Characterization of the submandibular gland microsomal calcium transport system.

Calcium accumulation by submandibular gland microsomes (first described by Selinger and Naim, ((1970) Biochim. Biophys. Acta 323, 337-341) has been further characterized. Accumulation was concentration dependent, had a Km of 25 microM added calcium and a Vmax of 12 nM calcium/mg protein per min. No accumulation was observed in the presence of either the calcium ionophore A23187, or the detergent Triton X-100 (0.05). The divalent cations Sr2+ and Mn2+ inhibited accumulation competitively with Ki values of 67 microM and 200 microM, respectively. The effect of various enzyme inhibitors were tested on the microsomal calcium transport system and it was found that chlorpromazine, trifluoperazine, and DIDS all inhibited. The mitochondrial transport inhibitors ruthenium red and CCCP had no effect on transport. Experiments directed at clarifying the cellular location of the system are described. It was found that the membrane vesicles responsible for transport show different purification properties than the membrane vesicles which contain the standard enzyme markers for total and rough endoplasmic reticulum, Golgi apparatus, plasma membrane, and lysosomes. These conclusions are based upon experiments using these properties for membrane purification, density, size, and electrophoretic mobility. Three possible explanations of the results are given and it is organelles. The significance of the results in: (1) understanding the biochemical properties of the submandibular gland microsomal calcium transport system, (2) clarifying the cellular location of the system, and (3) clarifying the function of the system in salivary secretion are discussed.

The rapid mode of calcium uptake into heart mitochondria (RaM): comparison to RaM in liver mitochondria.

A mechanism of Ca(2+) uptake, capable of sequestering significant amounts of Ca(2+) from cytosolic Ca(2+) pulses, has previously been identified in liver mitochondria. This mechanism, the Rapid Mode of Ca(2+) uptake (RaM), was shown to sequester Ca(2+) very rapidly at the beginning of each pulse in a sequence [Sparagna et al. (1995) J. Biol. Chem. 270, 27510-27515]. The existence and properties of RaM in heart mitochondria, however, are unknown and are the basis for this study. We show that RaM functions in heart mitochondria with some of the characteristics of RaM in liver, but its activation and inhibition are quite different. It is feasible that these differences represent different physiological adaptations in these two tissues. In both tissues, RaM is highly conductive at the beginning of a Ca(2+) pulse, but is inhibited by the rising [Ca(2+)] of the pulse itself. In heart mitochondria, the time required at low [Ca(2+)] to reestablish high Ca(2+) conductivity via RaM i.e. the 'resetting time' of RaM is much longer than in liver. RaM in liver mitochondria is strongly activated by spermine, activated by ATP or GTP and unaffected by ADP and AMP. In heart, RaM is activated much less strongly by spermine and unaffected by ATP or GTP. RaM in heart is strongly inhibited by AMP and has a biphasic response to ADP; it is activated at low concentrations and inhibited at high concentrations. Finally, an hypothesis consistent with the data and characteristics of liver and heart is presented to explain how RaM may function to control the rate of oxidative phosphorylation in each tissue. Under this hypothesis, RaM functions to create a brief, high free Ca(2+) concentration inside mitochondria which may activate intramitochondrial metabolic reactions with relatively small amounts of Ca(2+) uptake. This hypothesis is consistent with the view that intramitochondrial [Ca(2+)] may be used to control the rate of ADP phosphorylation in such a way as to minimize the probability of activating the Ca(2+)-induced mitochondrial membrane permeability transition (MPT).

The Ca2+ transport mechanisms of mitochondria and Ca2+ uptake from physiological-type Ca2+ transients.

Mitochondria contain a sophisticated system for transporting Ca2+. The existence of a uniporter and of both Na+-dependent and -independent efflux mechanisms has been known for years. Recently, a new mechanism, called the RaM, which seems adapted for sequestering Ca2+ from physiological transients or pulses has been discovered. The RaM shows a conductivity at the beginning of a Ca2+ pulse that is much higher than the conductivity of the uniporter. This conductivity decreases very rapidly following the increase in [Ca2+] outside the mitochondria. This decrease in the Ca2+ conductivity of the RaM is associated with binding of Ca2+ to an external regulatory site. When liver mitochondria are exposed to a sequence of pulses, uptake of labeled Ca2+ via the RaM appears additive between pulses. Ruthenium red inhibits the RaM in liver mitochondria but much larger amounts are required than for inhibition of the mitochondrial Ca2+ uniporter. Spermine, ATP and GTP increase Ca2+ uptake via the RaM. Maximum uptake via the RaM from a single Ca2+ pulse in the physiological range has been observed to be approximately 7 nmole/mg protein, suggesting that Ca2+ uptake via the RaM and uniporter from physiological pulses may be sufficient to activate the Ca2+-sensitive metabolic reactions in the mitochondrial matrix which increase the rate of ATP production. RaM-mediated Ca2+ uptake has also been observed in heart mitochondria. Evidence for Ca2+ uptake into the mitochondria in a variety of tissues described in the literature is reviewed for evidence of participation of the RaM in this uptake. Possible ways in which the differences in transport via the RaM and the uniporter may be used to differentiate between metabolic and apoptotic signaling are discussed.

An electron paramagnetic resonance study of Mn2+ uptake by the chick chorioallantoic membrane.

Mn2+ uptake in the chick chorioallantoic membrane, an embryonic epithelial tissue which transports Ca2+ in vivo was studied using electron paramagnetic resonance (EPR). Mn2+ was used as a paramagnetic analog for Ca2+, since there is evidence that Mn2+ is accumulated by the Ca2+ transport mechanism. After 1.5 h of uptake the EPR spectrum of the Mn2+ in the membrane indicated that 89% of the Mn2+ was in a spin-exchange form, indicating close packing of Mn2+. The Mn2+ spacing was estimated from the line width to be about 4.7 A. The remaining Mn2+ was very likely Mn2+ hexahydrate. At pH 7.4 the spin-exchange spectrum tended to broaden when uptake was inhibited, while at pH 5.0 the spin-exchange spectrum was completely abolished in the presence of inhibitors. The EPR spectrum of Mn2+ in the chorioallantoic membrane had a broader line width than that of Mn2+ in isolated mitochondria, suggesting that in this tissue mitochondria are not directly involved in divalent cation transport. These EPR studies support the concept that divalent cations are sequestered in high concentrations from the rest of the cell contents during transcellular active transport.

Energy-dependent Mn2+ and Ca2+ uptake by the embryonic chick chorioallantoic membrane.

The chick chorioallantoic membrane is an epithelial tissue which actively transports large amounts of Ca2+ during embryonic development. In this paper Mn2+ uptake by the tissue was studied and compared to Ca2+ uptake in parallel experiments. The purpose of these experiments was to determine if Mn2+ could be used to gain more information about the Ca2+ transport system. It was found that Mn2+ uptake was reduced significantly under conditions that reduced Ca2+ uptake and that Mn2+, like Ca2+, was taken up preferentially by the ectodermal side of the tissue. Mn2+ uptake showed saturation kinetics with a Km of 0.33 MM. Mn2+ uptake was also competitively inhibited by Ca2+, and Ca2+ uptake inhibited by Mn2+. Electron microprobe studies showed that Mn2+ was localized in the ectoderm of the tissue in the same way as Ca2+. It was concluded from these studies that significant amounts of Mn2+ were accumulated by the active Ca2+ transport mechanism and that Mn2+ could be useful paramagnetic probe of divalent cation transport in this tissue.

Mitochondrial calcium transport: mechanisms and functions.

Ca(2+)transport across the mitochondrial inner membrane is facilitated by transporters having four distinct sets of characteristics as well as through the Ca(2+)-induced mitochondrial permeability transition pore (PTP). There are two modes of inward transport, referred to as the Ca(2+)uniporter and the rapid mode or RaM. There are also two distinct mechanisms mediating outward transport, which are not associated with the PTP, referred to as the Na(+)-dependent and the Na(+)-independent Ca(2+)efflux mechanisms. Several important functions have been proposed for these mechanisms, including control of the metabolic rate for cellular energy (ATP) production, modulation of the amplitude and shape of cytosolic Ca(2+)transients, and induction of apoptosis through release of cytochrome c from the mitochondrial inter membrane space into the cytosolic space. The goals of this review are to survey the literature describing the characteristics of the mechanisms of mitochondrial Ca(2+)transport and their proposed physiological functions, emphasizing the more recent contributions, and to consider how the observed characteristics of the mitochondrial Ca(2+)transport mechanisms affect our understanding of their functions.

Cytosolic free calcium concentrations in avian growth plate chondrocytes.

Isolated avian growth plate chondrocytes convert the acetoxymethyl ester (AM) form of Fura-2 quickly and efficiently to the Ca2(+)-sensitive pentacarboxylic acid (FA) form. Control experiments indicate that the Kd for intracellular Fura-2/FA is very close to that of extracellular Fura-2/FA at the same ionic strength and pH and that the Fura-2/FA fluorescence from indicator converted by intracellular organelles is quite small. Correcting for the effects of extracellular Fura-2/FA and partial hydrolysis products has improved the accuracy of determination of intracellular [Ca2+] over earlier measurements in chondrocytes. Cytosolic [Ca2+] in isolated growth plate chondrocytes (containing cells from each maturational stage) is found to require approximately 9 hours to recover from the isolation process. After this recovery period, cytosolic [Ca2+] in these cells converges to approximately 70 nM regardless of the [Ca2+] of the recovery medium, suggesting regulation of cytosolic [Ca2+] to a set point. Chondrocytes that are separated into maturationally distinct fractions using countercurrent centrifugal elutriation show an increase in cytosolic [Ca2+] with cellular maturation. The least mature resting cells have a [Ca2+] near 57 nM, while the most mature hypertrophic cells are around 95 nM.

Activation of phosphoinositide metabolism by parathyroid hormone in growth plate chondrocytes.

Parathyroid hormone (PTH) is one of the most potent stimulators of growth plate chondrocyte mitogenesis that has been reported. However, study of the second messenger signaling mechanisms involved in the transduction of the hormone's effects on these cells is incomplete. Our data indicate that in addition to stimulating cyclic adenosine-3'5'-monophosphate metabolism, PTH also activates the phosphoinositide cascade, the pathway responsible for the generation of inositol-1,4,5-trisphosphate dependent Ca2+ signals. Our conclusion that PTH activates the phosphoinositide cascade is based on data that demonstrate: (1) the Ca2+ transients evoked by the hormone are dependent on intracellular Ca2+ stores; (2) the hormone stimulates the release of radiolabeled inositol from GPC plasma membranes; and (3) the hormone stimulates a greater than 8-fold increase in cytosolic inositol-1,4,5-trisphosphate pool size.

Manganese and calcium transport in mitochondria: implications for manganese toxicity.

Mn2+ is sequestered by liver and brain mitochondria via the mitochondrial Ca2+ uniporter. The mitochondrial Ca2+ uniporter is a cooperative transport mechanism possessing an external activation site and a transport site. Ca2+ binding to the activation site greatly increases the velocity of uptake of both Ca2+ and Mn2+. Electron paramagnetic resonance (EPR) shows that over 97% of the Mn2+ in the mitochondrial matrix is normally bound to the membrane or to matrix proteins. EPR measurements of manganese within living isolated mitochondria can be repeated for hours, and during this time most of the manganese remains in the Mn2+ state. Mn2+ is transported out of mitochondria via the very slow Na(+)-independent efflux mechanism, which is an active (energy-requiring) mechanism. Mn2+ is not significantly transported over the Na(+)-dependent efflux mechanism, which is the dominant efflux mechanism in heart and brain mitochondria. Mn2+ inhibits the efflux of Ca2+ through both of these efflux mechanisms, having an apparent Ki of 7.9 nmol/mg protein on the Na(+)-independent efflux mechanism and an apparent Ki of 5.1 nmol/mg on the Na(+)-dependent efflux mechanism. Mn2+ inhibition of Ca2+ efflux may increase the probability of the mitochondria undergoing the mitochondrial permeability transition (MPT). Intramitochondrial Mn2+ also inhibits State 3 mitochondrial respiration using either succinate or malate plus glutamate as substrate. The data suggest that Mn2+ depletes cellular energy supplies by interfering with oxidative phosphorylation at the level of the F1ATPase and at much higher concentrations, at Complex I. Effects such as these could lead to apoptosis in active neurons.

Mechanisms of mitochondrial calcium transport.

Mitochondria are known to possess a rapid calcium uptake mechanism or uniport and both sodium-dependent and sodium-independent efflux mechanisms. Whether sodium-independent calcium efflux is mediated and whether sodium-dependent calcium efflux can be found in liver mitochondria have been questioned. Kinetics results relevant to the answers of these questions are discussed below. A slow, mediated, sodium-independent calcium efflux mechanism is identified which shows second order kinetics. This mechanism, which shows "nonessential activation" kinetics, has a Vmax around 1.2 nmol calcium per mg protein per min and a half maximal velocity around 8.4 nmol calcium per mg protein. A slow, sodium-dependent calcium efflux mechanism is identified, which is first order in calcium and second order in sodium. This mechanism has a Vmax around 2.6 nmol of calcium per mg protein per min. The sodium dependence is half saturated at an external sodium concentration of 9.4 mM, and the calcium dependence is half saturated at an internal calcium concentration of 8.1 nmol calcium per mg protein. The cooperativity of the sodium dependence effectively permits a terreactant system to be fit by a bireactant model in which [Na] only appears as the square of [Na]. This liver system shows simultaneous, as opposed to ping-pong, kinetics. It is also found to be sensitive to inhibition by tetraphenyl phosphonium, magnesium, and ruthenium red. A model is proposed in which mitochondrial calcium transport could function to "shape the pulses" of cytosolic calcium. Simultaneously, mitochondria may mediate a "calcium memory" coupled perhaps to activation of cytosolic events through calmodulin or perhaps to activation of electron transport through the activation of specific dehydrogenases by intramitochondrial calcium.

Kinetics of mitochondrial calcium transport. II. A kinetic description of the sodium-dependent calcium efflux mechanism of liver mitochondria and inhibition by ruthenium red and by tetraphenylphosphonium.

Sodium-dependent calcium efflux from rat liver mitochondria has been studied as a function of mitochondrial calcium loads (2 to 40 nmol/mg) and extramitochondrial sodium concentrations (5 to 40 mM). The resulting data can be fit to a terreactant model which exhibits simultaneous kinetics (i.e. both sodium and calcium must be bound simultaneously for transport to occur). The Hill coefficients for the calcium and sodium dependences were 1.0 +/- 0.1 and 2.0 +/- 0.2, respectively. The cooperativity of the sodium dependence allows the terreactant model to be reduced to a bireactant model in which the sodium concentration only appears mathematically as the square of the sodium concentration. The data then fit the relationship (Formula: see text) The experimentally determined value of Vmax is found to be 2.6 +/- 0.5 nmol/mg/min, and the load of calcium (KCa) and concentration of sodium (KNa) necessary to stimulate the efflux to half its maximal calcium-dependent activity and sodium-dependent activity, respectively, were 8.1 +/- 1.4 nmol of Ca2+/mg and 9.4 +/- 0.6 mM Na+. This sodium-dependent calcium efflux from liver mitochondria was inhibited by magnesium, by ruthenium red, and by tetraphenylphosphonium. Fifty percent inhibition was obtained at 1.0-1.5 mM magnesium, at 12 nmol of ruthenium red/mg of protein, and at 0.2 microM tetraphenylphosphonium.

The efficiencies of the component steps of oxidative phosphorylation. I. A simple steady state theory.

Most earlier theoretical work on oxidative phosphorylation has emphasized the application of the formalism of nonequilibrium thermodynamics to the overall process. The resultant mathematical development and interpretation of some experimental data is complicated somewhat by the necessity of treating a system which is incompletely coupled (degree of coupling, q less than 1). Here a simple alternative approach is proposed which can be applied to many studies in the field. In this approach the overall process is broken up into sequential steps so that the product of the efficiencies of the steps is equal to the efficiency of the overall process. Steps of interest for which the degree of coupling may be quite close to unity can be "isolated" by this procedure. This approach results in a simple mathematical formalism emphasizing the power use (or energy use) at each step of the energy transduction process. The efficiencies of the steps of the process can be experimentally evaluated as is shown in the accompanying paper (B.D. Jensen, K. K. Gunter, and T. E. Gunter, 1986, Arch. Biochem. Biophys. 248, 305-323) where measurements are performed as dictated by the assumptions of the current theory. This alternative approach simplifies the analysis of changes induced in the process of oxidative phosphorylation as a result of agents added to the system or of changes in conditions. The locus (or loci) of such changes becomes rapidly apparent if the data is treated as suggested. Furthermore, the mathematical formalism lends itself both to the development of expressions and new experimental approaches which minimize the effects of a decrease in a value of q below unity and also to optimal statistical treatment of the data. As a concrete example of the use of this approach we reinvestigate the question of the equivalence of use of energy from the pH gradient and of the membrane potential in phosphorylation.

Mechanisms by which mitochondria transport calcium.

It has been firmly established that the rapid uptake of Ca2+ by mitochondria from a wide range of sources is mediated by a uniporter which permits transport of the ion down its electrochemical gradient. Several mechanisms of Ca2+ efflux from mitochondria have also been extensively discussed in the literature. Energized mitochondria must expend a significant amount of energy to transport Ca2+ against its electrochemical gradient from the matrix space to the external space. Two separate mechanisms have been found to mediate this outward transport: a Ca2+/nNa+ exchanger and a Na(+)-independent efflux mechanism. These efflux mechanisms are considered from the perspective of available energy. In addition, a reversible Ca2(+)-induced increase in inner membrane permeability can also occur. The induction of this permeability transition is characterized by swelling of the mitochondria, leakiness to small ions such as K+, Mg2+, and Ca2+, and loss of the mitochondrial membrane potential. It has been suggested that the permeability transition and its reversal may also function as a mitochondrial Ca2+ efflux mechanism under some conditions. The characteristics of each of these mechanisms are discussed, as well as their possible physiological functions.

Transport of calcium by mitochondria.

The identification of intramitochondrial free calcium ([Ca2+]m) as a primary metabolic mediator [see Hansford (this volume) and Gunter, T. E., Gunter, K. K., Sheu, S.-S., and Gavin, C. E. (1994) Am. J. Physiol. 267, C313-C339, for reviews] has emphasized the importance of understanding the characteristics of those mechanisms that control [Ca2+]m. In this review, we attempt to update the descriptions of the mechanisms that mediate the transport of Ca2+ across the mitochondrial inner membrane, emphasizing the energetics of each mechanism. New concepts within this field are reviewed and some older concepts are discussed more completely than in earlier reviews. The mathematical forms of the membrane potential dependence and concentration dependence of the uniporter are interpolated in such a way as to display the convenience of considering Vmax to be an explicit function of the membrane potential. Recent evidence for a transient rapid conductance state of the uniporter is discussed. New evidence concerning the energetics and stoichiometries of both Na(+)-dependent and Na(+)-independent efflux mechanisms is reviewed. Explicit mathematical expressions are used to describe the energetics of the system and the kinetics of transport via each Ca2+ transport mechanism.

Uptake of calcium by mitochondria: transport and possible function.

Vertebrate mitochondria contain a complex system for transport of Ca2+ and related ions, consisting of two saturable modes of Ca2+ influx and two separate, saturable mechanisms of Ca2+ efflux. The characteristics of the mechanisms of Ca2+ uptake, the uniporter and the RaM, are discussed here and suggestions are made about how the mechanisms may work together and separately to mediate the two physiological roles with which they are most commonly associated-control of the rate of cellular ATP production and induction of the permeability transition and apoptosis. It is argued that more subtlety of control of intramitochondrial free Ca2+ concentration ([Ca2+]m) must be used by the uniporter and the RaM to fulfill their physiological roles than has been commonly recognized. This is because an increase in [Ca2+]m is associated with both increased production of ATP which supports the continued life of the cell and with induction of the permeability transition and possibly apoptosis, which leads to cell death. The saturable mechanisms of mitochondrial Ca2+ efflux and the Ca2+-induced mitochondrial permeability transition, which can transport Ca2+ as well as other ions and molecules and is often considered as a Ca2+ transport mechanism, are being reviewed separately.