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Effect of Growth and Maturation on Membrane-Initiated Actions of 1,25-Dihydroxyvitamin D₃. I. Calcium Transport, Receptor Kinetics, and Signal Transduction in Intestine of Male Chickens

Department of Nutrition and Food Sciences and Biotechnology and Genomic Research Center, Utah State University, Logan, Utah 84322-8700

Address all correspondence and requests for reprints to: Dr. Ilka Nemere, Department of Nutrition and Food Sciences, Utah State University, Logan, Utah 84322-8700. E-mail: nemere{at}cc.usu.edu.

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 D₃ [1,25-(OH)₂D₃] in duodena from 7-, 14-, 28-, and 58-wk-old birds. Measurements of 1,25-(OH)₂D₃ (130 pM) responsiveness as a function of age, showed a decreased intestinal Ca²⁺ 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 [³H]1,25-(OH)₂D₃ 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 [³H]1,25-(OH)₂D₃ 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)₂D₃ was assessed by determination of protein kinase C and A activities. Stimulation of protein kinase C activity in response to 1,25-(OH)₂D₃ 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)₂D₃ as a function of age in duodena of male chickens, which can be correlated to a decreased affinity for 1,25-(OH)₂D₃, a reduced expression of membrane-associated rapid response binding protein, and a decreased protein kinase C activity.

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