Douglas R. Hunter

The Ca2+-induced membrane transition in mitochondria: I. The protective mechanisms☆☆☆

Some physiological factors which control the rate of induction of the Ca2+-induced membrane transition (the term “Ca2+-induced membrane transition” is defined in the introduction) have been systematically investigated. To exclude the complicating factors of electron flow and energization, the transition was studied in mitochondria depleted of endogenous substrate, in the presence of uncoupler. In these mitochondria the transition could be induced by Ca2+, whether entry was mediated by ruthenium red-sensitive permeation or by A23187 facilitated diffusion. The rate of the transition was reduced fivefold by any agent which caused complete reduction of endogenous NAD. The rate of the transition was increased threefold by the exchange of endogenous ADP for phosphoenolpyruvate. A further increase was found on the addition of atractyloside, but bongkrekic acid caused inhibition. Addition of uncoupler to energized mitochondria when the endogenous NAD was already fully oxidized caused a stimulation of the transition. From these observations we conclude that mitochondria have a set of protective mechanisms (the term “protective mechanism” refers to the means by which these agents inhibit the Ca2+-induced transition; such a mechanism could be through allosteric interactions between the sites of binding of inhibitor and Ca2+; it would, however, be premature to conclude this on the basis of this paper) involving endogenous NADH, ADP, and energization which regulate the rate of the Ca2+-induced transition. ADP appears to work at two sites: one site which is internal, and another at the ADP/ATP translocase. In addition, we conclude that the transition requires neither electron flow nor energy, but rather the mere accessibility of some internal site to Ca2+. Finally, the key roles played by the protective agents in metabolism give the cell great potential flexibility in regulating the Ca2+-induced transition. This degree of control suggests that the transition has substantial physiological significance.

The Ca2+-induced membrane transition in mitochondria: II. Nature of the Ca2+ trigger site☆

The permeability of isolated mitochondria which have undergone the Ca2+-induced transition can be modulated over a wide range simply by adjusting the concentration of free Ca2+ in the medium. The effect varies sigmoidally with respect to Ca2+ concentration, with an apparent Km of 16 μm at pH 7.0. It is concluded that the trigger site (by “trigger site” we mean the site of binding of Ca2+ which, when Ca2+ is bound, will allow the transition in permeability to occur) is possibly also the site for high-affinity Ca2+ uptake. Added ADP, NADH and Mg2+ inhibit the Ca2+-induced permeability of mitochondria which have undergone the Ca2+-induced transition. Mg2+ and other ions, including H+, act like competitive inhibitors of the Ca2+ effect. In the presence of Ca2+, both neutral and charged molecules of molecular weight <1000 pass readily through the membrane. This response to Ca2+ is interpreted as a gating effect at the internal end of hydrophilic channels which span the inner membrane.

The Ca2+-induced membrane transition in mitochondria: III. Transitional Ca2+ release

The efflux of Ca2+ from mitochondria respiring at steady state, and much of uncoupler-induced Ca2+ efflux, is shown to be a consequence of the Ca2+-induced membrane transition (the Ca2+-induced transition is the Ca2+-dependent sudden increase in the nonspecific permeability of the mitochondrial inner membrane which occurs spontaneously when mitochondria are incubated under a variety of conditions (D. R. Hunter, R. A. Haworth, and J. H. Southard, 1976, J. Biol. Chem.251, 5069–5077)). Ca2+ release from mitochondria respiring at steady state is shown to be transitional by four criteria: (1) Ca2+ release is inhibited by Mg2+, ADP, and bovine serum albumin (BSA), all inhibitors of the transition; (2) release is selective for Ca2+ over Sr2+, a selectivity also found for the transition; (3) the time course of Ca2+ release is identical to the time course of the change in the mitochondrial population from the aggregated to the orthodox configuration; and (4) from kinetics, Ca2+ release from individual mitochondria is shown to occur suddenly, following a lag period during which no release occurs. Ca2+ release induced by uncoupler is shown to be mostly by a transitional mechanism, as judged by four criteria: (1) release of Ca2+ is ruthenium red-insensitive and is an order of magnitude faster than Sr2+ release which is ruthenium red-sensitive; (2) release of Ca2+ is strongly inhibited by keeping the mitochondrial NAD+ reduced; (3) the kinetics of Ca2+ release indicates a transitional release mechanism; and (4) uncoupler addition triggers the aggregated to orthodox configurational transition which, at higher levels of Ca2+ uptake, occurs in the whole mitochondrial population at a rate equal to the rate of Ca2+ release. Na2+-induced Ca2+ release was not accompanied by a configurational change; we therefore conclude that it is not mediated by the Ca2+-induced transition.

Contracture in isolated adult rat heart cells. Role of Ca2+, ATP, and compartmentation.

Isolated intact quiescent myocytes from the adult rat were used as a model system for investigating the determinants of contracture induced by metabolic deprivation. The model simulated the pattern of contracture and ATP decline seen in the intact heart during Ischemia. Three new insights into the contracture process were gained: (1) in the quiescent cell system, the rate of onset of contracture was independent of external Cn2+, supporting the view that the Ca2+ dependence of the rate of onset in the whole heart is related to beat-dependent substrate utilization; (2) the second phase of ATP decline was paralleled by a decline In the percentage of cells which had not undergone contracture, suggesting that–in any cell–contracture is immediately preceded by a total loss of ATP; and (3) oligomycin delayed the onset of contracture by 55 ± 12%, suggesting that mitochondria ATPase activity ia a significant drain on energy resources in the quiescent ischemic heart.

Control of the Mitochondrial Permeability Transition Pore by High-Affinity ADP Binding at the ADP/ATP Translocase in Permeabilized Mitochondria

Low levels of ADP binding at the ADP/ATP translocase caused inhibition of the Ca2+-inducedpermeability transition of the mitochondrial inner membrane, when measured using the shrinkage assay on mitochondria, which have already undergone a transition. Inhibition was preventedby carboxyatractyloside, but potentiated by bongkrekic acid, which increased the affinity forinhibition by ADP. This suggests that inhibition was related to the conformation of thetranslocase. Ca2+ addition was calculated to remove most of the free ADP. Ca2+ added after ADPinduced a slow decay of the inhibition, which probably reflected the dissociation of ADP fromthe translocator. We conclude that the probability of forming a permeability transition pore(PTP) is much greater when the translocase is in the CAT conformation than in the BKAconformation, and, in the absence of CAT and BKA, the translocator is shifted between theBKA and CAT conformations by ADP binding and removal, even in deenergized mitochondria with no nucleotide gradients.

The isolation of Ca2+-resistant myocytes from the adult rat.

Abstract A method is described for the preparation of Ca2+-resistant myocytes from the adult rat. Ca2+-resistant cells were obtained in yields of 6.3 ± 1.5 × 106 cells/gm wet weight tissue, with a purity of 67.5 ± 8.8% cells excluding trypan blue. Of the cells which excluded trypan blue, over 90% were rod-shaped. The rodshaped cells when isolated were quiescent, but beat in response to electric field stimulation in the presence of Ca2+. Incubation of the cells at 37° in the presence of 2 m m Ca2+ caused only a slow decline in the percentage of cells able to exclude trypan blue or to beat when stimulated. The rate of decline was comparable to that of cells incubated without Ca2+. Incubation of the cells in the presence of EGTA (ethylene glycol bis (β-aminoethyl ether)-N,N′-tetraacetic acid) for 30 min had little effect on the ability of the cells to withstand Ca2+. The key step in preparing Ca2+ resistant cells was found to be the inclusion of trypsin and Ca2+ at the final stage of incubation of tissue pieces. The trypsin appears to act not only by selective removal of cells susceptible to Ca2+ but also by conferring Ca2+ resistance on cells which otherwise would be Ca2+ susceptible. The properties displayed by this preparation indicate that the cells are both mechanically and electrochemically intact. They therefore provide an excellent model system for the study of excitation-contraction coupling at the cellular level, and also provide a more realistic starting point for studies of pathology than previous Ca2+-susceptible preparations.

Cellular manganese uptake by the isolated perfused rat heart: a probe for the sarcolemma calcium channel ☆

Abstract The slow inward Ca channel with isolated working rat hearts was assayed by measuring the cellular uptake rate of 5 μ m 54Mn from the perfusate. This new method is successful because Mn taken up by the cell is not readily released and remains in the cell even after all the extracellular 54Mn is washed away. Reagents which have been shown to affect the Ca channel of isolated heart muscle using a voltageclamp technique, also affect similarly the rate of Mn uptake by the rat heart: Verapamil (2 μ m ) inhibited Mn uptake 73%, oligomycin (2 μ m ) inhibited 55% and isoproterenol (2 μ m ) stimulated 80%. In addition Mn uptake appeared to be inhibited by perfusate Ca because a drop of perfusate Ca from 2.5 to 0.2 m m resulted in a 137% stimulation of Mn uptake. The uptake of cellular Mn by mitochondria was also investigated. The uptake was found to be stimulated by over 300% when the activity of the heart was increased by either raising the level of perfusate Ca or by adding isoproterenol. It is possible that changes in the concentration of cellular Ca promoted the observed stimulation of mitochondrial Mn uptake by a mechanism of mitochondrial CaMn co-transport.

Inhibition of calcium influx in isolated adult rat heart cells by ATP depletion.

Using 45Ca, indo1, and quin2, calcium uptake was measured in isolated quiescent adult rat heart cells under different metabolic conditions. Exposure of cells in a medium containing 1 mM CaCl2 to rotenone and uncoupler resulted in adenosine triphosphate (ATP) depletion from 17.08 +/- 2.26 to 0.63 +/- 0.11 nmol/mg within 8 minutes, and the cells went into contracture. In this time, the cells lost 1.65 +/- 0.1 nmol Ca/mg of total rapidly exchangeable cellular calcium, and the level of free cytosolic calcium as measured by indo1 rose from 47.4 +/- 16.3 nM to 79.8 +/- 27.6 nM. The subsequent rate of rise of intracellular free calcium concentration was just 4 nM/min for at least 40 minutes. Therefore, we investigated the effect of ATP depletion on the rate of calcium entry. In cells loaded with sodium by ouabain treatment without calcium, the initial rate of calcium influx on calcium addition was inhibited by 82-84% when cellular ATP was depleted, as measured by 45Ca or indo1. Quin2 also showed a strong inhibition of calcium influx by ATP depletion, but itself also caused a strong inhibition of calcium influx. The rate of calcium influx declined even further in ATP-depleted cells after the initial influx: Between 1 and 12 minutes after calcium addition, the residual 45Ca uptake rate of the first minute was inhibited by an additional 90%. We conclude that ATP depletion per se does not quickly elevate cytoplasmic free calcium and that such an elevation is prevented by a very strong inhibition of the rate of calcium entry.

Na+ releases Ca2+ from liver, kidney and lung mitochondria.

Intracelhrlar CaZ’ has assumed increasing importance as a regulator of a variety of cellular processes, including muscle contraction, secretion and cell division [l]. A prime candidate for a controller of intracellular Cazf levels, by virtue of its high affinity Ca2* uptake mechanism, is the mitochondrion. Such a role for the mitochondrion has gamed support from the demonstration that mitochondri~ Ca*” release is under separate control from uptake [2]. Two systems capable of releasing mitochondria Ca”’ are the Na+induced release system [3] and the Ca2+ -induced transition in permeability of the inner membrane 243. In elucidating the role of these release systems in Ca’* homeostasis it is important to know the extent of their occurence in mitochondria from different tissues. Such a study for the Na+ system has concluded that this system was most active in brain, heart and adrenal cortex, present in striated muscle, but absent in kidney, liver, lung, smooth muscie and hepatoma [5]. We report that the Na+ system is indeed present in mitochondria from liver, kidney and lung. We have not studied smooth muscle or hepatoma.

Modulation of cellular calcium stores in the perfused rat heart by isoproterenol and ryanodine.

The inhibitory action of procaine on cellular calcium release was utilized to define a new cellular calcium pool which, under physiological conditions, is present only during catecholamine stimulation. Rat hearts labeled with 45Ca++ were perfused with medium containing procaine and EGTA at 23 degrees C to remove extracellular calcium, and then cellular calcium was released by removal of procaine and restoration of calcium. By this method we have identified a cellular calcium pool (pool C) whose release is inhibited by procaine, but which does not require extracellular calcium for its release. Release of pool C can also be triggered by caffeine. [We have previously identified a cellular calcium pool (pool A) whose release is triggered by caffeine, inhibited by procaine, and which does require extracellular calcium for its release.] When hearts were labeled for 3 minutes with perfusate containing 1 mM 45Ca++, 48 +/- 6 nmol Ca++/g wet weight was found in pool A, but only 3 +/- 1 nmol Ca++/g in pool C. However, if isoproterenol was present during labeling, the hearts contained 72 +/- 5 nmol Ca++/g in pool A and 42 +/- 6 nmol Ca++/g in pool C. When calcium concentration in the labeling perfusate was varied, with and without isoproterenol, it was found that pool C does not begin to fill until pool A is almost full. The same effect was seen when excess cellular calcium uptake was induced by removing sodium from the perfusate. Ryanodine (0.2 microM) induced contractile failure (t1/2 = 3.4 +/- 0.4 min) and depleted pool A in control hearts by 85%. Ryanodine also similarly depleted pools A and C in isoproterenol-treated hearts. When contractility was monitored at the same time as the hearts were labeled, a linear relationship between dP/dt and the sum of pools A and C was observed over a wide range of conditions. Pools A and C both selected strongly for calcium over barium. These observations suggest that both pools A and C are located in the sarcoplasmic reticulum and are intimately involved in the regulation of contractility.