Elizabeth Murphy

Role of Lipoxygenase Metabolites in Ischemic Preconditioning

Preconditioning with brief intermittent periods of ischemia before a sustained period of ischemia has been shown to reduce infarct size and improve recovery of function in rat hearts. The mediators of this protective response are unknown in rats. We tested the hypothesis that a lipoxygenase metabolite might be involved in preconditioning, since lipoxygenase metabolites such as 12-hydroperoxyeicosatetraenoic acid have been shown to increase K+ channel activity and to decrease Ca2+ channel activity, which could have a protective effect on ischemic injury. In support of this hypothesis, we report that the lipoxygenase inhibitors nordihydroguaiaretic acid (NDGA, 5 mumol/L) and eicosatetraynoic acid (7 mumol/L) added just before and during preconditioning blocked the protective effects of preconditioning on recovery of function during reflow after 30 minutes of global ischemia. In addition, these lipoxygenase inhibitors partially blocked the ability of preconditioning to attenuate the rise in cytosolic free calcium during sustained ischemia. We also investigated the effects of preconditioning on eicosanoid metabolism by using high-performance liquid chromatography and found that 12-hydroxyeicosatetraenoic acid (12-HETE), the stable product of the lipoxygenase pathway, was made during the preconditioning protocol and that 12-HETE accumulation was blocked by NDGA. Thus, there is a correlation between functional recovery after ischemia and stimulation of the lipoxygenase pathway of arachidonic acid metabolism before the sustained period of ischemia; inhibition of the lipoxygenase pathway eliminates the protective effect of preconditioning on recovery of function after ischemia.

Cytosolic free calcium in chick heart cells its role in cell injury

The role of cytosolic free Ca2+ (Caf) in cell injury was investigated using two methods for measuring Caf in freshly disaggregated embryonic chick heart cells. The null-point method, using arsenazo III, is based on determining the extracellular Ca2+ concentration at which no net Ca2+ movement occurs when plasma membrane permeability is increased. With this technique, the null point Caf averaged 0.23 +/- 0.07 microM (n = 6) in the basal state. Using quin2, an intracellular fluorescent dye, to measure Caf a value of 0.05 +/- 0.01 microM (n = 5) was obtained. Elevation of Caf by various agents was associated with an increase in cell injury as measured by the release of the cytosolic enzyme, LDH. However, the relationship between Caf and LDH release was not a direct one under all experimental conditions, indicating that the level of Caf is not the sole determinant of cell injury.

Magnesium homeostasis in cardiac cells

Several aspects of Mg2+ homeostasis were investigated in cultured chicken heart cells using the fluorescent Mg2+ indicator, FURAPTRA. The concentration of cytosolic Mg2+ ([Mg2+]i) is 0.48 ± 0.03 mM (n = 31). To test whether a putative Na/Mg exchange mechanism controls [Mg2+]i below electrochemical equilibrium, we manipulated the Na+ gradient and assessed the effects on [Mg2+]i. When extracellular Na+ was removed, [Mg2+]i increased; this increase was not altered in Mg-free solutions, but was attenuated in Ca-free solutions. A similar increase in [Mg2+]i, which was dependent upon extracellular Ca2+, was observed when intracellular Na+ was raised by inhibiting the Na/K pump with ouabain. These results do not provide evidence for Na/Mg exchange in heart cells, but they suggest that Ca2+ can modulate [Mg2+]i. In addition, removing extracellular Na+ caused a decrease in intracellular pH (pHi), as measured by pH-sensitive microelectrodes, and this acidification was attenuated when Cat+ was also removed from the solution. These results suggest that Ca2+ and H+ interact intracellularly. Since changes in the Na+ gradient can also alter pHi, we questioned whether pH can modulate [Mg2+]i. pHi was manipulated by the NH4Cl prepulse method. NH4+-evoked changes in pHi, as measured by the fluorescent indicator BCECF, were accompanied by opposite changes in [Mg2+]i; [Mg2+]i changed by −0.16 mM/unit pH. These NH4+-evoked changes in [Mg2+]i were not caused by movements of Mg2+ or Ca2+ across the sarcolemma or by changes in cytosolic Ca2+. Additionally, pHi was manipulated by changing extracellular pH (pHo). When pHo was decreased from 7.4 to 6.3, pHi decreased by 0.64 units and [Mg2+]i increased by 0.12 mM; in contrast, when pHo was raised from 7.4 to 8.3, pHi increased by 0.6 units and [Mg2+]i did not change significantly. The results of our investigations suggest that Ca 2+ and H+ can modulate [Mg2+]i, probably by affecting cytosolic Mg2+ binding and/or subcellular Mg2+ transport and that such redistribution of intracellular Mg2+ may play an important role in Mg2+ homeostasis in cardiac cells.

Effects of sodium‐potassium pump inhibition and low sodium on membrane potential in cultured embryonic chick heart cells.

1. When the Na+‐K+ pump of cultured embryonic chick heart cells was inhibited by addition of ouabain with or without removal of external K+, the membrane potential rapidly depolarized to ‐40 mV and the Na+ content approximately doubled within 3 min. 2. After this, exposure to an [Na+]o of 27 mM caused a fall in Na+ content, a gain in Ca2+ content and a hyperpolarization. The hyperpolarization was approximately 25 mV in a [K+]o of 0 or 5.4 mM after 3 min of pump inhibition. After approximately 10 min of pump inhibition, the same hyperpolarization was observed in a [K+]o of 5.4 mM but in K+‐free solution the hyperpolarization increased to approximately 44 mV. 3. Varying [K+]o during the 10 min period of Na+‐K+ pump inhibition showed that the increase in hyperpolarization was associated with the period of exposure to K+‐free solution rather than the [K+]o at the time of lowering [Na+]o. 4. Changes in Na+ and Ca2+ content induced by exposure to an [Na+]o of 27 mM in K+‐free solution were similar at 3 and 10 min. This and the above observations suggest that the increased hyperpolarization was due to an increased membrane resistance. 5. 10 mM‐Cs+ reduced the low‐[Na+]o hyperpolarization by 26% but did not significantly affect the movements of Na+ and Ca2+. 1 mM‐La3+ reduced the low‐[Na+]o hyperpolarization by 15%: it also totally blocked the rise in Ca2+ content and partially blocked the fall in Na+ content. 1 mM‐Ba2+ reduced the low‐[Na+]o hyperpolarization by 20%. 6. Raising [Ca2+]o from 2.7 to 13.5 mM produced similar but smaller hyperpolarizations (approximately 6 mV after 3 min pump inhibition). High [Ca2+]o caused a rise in Ca2+ content but no significant drop in Na+ content. The hyperpolarization in high [Ca2+]o was insensitive to verapamil (20 microM) and 10 mM‐Cs+. 7. We conclude from the disparities between the magnitudes of the hyperpolarizations and the changes in ion contents that Na+‐Ca2+ exchange cannot be unequivocally identified as electrogenic solely from the low‐[Na+]o hyperpolarizations.

Magnesium homeostasis and cardiac cell function

Nearly 40 years ago, Carl Wiggers introduced the monograph entitled “Excitability of the Heart” [1] with the comment that “progress in basic research and in its practical application rests chiefly on the development of new tools for digging out nature’s secrets.” At the time, abnormalities in cardiac function resulting directly from changes in blood levels of magnesium (Mg2+) were not recognized. Similarly, altering intracellular magnesium (Mg2+ i) was thought to have a negligible effect on the excitability of the heart based on observations that neither high nor low extracellular magnesium concentration ([Mg2+]o) caused a noticeable change of the cardiac resting potential or action potential, although Brooks et al. [1] demonstrated the effects of calcium-magnesium interaction on the cardiac action potential.