Aluminum inhibits calpain-mediated proteolysis and induces human neurofilament proteins to form protease-resistant high molecular weight complexes.

We studied the effects of aluminum salts on the degradation of human neurofilament subunits (NF-H, NF-M, and NF-L, the high, middle, and low molecular weight subunits, respectively) and other cytoskeletal proteins using calcium-activated neutral proteinase (calpain) purified from human brain. Calpain-mediated proteolysis of NF-L, tubulin, and glial fibrillary acidic protein (GFAP), three substrates that displayed constant digestion rates in vitro, was inhibited by AlCl3 (IC50 = 200 microM) and by aluminum lactate (IC50 = 400 microM). Aluminum salts inhibited proteolysis principally by affecting the substrates directly. After exposure to 400 microM aluminum lactate and removal of unbound aluminum, human cytoskeletal proteins were degraded two- to threefold more slowly by calpain. When cytoskeleton preparations were exposed to aluminum salt concentrations of 100 microM or higher, proportions of NF-M and NF-H formed urea-insoluble complexes of high apparent molecular mass, which were also resistant to proteolysis by calpain. Complexes of tubulin and of GFAP were not observed under the same conditions. Aluminum salts irreversibly inactivated calpain but only at high aluminum concentrations (IC50 = 1.2 and 2.1 mM for aluminum lactate and AlCl3, respectively), although longer exposure to the ion reduced by twofold the levels required for protease inhibition. These interactions of aluminum with neurofilament proteins and the effects on proteolysis suggest possible mechanisms for the impaired axoplasmic transport of neurofilaments and their accumulation in neuronal perikarya after aluminum administration in vivo.

Extra-adrenal chromaffin cells grown in tissue culture.

1. Extra-adrenal chromaffin cells from adult frogs were grown in tissue culture and their morphology and behaviour observed with both light and electron microscopy. 2. Two types of chromaffin cells were distinguished: Type A cells contain large, electron dense vesicles (2000-6000 A) and are equated to Type I chromaffin cells seen in vivo, i.e. they contain noradrenaline; Type B cells contain smaller vesicles (700-2000 A) which are incompletely filled with an electron dense material and are equated to Type III chromaffin cells seen in vivo, i.e. cells depleted of their catecholamines by stimulation. No cells comparable to Types II and IV cells in vivo were seen. 3. Close associations between the cultured chromaffin cells and sympathetic neurons were observed with the light microscope, but no examples of synaptic structures were seen in the material examined with electron microscopy in this study.