Subcellular calcium pools of ischaemic and reperfused myocardium characterised by electron probe.

OBJECTIVE: The subcellular redistribution of calcium and other electrolytes was analysed in myocardium subjected to global ischaemia and reperfusion in order to establish possible causes of reperfusion injury. METHODS: Isolated ferret papillary muscles (maintained at 37 degrees C, 1.2 Hz stimulation) were exposed to ischaemic conditions for 1 h by isolating from room air, removing buffer, and exposing to a constant flow of water-saturated 95% N2/5% CO2 gas. Some muscles were "reperfused" for 5 min by exposing to control buffer. Electron probe microanalysis was used to measure subcellular electrolyte content. RESULTS: Ischaemia caused severe swelling and doubling of cell sodium [84(SEM 5) to 156(9) mmol.kg-1 dry weight (dw), P < 0.05]. Mitochondrial electrolyte concentrations were generally increased, most notably calcium [0.40(0.15) to 1.99(0.32) mmol.kg-1 dw, P < 0.05]. Electrolytes in other structures were more moderately affected. Reperfusion resulted in two general myocyte populations. Irreversibly injured cells exhibited contracture knots and an influx of extracellular fluid. Analysis was restricted to moderately injured cells, which had reduced swelling and varying numbers of vacuolated mitochondria. In the moderately injured cells, sodium remained high [167(11) mmol.kg-1 dw] and magnesium had increased from 39(2) to 52(2) mmol.kg-1 dw (P < 0.05). Mitochondrial calcium was near control levels [0.64(0.42) mmol.kg-1 dw]. Junctional sarcoplasmic reticular calcium significantly increased [6.56(0.59) to 18.53(1.64) mmol.kg-1 dw in control and reperfused cells, respectively, P < 0.05], while calcium had not changed significantly in T tubule lumen or "3rd compartment", composed of sarcoplasmic reticulum, T tubules, and free sarcoplasm. "3rd compartment" and junctional sarcoplasmic reticulum results suggest that sarcolemma bound calcium had decreased. CONCLUSIONS: Surviving myocytes had no change in total calcium, but showed a large degree of calcium redistribution. Mitochondria accumulated calcium during ischaemia, but released it upon reperfusion. Sarcoplasmic reticulum in reperfused cells appeared functional and may have helped maintain physiological [Ca2+]i by storing calcium released from mitochondria and the sarcolemma. Mitochondrial and sarcolemmal damage are proposed as critical factors in reperfusion injury.

Calcium displacement by lanthanum in subcellular compartments of rat ventricular myocytes: characterisation by electron probe microanalysis.

OBJECTIVE: Much of the calcium in cardiac myocytes resides in a kinetic compartment defined by rapid exchange and rapid displacement by La3+. The aim of this study was to ascertain the subcellular location of this calcium pool. METHODS: Electron probe microanalysis (EPMA) was used to measure redistribution of total calcium in rat cardiac myocytes after 30 s in 1 mM lanthanum. "Cells", mitochondria, and myofibrils were separately analysed. The data permitted calculating a third (difference) compartment, containing primarily sarcoplasmic reticulum, sarcolemma, and T tubule lumen. Total calcium levels in junctional sarcoplasmic reticulum and T tubule lumen were also measured directly. RESULTS: Lanthanum decreased total "cell" calcium from 1.4 (SEM 0.3) to 0.5(0.4) mmol.kg-1 dry weight (p < 0.05); loss from the third (difference) compartment was primarily responsible. Simultaneously, junctional sarcoplasmic reticulum increased from 5.1(0.6) to 8.2(0.9) mmol.kg-1 dry weight (p < 0.05), while the T tubule lumen was unchanged. CONCLUSIONS: These results suggest that lanthanum displaced calcium primarily from sarcolemmal sites and that calcium redistributed to sarcoplasmic reticulum from an intracellular source.

Subcellular electrolyte shifts during in vitro myocardial ischemia and reperfusion.

Isolated perfused rabbit right ventricular wall was studied with electron probe microanalysis (EPMA) under three conditions: 1) control (37 degrees C, 1.2 Hz), 2) 60 min global ischemia, and 3) ischemia plus 5 min of reperfusion. After 60 min of ischemia, only one cell population was evident; the variance of intracellular electrolyte concentrations was the same as in controls. When compared with controls, there was no change in Ca concentration within any region of the cell, but mitochondria were swollen with K-rich fluid. Two cell populations were evident after 5 min of reperfusion. The severely injured cells were markedly swollen, exhibited hypercontraction bands, and had electrolyte profiles similar to extracellular fluid. The moderately injured cells were normal in appearance, still retained electrolyte gradients, but had elevated Na and Cl concentrations in all compartments. Cell Ca did not increase in the moderately injured cells, but the region of the cell containing the sarcoplasmic reticulum (SR) lost 90% of its Ca. Accompanying this loss were large increases in myofibrillar and mitochondrial Ca concentration. It appears that release of SR Ca, loss of SR Ca-accumulating capacity, and increased intracellular Na are the principal electrolyte shifts in functional cells during early reperfusion.

Cellular compartmentation in ischemic myocardium: indirect analysis by electron probe.

Electron probe microanalysis (EPMA) was carried out directly on myocardial cells and on the myofibrils and the mitochondria within them. A third subcellular compartment, which contains sarcoplasmic reticulum (SR), was measured indirectly. The percent of the total cell calcium content that resides within this "hidden" compartment was calculated from cell data minus weighted myofibril and mitochondria data. This approach was applied to control, ischemic, and reperfused myocardium, and other elements were also quantified. We found that the calcium content of this third compartment is little changed during global ischemia but is markedly depleted after 5 min reperfusion. We conclude that these changes are ascribable to changes in SR function.

Electron probe x-ray microanalysis of sarcolemma and junctional sarcoplasmic reticulum in rabbit papillary muscles: low sodium-induced calcium alterations.

This project was undertaken to determine whether electron probe x-ray microanalysis (microprobe analysis) could be utilized to determine the subcellular sites responsible for low sodium-induced calcium accumulation in myocardium. Ultrathin cryosections of rabbit papillary muscles were analyzed using microprobe analysis, and the concentrations (mmol/kg dry wt) of Na, Mg, I, S, Cl, K, and Ca were compared against low sodium (36 mM) and control (139 mM NaCl) muscle groups. Visual resolution of junctional sarcoplasmic reticulum in freeze-dried myocardial sections was achieved, and systematic analysis of junctional sarcoplasmic reticulum and sarcolemma was performed. Myofibrils and mitochondria were also analyzed. Reductions in Na and Cl concentration were measured in virtually all compartments of muscles bathed in low sodium. In addition, low sodium produced a doubling of junctional sarcoplasmic reticulum and sarcolemmal calcium concentrations (p less than 0.01). No significant changes in calcium were observed at other analyzed sites. The increased calcium at the junctional sarcoplasmic reticulum and sarcolemma correlates with (but may not completely account for) the threefold increase in contractility measured after 40 minutes in low sodium concentrations. This work demonstrates that elemental changes associated with the myocardial junctional sarcoplasmic reticulum and sarcolemma are amenable to direct, in situ microprobe analysis and further defines these structures as primary sites of calcium accumulation in low sodium concentrations.

Origin of artifactual quantitation of electrolytes in microprobe analysis of frozen sections of erythrocytes.

Frozen sections of erythrocytes have been used to validate microprobe X-ray analysis of diffusible elements in biological samples. At this meeting last year we reported that intracellular Na concentrations measured by microprobe were much higher than those measured by bulk chemical methods. It was suggested that this might be due to the movement of extracellular material over the cells by microtomy. We now present evidence that such results are better explained by electron scattering within the sample during analysis. This evidence includes observations that the excess measured Na varies directly with section thickness and inversely with accelerating voltage. At 80 kV accelerating voltage there is excellent agreement between microprobe and chemical analysis. It may be concluded that specimen preparation techniques such as used in our laboratory are satisfactory for reliably localizing diffusible elements in small volumes, but that instrumental factors can seriously affect their quantitation.

Localization of Na/K-ATPase sites in the secretory and reabsorptive epithelia of perfused eccrine sweat glands: a question to the role of the enzyme in secretion.

Na/K-ATPase sites in both the secretory and reabsorptive epithelia of isolated and microperfused human eccrine sweat glands are localized cytologically. Localization was accomplished through autoradiography of bound 3H-ouabain, a specific inhibitor of the enzyme. Ouabain binding characteristics were determined to ensure maximum specific binding. Enzyme sites are localized only on the basolateral surface of both epithelia in spite of the fact that sodium transport is reversed, i.e., secretory: blood to lumen and reabsorptive: lumen to blood. In view of these findings and in comparison with other recent observations, the role of Na/K-ATPase in secretory electrolyte transport is questioned.

The ultrastructural route of fluid transport in rabbit gall bladder.

The route of fluid transport across the wall of the rabbit gall bladder has been examined by combined physiological and morphological techniques. Fluid transport was either made maximal or was inhibited by one of six physiological methods (metabolic inhibition with cyanide-iodoacetate, addition of ouabain, application of adverse osmotic gradients, low temperature, replacement of Cl by SO(4), or replacement of NaCl by sucrose). Then the organ was rapidly fixed and subsequently embedded, sectioned, and examined by light and electron microscopy. The structure of the gall bladder is presented with the aid of electron micrographs, and changes in structure are described and quantitated. The most significant morphological feature seems to be long, narrow, complex channels between adjacent epithelial cells; these spaces are closed by tight junctions at the luminal surface of the epithelium but are open at the basal surface. They are dilated when maximal fluid transport occurs, but are collapsed under all the conditions which inhibit transport. Additional observations and experiments make it possible to conclude that this dilation is the result of fluid transport through the spaces. Evidently NaCl is constantly pumped from the epithelial cells into the spaces, making them hypertonic, so that water follows osmotically. It is suggested that these spaces may represent a "standing-gradient flow system," in which osmotic equilibration takes place progressively along the length of a long channel.