Histochemical demonstration of an ATP-dependent Ca2+-pump in bullfrog myocardial cells

In the present investigation different finestructural and histochemical procedures were employed to demonstrate normal morphology and Ca2+-transport mechanism in bullfrog atrial myocardium. For normal morphology specimens were fixed in 1% OsO4, 2.5% glutaraldehyde or liquid propane at -185 degrees C before they were prepared for conventional embedding and freeze-etching, respectively. Special interest was focussed on the caveolae system, which is composed of single, spherical membrane invaginations (diameter, 85 nm), randomly distributed at the entire cell periphery. The caveolae enlarge the cell surface by 24.5% and occupy 8.5% of the cellular volume. The caveolae membrane contains a few intramembraneous particles with a diameter of 8.4 nm comparable to the size of ATPases as found in other cells. For histochemistry the specimens were first stabilized by treatment with 50% glycerol, 0.025% glutaraldehyde or 0.15% formaldehyde and then incubated in a medium containing 4 mM CaCl2, 4 mM EGTA, 5 mM MgCl2, 5 mM K2C2O4, 5 mM ATP and 20 mM histidine at pH 7.0. This incubation always succeeded in the formation of electron-dense deposits with elliptical shape, measuring 100-200 nm in length and 10-30 nm in diameter. According to X-ray spectra they deliver a characteristic calcium-peak and can be found within two different cellular compartments: in small invaginations of the sarcolemma, i.e. the caveolae-system, and in the intrafibrillar sarcoplasmic reticulum. The elliptical deposits can be clearly distinguished from round electron-dense granules measuring 16 nm in diameter, which are located within randomly distributed small vesicles and composed of potassium and phosphate. Contrary to the elliptical deposits the round granules are also present in controls and seem to be identical with the so-called atrial granules. In comparison to observations obtained with the same method in other muscular systems and derived from various control experiments the results of this study favour the existence of an ATP-dependent Ca2+-pumping mechanism in frog atrial muscle bound to both, the sarcoplasmic reticulum and the caveolae system.