Regulation of phosphatidylinositide transduction system in the rat spinal cord during aging.

Abstract

Age-related functional alterations in a variety of neurotransmitter systems result in modulation of interneuronal communications which has some relevance in neurological deficits observed in the aging process. The synergistic interactions between protein kinase and inositol 1,4,5-trisphosphate (insP3)/Ca2+ pathways underlie a variety of cellular responses to external stimuli. To determine whether age-dependent changes occur in the regulation of protein kinase C and inositol 1,4,5-trisphosphate/Ca2+ pathways, insP3 contents as a marker for the release of intracellular calcium, saturation binding analysis of Ins P3 receptor using [3H]inositol 1,4,5-trisphosphate, slot/northern blot analysis of Ins P3 receptor-encoding mRNA transcripts, and the activities of Ca2+/phospholipid-dependent protein kinase C isozymes were investigated in the rat spinal cord. Inositol 1,4,5-trisphosphate content and [3H]inositol 1,4,5-trisphosphate binding site density (Bmax) were quantified in the spinal cords of young (three months old), adult (12 months old) and senescent (25 months old) male Fischer 344 rats. Spinal cord content of inositol 1,4,5-trisphosphate was increased (P < 0.01) in the 25-month old compared to the three- and 12-month old animals. The density of Ins P3 receptor in particulate membranes derived from the 25-month old rats was reduced (P < or = 0.01), but the binding affinity (Kd) was increased (P < or = 0.04) by a factor of 2.2 and 3.2 at 25 months of age when compared with three- and 12-month old animals, respectively. Young and middle-aged animals showed no differences in both inositol 1,4,5-trisphosphate contents and [3H]inositol 1,4,5-trisphosphate binding site density. The quantity of Ins P3 receptor mRNA was significantly increased with age in the order 25 >> 12 > 3 months of age. Total functional cytosolic and membrane-associated PKC activities were decreased (P < or = 0.05) in the 25-month compared to the three- and 12-month old rats in which activity remained unchanged. Total membrane/cytosolic activity ratios were unchanged by the aging process. In all cases, the activities of membrane-associated conventional protein kinase C isozymes (alpha, beta and gamma), determined by immunoprecipitation followed by in situ quantification of protein kinase C activities in the immunoprecipitates, showed age-dependent decline. The activities of protein kinase C-alpha and beta were significantly decreased in age-related manner. However, the activity of the gamma-isozyme was not significantly changed at 12- and 25-months of age, although it was higher (P < or = 0.03) in young rats. Western blot analyses using affinity purified polyclonal antibodies specific for each isozyme indicated a single protein with an apparent molecular mass of approximately 80 x 10(3) molec. weight for all isozymes except for the beta isozyme that also had an appreciable immunoreactive band at approximately 36 x 10(3) molec. weight. Overall, the aging process did not affect the electropheretic mobility of each isozyme. With decreased protein kinase C activity, the present data suggest that the aging process would decrease protein kinase C-induced phosphorylation of membrane proteins including Ins P3 receptor. A significant change in Ins P3 receptor affinity combined with increased levels of Ins P3 receptor mRNA-encoding transcripts in senescent rats suggests not only a modification (possibly by phosphorylation) of Ins P3 receptor protein but also the existence of multiple (spliced) variants of Ins P3 receptor in spinal neurons with increasing age. The present data indicate that the spinal contents of inositol 1,4,5-trisphosphate increased with age, but with decreased efficacy and number of inositol 1,4,5-trisphosphate-activatable Ca2+ channels in the spinal cord of senescent rats. These age-related changes may contribute to the attenuated responsiveness of spinal cord neurons by phosphoinositide-coupled receptors during the aging process.