Marianne S. Jurkowitz

Adenosine, Inosine, and Guanosine Protect Glial Cells During Glucose Deprivation and Mitochondrial Inhibition: Correlation Between Protection and ATP Preservation

Abstract: The purpose of this study was to determine the mechanism by which adenosine, inosine, and guanosine delay cell death in glial cells (ROC‐1) that are subjected to glucose deprivation and mitochondrial respiratory chain inhibition with amobarbital (GDMI). ROC‐1 cells are hybrid cells formed by fusion of a rat oligodendrocyte and a rat C6 glioma cell. Under GDMI, ATP was depleted rapidly from ROC‐1 cells, followed on a much larger time scale by a loss of cell viability. Restoration of ATP synthesis during this interlude between ATP depletion and cell death prevented further loss of viability. Moreover, the addition of adenosine, inosine, or guanosine immediately before the amobarbital retarded the decline in ATP and preserved cell viability. The protective effects on ATP and viability were dependent on nucleoside concentration between 50 and 1,500 µM. Furthermore, protection required nucleoside transport into the cell and the continued presence of nucleoside during GDMI. A significant positive correlation between ATP content at 16 min and cell viability at 350 min after the onset of GDMI was established (r = 0.98). Modest increases in cellular lactate levels were observed during GDMI (1.2 nmol/mg/min lactate produced); however, incubation with 1,500 µM inosine or guanosine increased lactate accumulation sixfold. The protective effects of inosine and guanosine on cell viability and ATP were >90% blocked after treatment with 50 µM BCX‐34, a nucleoside phosphorylase inhibitor. Accordingly, lactate levels also were lower in BCX‐34‐treated cells incubated with inosine or guanosine. We conclude that under GDMI, the ribose moiety of inosine and guanosine is converted to phosphorylated glycolytic intermediates via the pentose phosphate pathway, and its subsequent catabolism in glycolysis provides the ATP necessary for maintaining plasmalemmal integrity.

Inosine and guanosine preserve neuronal and glial cell viability in mouse spinal cord cultures during chemical hypoxia

Murine spinal cord primary mixed cultures were treated with the respiratory inhibitor, rotenone, to mimic hypoxic conditions. Under these conditions neurons rapidly underwent oncosis (necrosis) with a complete loss in viability occurring within 260 min; however, astrocytes, which accounted for most of the cell population, died more slowly with 50% viability occurring at 565 min. Inosine preserved both total cell and neuronal viability in a concentration-dependent manner. The time of inosine addition relative to hypoxic insult was critical with the most effective protection occurring when inosine was added just prior to or within 5 min after insult. Inosine was ineffective when added 30 min after hypoxic insult. The effect of guanosine was similar to that of inosine. Treatment of cultures with BCX-34, a purine nucleoside phosphorylase inhibitor, prevented protection by inosine or guanosine, suggesting involvement of a purine nucleoside phosphorylase in the nucleoside protective effect.

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.

Mg2+ and the permeability of heart mitochondria to monovalent cations

Abstract EDTA activates respiration-dependent ion accumulation and osmotic swelling in beef heart mitochondria and alters the monovalent cation selectivity of the reaction from K+ = Li+ > Na+ to Li+ > Na+ > K+. The chelator does not penetrate the matrix of the mitochondrion at neutral pH and the small amount of Mg2+ which it removes appears to be located outside of and on the outer surface of the inner membrane. Mitochondrial Mg2+ can be reduced from 30 to 2–4 nmol/mg protein by EDTA under conditions which permit the chelator to enter the matrix (pH 8 and swelling of the matrix). Mitochondrial Mg2+ also can be reduced to these low values in the absence of EDTA by addition of the divalent ionophore A23187. Respiration-dependent cation uptake is activated by EDTA in the A23187-treated mitochondria. EDTA is not bound to the membrane and does not penetrate the matrix under these conditions and it appears that removal of 1 nmol of Mg2+/mg (or less) by the chelator is responsible for an increase in transmembrane, electrophoretic permeability to monovalent cations and the change in the selectivity pattern. EDTA treatment and the level of endogenous mitochondrial Mg2+ have little effect on the Na + H + exchange reaction of the membrane. The studies suggest that specific pathways for electrophoretic penetration of monovalent cations are present in the inner membrane of the mitochondrion and that Mg2+ bound by a limited number of high-affinity sites in or near these pathways can control monovalent cation permeability.

Ion transport by heart mitochondria. XXII. Spontaneous, energy-linked accumulation of acetate and phosphate salts of monovalent cations.

Abstract The accumulation of monovalent cations by isolated beef heart mitochondria has been studied by evaluating the efficiency of energy-dependent osmotic swelling. Extensive osmotic swelling occurs spontaneously when isolated heart mitochondria are suspended in 0.1 m acetate or phosphate salts. The swelling and ion uptake depend on either respiration or the presence of exogenous ATP, and the initial rate of swelling is proportional to the initial rate of respiration or ATP hydrolysis, respectively. The efficiency of the reaction varies somewhat from preparation to preparation but approaches a limit of about 2 cations accumulated per pair of electrons traversing a phosphorylation site. All monovalent cations tested support the reaction, but the most efficient energy-dependent swelling occurs with K + . Weak acid anions are required for the ion accumulation and swelling and the reaction appears to depend on the amount of free acid available in the suspension. Permeant strong acid anions, such as NO 3 − , fail to support the swelling reaction in the presence of energy. Valinomycin increases both the amount and the efficiency of ion uptake under these conditions. Mg 2+ decreases both of these values whereas p -chloromercuriphenyl sulfonate increases both. These responses are discussed in terms of current models of mitochondrial ion transport.

Ion transport by heart mitochondria. Retention and loss of energy coupling in aged heart mitochondria.

Abstract The retention and loss of energy-coupling reactions in isolated beef heart mitochondria have been examined under anaerobic conditions using suspending media chosen to mimic the intracellular milieu. In long-term incubations at 37 °C, a loose coupling develops which can be controlled by adding serum albumin. This lesion closely resembles that produced by addition of free fatty acids which has been described in previous studies. Shorter incubation times produce an increased susceptibility to hydrogen peroxide which is characterized by elevated ATPase activity, increased permeability to monovalent cations, and increased proton ejection on transition from the anaerobic to the aerobic state. This peroxide sensitivity is prevented by chelators such as EGTA and appears to involve a time-dependent release of metal ions. Of the metabolites which are known to increase in concentration in the ischemic heart cell, Na + , P 1 , lactate, and H + all promote swelling of isolated heart mitochondria and contribute to a decline in energy coupling. The relationship of these results to the pathological deterioration of mitochondria in ischemic heart tissue is discussed.

Osmotic swelling of heart mitochondria in acetate and chloride salts. Evidence for two pathways for cation uptake.

Abstract Swelling of nonenergized heart mitochondria suspended in acetate salts appears to depend on the activity of an endogenous cation/H + exchanger. Passive swelling in acetate shows a characteristic cation selectivity sequence of Na + >Li + >K + , Rb + , Cs + , or tetramethylammonium, a sharp optimum at pH 7.2–7.3, activation by Ca 2+ , and loss of activity on aging which can be related to loss of endogenous K + . The reaction is nearly insensitive to either addition of exogenous Mg 2+ or removal of membrane Mg 2+ with EDTA. Each of these characteristics of passive swelling in acetate salts is duplicated in chloride media when tripropyltin is added to induce Cl − /OH − exchange. In contrast to nonenergized mitochondria, swelling of respiring mitochondria has been postulated to depend on electrophoretic uptake of cations in response to an interior negative membrane potential. Respiration-dependent swelling in acetate shows an indistinct cation selectivity sequence with Li + and Na + supporting higher rates of swelling at higher efficiency than K + , Rb + , and Cs + . The high rates of respiration-dependent swelling in Li + and Na + are inhibited by low levels of exogenous Mg 2+ ( K i of 5–10 μ m ), but a significant swelling with almost no cation selectivity persists in the presences of 2 m m Mg 2+ . Removal of membrane Mg 2+ by addition of EDTA strongly activates the rate of respiration-dependent swelling and converts a sigmoid dependency of swelling rate on Li + concentration to a hyperbolic one with a Km of about 14 m m Li + . The cation selectivity and Mg 2+ dependence of the reaction induced in chloride salts by tripropyltin are identical to these properties in acetate. Energy-dependent swelling in acetate shows optimum activity at pH 6.5 which appears related to the availability of free acetic acid, since the corresponding reaction induced in chloride shows a broad optimum at about pH 7.5. These studies support the concept that monovalent cations enter nonenergized mitochondria by electroneutral exchange with protons but penetrate respiring mitochondria by electrophoretic movement through one or more uniport pathways. They further suggest that both a Mg 2+ -sensitive uniport with high activity for Na + and Li + and a Mg 2+ -insensitive pathway with little cation discrimination are available in the membrane.

Inhibition of Na+‐dependent Ca2+ efflux from heart mitochondria by amiloride analogues

The Na+‐induced release of accumulated Ca2+ from heart mitochondria is inhibited by amiloride, benzamil and several other amiloride analogues. These drugs do not affect uptake or release of Ca2+ mediated by the ruthenium red‐sensitive uniporter and their effects, like those of diltiazem and other Ca2+‐antagonists, appear to be localized principally at the Na+/Ca2+ antiporter of the mitochondrion. Benzamil inhibits Na+/Ca2+ antiport non‐competitively with respect to [Na+] with a K i of 167 μM. In the presence of 1.5 mM Pi the K i for benzamil inhibition of this reaction is decreased to 87 μM.

Ruthenium red-sensitive and -insensitive release of Ca2+ from uncoupled heart mitochondria.

The uncoupler-induced release of accumulated Ca2+ from heart mitochondria can be separated into two components, one sensitive and one insensitive to ruthenium red. In mitochondria maintaining reduced NAD(P)H pools and adequate levels of endogenous adenine nucleotides, the release of Ca2+ following addition of an uncoupler is virtually all inhibited by ruthenium red and can be presumed to occur via reversal of the Ca2+ uniporter. When ruthenium red is added to block efflux via this pathway, high rates of Ca2+ efflux can still be induced by an uncoupler, provided either NADH is oxidized or mitochondrial adenine nucleotide pools are depleted by prior treatment. This ruthenium red-insensitive Ca2+-efflux pathway is dependent on the level of Ca2+ accumulated and is accompanied by swelling of the mitochondria and loss of endogenous Mg2+. Loss of Ca2+ by this relatively nonspecific pathway is strongly inhibited by Sr2+ and by nupercaine, as well as by oligomycin and exogenous adenine nucleotides. The loss of Ca2+ from uncoupled heart mitochondria occurs via a combination of these two mechanisms except under conditions chosen specifically to limit efflux to one or the other pathway.

Ion transport by heart mitochondria: XXV. Activation of energy-linked K+ uptake by non-ionic detergents

Abstract The energy-dependent uptake of K + salts by isolated heart mitochondria is markedly activated by Triton X-100 and several other nonionic detergents. The detergent-mediated reaction closely resembles that seen in the presence of low concentrations of the ionophore valinomycin. Triton increases the efficiency of energylinked K + uptake, induces passive permeability to K + , and affects respiration, phosphorylation, O 2 pulses, and ATPase activity in a manner similar to valinomycin. Studies of equilibrium extraction of cations into organic solvents indicate that detergents with the poly(ethyleneoxy) ethanol structure can function as ionophores with properties roughly analogous to the cyclic polyethers. The effects of detergents with the ability to act as ionophores are compared with those of other classes of surface-active reagents.