(i) is not formed from the adenine nucleotide translocase although the translocase conformation is one of several factors which regulate the pore. The matrix Mg2+ concentration is also one of these factors. Formation of the pore by a Ca2+ and ADP binding protein is one model which is consistent with the present data.

Environmental induction of tumor phenotype in a putative Kaposi sarcoma progenitor cell.

Many features of AIDS-related Kaposi sarcoma (AIDS-KS), e.g., multifocal lesional presentation at sites perfused by the microvasculature, suggest that AIDS-KS is initially a hyperplasia that subsequently progresses to a neoplasia. We propose that the unique AIDS environment, which contains high levels of circulating factors such as viral cytokines, is key in initiating the KS lesion. Further, we maintain that due to their physiological function, human microvascular endothelial cells (HMECs) are both likely target cells for the AIDS-related cytokines, and are putative AIDS-KS progenitor cells. Previously, we have shown that as a component of HMEC transition between proliferative and differentiated growth, HMECs modulate their nucleotide and glutathione levels. After attaining contact inhibition, HMECs enter a state of differentiation, which is characterized by cellular entrance into a G0, quiescent growth state, a decrease in cellular bioenergetic profiles, and spontaneous formation of microtubules. In contrast, when cultured in a "KS milieu", HMECs fail to differentiate. Instead, the "KS milieu" cultured cells assume a "growth relaxed" phenotype and demonstrate a lack of contact inhibition, loss of anchorage dependence, and retention of a "proliferative" bioenergetic profile despite culture confluence. Our results imply both that HMECs are responsive to AIDS-related cytokines, and that the local environment is key to instigating a relaxation of cellular growth controls.

Evidence against norepinephrine-stimulated efflux of mitochondrial Mg2+ from intact cardiac myocytes.

We investigated the hypotheses that norepinephrine stimulates Mg2+ efflux from intact isolated adult rat ventricular cardiomyocytes and that adenosine 3',5'-cyclic monophosphate stimulates Mg2+ efflux from permeabilized myocytes and isolated mitochondria. Norepinephrine stimulation of Mg2+ release from cardiac myocytes was observed only when cells at approximately 20 mg protein/ml in Mg(2+)-containing buffer were diluted 50- to 60-fold into an Mg(2+)-free medium. Under these conditions, > 30% of total cellular lactic acid dehydrogenase activity was also released, indicating that a significant portion of the cells had died. In other protocols, where Mg2+ efflux from myocytes was not observed, extracellular Mg2+ removal and administration of 10 microM norepinephrine increased 45Ca2+ accumulation by cells in suspension. In single myocytes, Mg2+ removal and norepinephrine administration increased intracellular free [Ca2+] as measured by fura-2 fluorescence microscopy, and this was accompanied by vigorous spontaneous contractile activity followed by Ca2+ overload hypercontracture. With permeabilized myocytes and isolated mitochondria from a variety of sources, adenosine 3',5'-cyclic monophosphate did not stimulate Mg2+ efflux. These results suggest that recent evidence for direct hormonal regulation of myocardial Mg2+ homeostasis may need to be reevaluated.

Na(+)-dependent Ca2+ efflux mechanism of heart mitochondria is not a passive Ca2+/2Na+ exchanger.

Net Ca2+ flux across the inner membrane of respiring heart mitochondria was evaluated under conditions in which virtually all Ca2+ movement can be attributed to the Na+/Ca2+ antiport. If this antiport promotes a passive electroneutral exchange of Ca2+ for 2Na+, the Ca2+ gradient should be equal to the square of the Na+ gradient at equilibrium. Because the mitochondrial Na+/H+ antiport equilibrates the Na+ and H+ gradients, the Ca2+ gradient should also equal the square of the H+ gradient. In a series of > 20 determinations at different matrix [Ca2+], different delta pH, and varying membrane potential, it was found that Ca2+ is transported out of the mitochondrion against gradients from 15- to 100-fold greater than the value predicted for passive electroneutral exchange. It is concluded that the observed gradients are too large to be sustained by passive Ca2+/2Na+ exchange. The observed gradients are compatible with an electrogenic Ca2+/3Na+ exchange. Alternatively another source of energy is available to support these gradients.

Modulation of human microvascular endothelial cell bioenergetic status and glutathione levels during proliferative and differentiated growth.

During angiogenesis, formerly differentiated human microvascular endothelial cells (HMECs) return to a proliferative growth state. Many fundamental questions regarding HMEC function, such as how HMECs adapt to changes in bioenergetic requirements upon return to proliferative growth, remained unanswered. In this study, we evaluated whether modifications in HMEC bioenergetic profiles and glutathione (GSH) levels accompanied the cellular transition between differentiated and proliferative growth. To provide insight into the continuum of cellular adaptations that occur during this transition, we used a method recently developed in our laboratory that induces a state of morphological and functional predifferentiation in HMECs. Cellular morphology, in conjunction with flow cytometric DNA analyses and HMEC functional assays (the directed migration and intercellular association involved in microtubule formation) were employed to validate the HMEC culture state of growth. Analysis of the HPLC nucleotide profiles disclosed several findings common to all culture growth states. These uniform findings, e.g., cellular energy charges > 0.90, and highly reduced redox states, revealed that cultured HMECs maintain high rates of oxidative metabolism. However, there were also significant, culture growth state related differences in the nucleotide profiles. Proliferative HMECs were shown to possess significantly higher (relative to both large vessel endothelial cells, and differentiated HMECs) levels of GSH and specific nucleotides which were related with a return to the active cell cycle-ATP, GTP, UTP, and CTP, and NADPH. Further, the nucleotide profiles and GSH levels of the predifferentiated HMECs were determined to be intermediate between levels obtained for the proliferative and differentiated HMECs. The results of this study demonstrate that the capacity to modulate their cellular bioenergetic status during growth state transitions is one of the adaptations that enable HMECs to retain a growth state reciprocity. In addition, our findings also show that HMECs, especially during the proliferative growth state, are biochemically distinct from endothelial cells harvested from large vessels, and therefore suggest that HMECs are the cells of choice to employ when studying diseases that affect the human microvasculature.

The permeability transition in heart mitochondria is regulated synergistically by ADP and cyclosporin A.

Heart mitochondria respiring in a sucrose medium containing P(i) show a permeability transition when challenged with Ca2+ and an oxidant such as cumene hydroperoxide. The transition results from the opening of a Ca(2+)-dependent pore and is evidenced by loss of membrane potential (delta psi) and osmotic swelling due to uptake of sucrose and other solutes. In the absence of oxidant, high concentrations of Ca2+ (100-150 microM) are necessary to induce loss of delta psi and initiate swelling. Cyclosporin A delays the loss of delta psi but enhances swelling under these conditions, apparently by promoting better retention of accumulated Ca2+. Cyclosporin A and ADP together restore delta psi in respiring mitochondria that have undergone the permeability transition at levels that are not effective when either is added alone. When the state of the Ca(2+)-dependent pore is assessed using passive osmotic contraction in response to polyethylene glycol (Haworth, R. A., and Hunter, D. R. (1979) Arch. Biochem. Biophys. 195, 460-467), cyclosporin A is found to be a partial inhibitor of solute flow through the open pore. Cyclosporin A decreases the Vmax of passive contraction and increases the Km for Ca2+ without affecting the Hill slope. ADP in the presence of carboxyatractyloside closes the pore almost completely even in the presence of 40 microM Ca2+. ADP shows mixed type inhibition of the Ca(2+)-dependent pore, and cyclosporin A increases the affinity of the pore for ADP. It is concluded that cyclosporin A and ADP act synergistically to close the Ca(2+)-dependent pore of the mitochondrion and that the pore is probably not formed directly from the adenine nucleotide transporter.

Cyclosporin inhibits mitochondrial calcium efflux in isolated adult rat ventricular cardiomyocytes.

Exchangeable intracellular Ca2+ as measured by 45Ca2+ uptake more than doubled when isolated adult rat ventricular cardiomyocytes were incubated 30 min with 8 microM cyclosporin; nevertheless the cells retained a normal rod-shaped morphology. High concentrations of ouabain caused a similar increase in 45Ca2+ uptake, but in this case the Ca2+ overload caused nearly all cells to hypercontract into a round disorganized form. The response to cyclosporin was concentration dependent with an apparent half-maximal effective concentration of 0.5 microM for enhancement of net 45Ca2+ accumulation. Verapamil (1 microM) could not inhibit this cyclosporin effect, but it was abolished by a 5-min preincubation with 12 microM crude ruthenium red. Cyclosporin also decreased the rate of 45Ca2+ efflux from prelabeled myocytes into Ca(2+)-containing and Ca(2+)-free media. These data are consistent with inhibition of mitochondrial 45Ca2+ efflux through the cyclosporin-sensitive mitochondrial inner membrane pore. It would appear that periodic transient increases in mitochondrial inner membrane permeability provide a pathway for mitochondrial Ca2+ extrusion under relatively normal conditions in isolated adult rat heart cells.

Transmembrane gradients of free Na+ in isolated heart mitochondria estimated using a fluorescent probe.

The concentration of free Na+ in the matrix of isolated pig heart mitochondria has been monitored using the fluorescent probe sodium-binding benzofuran isophthalate (SBFI) developed by Minta and Tsien (J. Biol. Chem. 264: 19449-19457, 1989). SBFI was sequestered in the matrix by hydrolysis of the permeant acetoxymethyl ester. The sequestered probe showed altered quantum efficiency and excitation spectra in the presence and absence of Na+ when compared with SBFI free acid in solution. Fluorescence was calibrated in situ by using ionophores to equilibrate matrix [Na+] with external [Na+]. SBFI fluorescence showed that matrix [Na+] increased linearly as external [Na+] was increased to 95 mM in the presence or absence of respiration. Respiring mitochondria maintained a Na+ gradient (Na+ out greater than Na+in) of approximately 8.0. The corresponding gradient in nonrespiring mitochondria was approximately 2.0. The Na+ gradient was nearly equivalent to the H+ gradient in the presence or absence of respiration. The uptake of Pi by respiring mitochondria decreased matrix pH and increased matrix [Na+]. It is concluded that isolated mitochondria maintain a Na+ gradient across the inner membrane as a result of the activity of the endogenous Na(+)-H+ antiport.