1,25-Dihydroxy-vitamin D3 (calcitriol)-dependent protein phosphorylation in rat duodenum:: effects of ageing

We have examined the ability of 1,25(OH)(2)-vitamin D-3 [1,25(OH)(2)D-3: calcitriol]. the hormonal form of vitamin D-3, to stimulate the phosphorylation of proteins in rat duodenum from young (3 months) and aged (22-24 months) rats. Brief (30 s) exposure of duodenum preincubated with P-32-orthophosphate to the hormone increased the labeling of whole tissue proteins, an effect that was greatly diminished in aged animals. The response was dose-dependent, with maximal stimulation achieved at 1 nM calcitriol (+113% and +10% for young and aged rats, respectively). Phospho- proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by autoradiography. The hormone potentiated the phosphorylation predominantly on serine, threonine, and tyrosine residues of five acidic proteins of relative molecular masses of 66, 48, 45, 28, and 16 kDa. Moreover, the effects of calcitriol were exerted at the membrane level and varied as a function of exposure time. Direct treatment of purified basal lateral membranes fur 30 s with the hormone (1 nM) stimulated the incorporation of P-32 of a 66kDa protein by 75% and of a 48 and 45kDa proteins by 60%. The effects of the hormone on basal lateral membrane protein phosphorylation were suppressed by the PKA, PKC, and tyrosine kinase inhibitors, Rp-cAMPS, bisindolylmaleimide, and genistein, respectively. In basal lateral membrane isolated from old animals, only minor changes in calcitriol-induced protein phosphorylation of the 66-kDa protein were observed, Taken together, these results suggest that calcitriol modulates duodenal membrane protein phosphorylation, at least in part through PKA, PKC, and tyrosine kinases, and that this mechanism is severely altered with ageing. The identity of the proteins whose phosphorylation was stimulated by calcitriol and their physiological role is currently under investigation. (C) 1999 Elsevier Science Inc. All rights reserved.