Effect of growth and maturation on membrane-initiated actions of 1,25-dihydroxyvitamin D(3). I. Calcium transport, receptor kinetics, and signal transduction in intestine of male chickens.

Abstract

To study the physiological relevance of membrane-initiated steroid signaling, we investigated the correlation of age in male chickens with the magnitude of responses to 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] in duodena from 7-, 14-, 28-, and 58-wk-old birds. Measurements of 1,25-(OH)(2)D(3) (130 pM) responsiveness as a function of age, showed a decreased intestinal Ca(2+) transport. Western analyses of isolated basal lateral membranes indicated a decreased expression of the membrane-associated rapid response binding protein with increasing age. Saturation analyses of [(3)H]1,25-(OH)(2)D(3) binding to basal lateral membranes, revealed an allosteric interaction identified as cooperative binding. A significant increase in K(d) was observed with increasing age, indicating decreasing affinity. Determinations of the number of binding sites yielded a binding capacity of 190-250 fmol/mg protein during growth and maturation, whereas in adulthood (58 wk) saturable binding was no longer observed. Data obtained in parallel analyses of binding of [(3)H]1,25-(OH)(2)D(3) to nuclear fraction vitamin D receptor, in contrast, indicated an absence of cooperative binding and an absence of significant changes in K(d) or binding capacity with age. Membrane-initiated signal transduction by 1,25-(OH)(2)D(3) was assessed by determination of protein kinase C and A activities. Stimulation of protein kinase C activity in response to 1,25-(OH)(2)D(3) decreased with age, whereas no age-correlated changes in steroid-stimulated protein kinase A activities were observed. Thus, in conclusion, our experiments demonstrate that there is a decrease in responsiveness to exogenous 1,25-(OH)(2)D(3) as a function of age in duodena of male chickens, which can be correlated to a decreased affinity for 1,25-(OH)(2)D(3), a reduced expression of membrane-associated rapid response binding protein, and a decreased protein kinase C activity.