Virchows Arch [Cell Pathol] (1982) 39:267-272 V/reho e/ B 9 Springer-Verlag 1982 Ultrastructural Alterations of the Mitochondrial ATPase in the Calcium Paradox as Revealed by Negative Staining P.M. Rahamathulla, M. Ashraf, S. Sato and John Benedict Department of Pathology and Laboratory Medicine, University of Cincinnati Medical Center, 231 Bethesda Avenue, Cincinnati, OH 45267, USA Summary. We have used a simple negative staining technique to study the structural alterations of mitochondria from biopsies of hearts subjected to the calcium paradox and treatment with diltiazem, a calcium channel blocker. A significant (P < 0.05) decrease in the number of spheres on the mitochondri- al membranes occurs during the calcium paradox (58.0_+ 4.1/lam vs. control 80.5___6.5). Treatment with diltiazem prevented the loss of spheres from mitochondrial membranes during the calcium paradox (75.5+_ 5.0 ~tm). We found that this negative staining technique can be used for quick assessment of the condition of mitochondria in biopsies from normal and pathological organs. Key words: Calcium paradox - Mitochondria - ATPase - Negative staining - Diltiazem - Calcium channel blocker - Electron microscopy Introduction The calcium paradox described first by Zimmerman and Hulsmann (1966) refers to the irreversible injuries occurring in the hearts that are reperfused with calci- um-containing medium after a perfusion with calcium-free solution. It is general- ly accepted that reintroduction of calcium into the cells previously perfused with calcium-free solution is accompanied by a deterioration of mitochondrial function and decline in tissue stores of adenosine triphosphate (Nayler 1981; Ashraf et al. 1982). An earlier study [Ashraf et al. (in press)] conducted in our laboratory on isolated rat hearts subjected to the calcium paradox have revealed a reduction in ATP level in the cardiac muscle, and this reduction in cardiac ATP was considerably reduced after treatment with a calcium channel blocker, diltiazem (DTZ). The morphological correlation of functional changes in the mitochondria are generally determined either by examination of thin section of the fixed tissue or by isolation of mitochondrial pellets. In thin sections, cell organelles are subject to several chemical treatments and may not represent in vivo configurations of the cellular components. In isolation Offprint requests to : M. Ashrafat the above address 0340-6075/82/0039/0267/$01.60