Gerald P. Brierley

Adenine nucleotide metabolism and compartmentalization in isolated adult rat heart cells.

The metabolism and intracellular compartmentalization of adenine nucleotides in a preparation of adult rat heart myocytes showing good morphology, viability, and tolerance to calcium ion has been examined by high performance liquid chromatography. These myocytes contain an average of 23 nmol adenine nucleotide per milligram protein which is about 60% of the adenine nucleotide content of intact rat heart tissue. The loss of adenine nucleotide occurs during the incubation and washing steps that increase the yield of viable cells, rather than during the collagenase perfusion. An analysis of cellular compartments shows that the adenine nucleotide of the cell consists of 17 nmol adenine nucleotide in the cytosol, 5 nmol in the mitochondria, and 1.3 nmol adenosine diphosphate bound to myofibrils per milligram cell protein. Myocytes lose both adenosine triphosphate and adenine nucleotide when incubated anaerobically in the absence of glucose, and the lost adenine nucleotide can be accounted for as increased inosine, adenosine, and inosine monophosphate. Myocytes that contain less than 0.1 nmol of cytosol adenosine triphosphate per milligram cell protein maintain an intact sarcolemma, but are unable to carry out anaerobic glycolysis. Reoxygenation of anaerobic cells results in restoration of energy charge and a net resynthesis of about 2 nmol adenine nucleotide per milligram protein. Adenosine and inosine monophosphate decrease on reaerarion of anaerobic cells, whereas inosine levels increase. When iodoacetate is added to block glycolysis, the decline in adenine nucleotide and production of inosine monophosphate are accelerated and there is no resynthesis of adenine nucleotide when anaerobic cells are reoxygenated. Large accumulations of inosine monophosphate are also seen in myocytes treated with an uncoupler of oxidative phosphorylation.

Ion transport by heart mitochondria: XI. The spontaneous and induced permeability of heart mitochondria to cations

Abstract 1. 1. Isolated heart mitochondria show low permeability to most cations in the absence of a source of energy and of inducers of ion transport. Mitochondria are rapidly penetrated by NH4+ and to a lesser degree by Na+ under these conditions, however. In the presence of a source of energy, all cations tested appear to enter the mitochondrion by an energy-linked process. The extensive penetration of the mitochondrion by cations which results in osmotic swelling is dependent on the presence of a permeant anion. These studies suggest that the mitochondrial mechanism for cation uptake shows relatively little specificity as to cation. 2. 2. Gramicidin, an inducer of Na+ and K+ transport, increases the rate of swelling in isotonic Na+ or K+ acetate in the absence of an energy source. Addition of an uncoupler of oxidative phosphorylation lowers by two orders of magnitude the concentration of gramicidin which brings about this increased permeability. Gramicidin also releases the endogenous K+ of mitochondria suspended in K+-free media in the absence of a source of energy. These observations are compatible with the suggestion of a number of authors that gramicidin increases the permeability of the mitochondrial membrane to Na+ and K+. 3. 3. In contrast to gramicidin, Zn++ appears to have little effect on the permeability of the mitochondrion to cations in the absence of an energy source. However, once bound by an energy-linked reaction in the presence of phosphate, Zn++ causes an increase in passive permeability to K+, Na+, and Cl−. Mitochondria treated with Zn++ under these conditions remain impermeable to sucrose.

The uptake and extrusion of monovalent cations by isolated heart mitochondria

SummaryThe factors involved in the movement of monovalent cations across the inner membrane of the isolated heart mitochondrion are reviewed. The evidence suggests that the energy-dependent uptake of K+ and Na+ which results in swelling of the matrix is an electrophoretic response to a negative internal potential. There are no clear cut indications that this electrophoretic cation movement is carrier-mediated and possible modes of entry which do not require a carrier are examined. The evidence also suggests that the monovalent cation for proton exchanger (Na+ > K+) present in the membrane may participate in the energy-dependent extrusion of accumulated ions. The two processes, electrophoretic cation uptake (swelling) and exchange-dependent cation extrusion (contraction) may represent a means of controlling the volume of the mitochondrion within the functioning cell. A number of indications point to the possibility that the volume control process may be mediated by the divalent cations Ca+2 and Mg+2. Studies with mercurial reagents also implicate certain membrane thiol groups in the postulated volume control process.

Energy-dependent exchange of K+ in heart mitochondria K+ influx

The efflux 42K+ from isolated beef heart mitochondria under conditions of near steadystate K+ is increased by repiration and is sensitive to uncouplers and to exogenous Mg2 The respiration-dependent efflux is strongly activated by inorganic phosphate in the presence of external K+, but not Na+, and is inhibited by oxidative phosphorylation. Low concentrations of mersalyl also activate respiration-dependent efflux of 42K+ in the absence of net alteration in matrix K+. Acetate in the presence of mersalyl brings about net accumulation of K+ with retention of internal 42K+. The results are consistent with a model in which nearly constant matrix K+ is maintained by the regulated interplay between a K+ uniport (which is responsive to membrane potential and which is the pathway for K+ influx) and a K+H+ exchanger (which responds to the transmembrane pH differential and which is the pathway for net K+ efflux).

Estimation of intramitochondrial pCa and pH by fura-2 and 2,7 biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) fluorescence

Isolated heart mitochondria hydrolyze the acetoxymethyl esters of the Ca2+-sensitive fluorescent probe fura-2 and the fluorescent pH indicator biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). The free acid forms of both probes are retained in the matrix and their fluorescence can be used to monitor the pCa and pH, respectively, of this compartment. When fura-2 loaded rat heart myocytes are lysed with digitonin, a portion of the dye is retained in the mitochondrial fraction and its fluorescence reports the uptake and release of Ca2+ by the mitochondria. It is concluded that fura-2 and BCECF may report mitochondrial as well as cytosol parameters when the probes are used in intact cells.

Ion transport in heart mitochondria. XIII. The effect of ethylenediaminetetraacetate on monovalent ion uptake

Abstract 1. Ethylenediaminetetraacetate (EDTA) markedly activates the accumulation of Na + and Li + and the swelling which accompanies the ion uptake by isolated heart mitochondria. This activation is reflected in the removal of limited amounts of endogenous Mg 2+ and extensive loss of K + . The removal of these cations requires the presence of Na + , a source of energy, and a permeant anion as well as EDTA. The effects of EDTA on the activation of Na + uptake and cation removal are duplicated by chelators with a high affinity for Mg 2+ , but not by ethyleneglycol-bis-(β-aminoethylether)-N, N′-tetraacetic acid. Mg 2+ at concentrations 5 to 6 times less than EDTA prevents both activation of Na + uptake and cation removal. 2. EDTA does not appear to be bound by heart mitochondria. At neutral pH the chelator penetrates into the mitochondrial water volume to the same extent as sucrose and mannitol. At pH 8.1 where the removal of mitochondrial Mg 2+ by EDTA is more effective, EDTA penetrates virtually the entire water volume. This penetration requires the presence of a source of energy, a transported cation such as Na + , and a permeant anion. It appears possible that the oscillations in ion uptake and swelling observed in the presence of EDTA at pH 8.1 may be related to the presence of the chelator in the interior compartment under these conditions.

Response of isolated rat heart cells to hypoxia, re-oxygenation, and acidosis.

Responses of isolated adult rat heart cells to conditions that emphasize various aspects of ischemia have been evaluated. Cells maintained in hypoxic media with limited gubstrate deteriorate mort rapidly than aerobic controls supplemented with glucose. Two distinct irreversible pathways for cell alteration can be distinguished as follows: (1) continued anaerobic aging in the absence of glucose results in the production of large numbers of cells which retain the rod-shaped morphology of heart cells in situ, but which have lost saxcolemmal integrity, and (2) after a period of anaerobic aging, reaeration of the cells produces large numbers of rounded cells in irreversible contracture. These cells maintain an intact sarcolemma and are indistinguishable from those produced by addition of 1 mM Ca2+ to Na+-loaded, aerobic cells. Contracture of isolated cells on re-aeration is at least superficially analogous to the oxygen paradox in situ, but since the isolated cells maintain an intact sarcolemma, there is no loss of creatine phosphokinase or other components of the cytosol. Incubation of isolated heart cells at acid pH (pH 8.8 to 6.2) largely prevents both Ca2+-dependent contracture and a Ca2+- dependent loss of respiratory capacity. The acidic conditions virtually eliminate the net influx of 40Ca2+ into isolated cells that occurs at neutral pH, and the inhibition appears to be localized at the sarcolemma.

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 Ca2+-extruding mechanism that permits the mitochondrion to regulate matrix [Ca2+] by balancing Ca2+ efflux against influx on the Ca2+-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.

Calcium tolerance of isolated rat heart cells.

Abstract Freshly isolated adult rat heart cells, which initially show the elongated, rod-shaped morphology typical of heart cells in situ , are almost quantitatively converted to rounded contracture forms by exposure to 1 m m Ca 2+ . These Ca 2+ -sensitive cells became Ca 2+ -tolerant following a short period of metabolic activity in a low-Ca 2+ medium, in that they retain their rod-shaped configuration when challenged with Ca 2+ after this preincubation step. Tolerance to Ca 2+ develops in parallel with the establishment of low Na + /K + ratios in these cells and both processes are sensitive to ouabain. The initial net uptake of Ca 2+ is greater in Ca 2+ -sensitive than in Ca 2+ -tolerant cells. These results suggest that contracture in the Ca 2+ -sensitive cells is a consequence of the rapid entry of excessive amounts of Ca 2+ in exchange for internal Na + .

Ion transport by heart mitochondria: XXIII. The effects of lead on mitochondrial reactions

Abstract The interaction of lead with isolated beef heart mitochondria and submitochondrial particles has been investigated. The amount of lead bound by the membrane, and consequently the effects of lead on a number of mitochondrial parameters, depends on the anionic composition of the suspending medium. Up to about 140 nmoles of lead per milligram of protein can be bound passively in the absence of phosphate. The binding of increasing amounts of lead alters the passive permeability of the mitochondrial membrane to cations and to anions differentially. Lead has been shown to activate the energy-linked uptake of ions by the mitochondrion, and lead itself is accumulated by an energy-dependent reaction under a variety of conditions. The energy-dependent uptake of lead shares many of the features of the Ca2+ accumulation reaction. The effects of lead on mitochondrial respiration appear to result from an interplay between, (a) the intrinsic susceptibility of certain enzyme systems, such as succinic dehydrogenase, to inhibition by the heavy metal, (b) activation of energy-dependent ion movements by lead and the resulting increased respiration which supports these movements, (c) energy-dependent movements of lead itself, and (d) effects of lead on substrate uptake and retention.