Ca2+-mediated degradation of central nervous system (CNS) proteins: topographic and species variation.

Incubation of homogenates of rat, rabbit, and bovine spinal cord and of bovine brain white and gray matter in the presence of calcium (5 mM) produced an extensive degradation of the neurofilament triplet proteins (NFP; 200 K, 150 K, and 69 K). The breakdown products of the NFPs were identified by immunoblot. The glial fibrillary acidic protein (GFAP), microtubular proteins (MTP), and myelin proteins were also degraded. The 150 K NFP was more susceptible than the other NFPs. The extent of calcium-mediated degradation was slightly greater with rat spinal cord than the others. Bovine brain white matter had more activity than gray, which had no appreciable degradative activity. The breakdown was prevented by both EGTA and leupeptin but a similar concentration of MgCl2 (5 mM) had no effect. These results suggest that NFPs are degraded by a Ca2+-activated neutral proteinase in the central nervous system (CNS) of several species. The lesser activity in gray matter suggests that the enzyme is enriched in axons, myelin, and/or oligodendroglial cells.

Tissue calcium levels in CaCl2-induced myelopathy.

CaCl2-induced myelopathy was produced in rats by the application of a solution of CaCl2 to exposed leptomeninges of lumbosacral spinal cord. Equal lengths of remote cervical and lumbar cord were removed at intervals following Ca2+ application. The total Ca2+ in tissue from lumbar cord was significantly elevated over autologous cervical cord and homologous lumber controls after after 2 h. The maximum Ca2+ increase in lumbar cord was 3.9-fold that of homogolous control and was reached by 8 h post Ca2+ application. The time course for the elevation of Ca2+ as measured by atomic absorption spectrophotometry, resembles that found in spinal cord following direct physical trauma. These findings as well as similar changes in morphology and neural proteins from previous studies suggest that Ca2+ is involved in and may potentiate the degeneration of axons and myelin via Ca2+-activated neutral proteinases.

Distribution of mitochondrial calcium: pyroantimonate precipitation and atomic absorption spectroscopy.

Mitochondrial function is highly dependent upon the concentrations of cations within the various compartments of this organelle. Utilizing variants of the pyroantimonate precipitation method, we have undertaken to localize cations, particularly calcium, in isolated mitochondria. Precipitated cations were present in two major compartments: 1) within the intracristal space and 2) in the matrix, frequently in association with the matrix face of cristal membranes. Precipitated cations were abundant in mitochondrial samples fixed with most pyroantimonate-containing fixatives, but were usually absent in samples fixed in phosphate-buffered, pyroantimonate-containing fixative solutions. Atomic absorption spectroscopy demonstrated that Ca2+ added to mitochondrial suspensions prior to fixation was retained in the mitochondrial pellet if 0.1 M collidine was present as the fixative buffer. When the same concentration of phosphate-buffered pyroantimonate-osmium fixative was employed, however, much of the added Ca2+ was lost to the supernatant. The suborganellar localization of cations was influenced by the presence of added respiratory substrate. Incubation of mitochondria in an isoosmotic buffered sucrose medium without succinate favored localization of precipitates in the intracristal space of the outer compartment. In the presence of succinate, the localization of precipitates was shifted toward the matrix compartment. This redistribution was particularly obvious in samples to which Ca2+ had been added and was blocked by the addition of antimycin. The observation of precipitated cations in the outer mitochondrial compartment supports previous evidence for a calcium-binding protein in this compartment.

Ca2+-accumulation in experimental spinal cord trauma.

Quantitative measurements of the time course of calcium levels in experimental spinal cord trauma have been made. The injury was produced in rats by dropping a 10 g weight from 30 cm upon exposed dura-invested spinal cord. Lumbar sections of traumatized spinal cord and internal controls from remote cervical cord were excised and analyzed for calcium using atomic absorption spectroscopy. Total calcium levels in the lesioned cord were significantly elevated over control values within 45 min post-trauma (P less than 0.005), with maximal increase at 8 h. The increased levels of calcium in the lesion tissue confirm the previous morphologic finding of calcium deposits within axons in the lesion.