Studies on the Mechanism of Action of Calciferol

Abstract

Abstract Intestinal transport or flux, J, of calcium was studied from both the mucosal to serosal sides and from the serosal to mucosal sides in ileal tissue obtained from vitamin D-treated (+D) and -deficient (-D) chicks. It was found that intestinal calcium transport in the +D ileum was an active, cation-oriented process, while calcium was not actively transported in the -D chick. Other parameters which are obligatorily associated with the +D calcium transport system are (a) sensitivity to cold, (b) inhibition by N-ethylmaleimide, and (c) an effective calcium carrier at the microvilli side of the cell. The polyene antibiotic, filipin, was found to be a specific tool with which to study calcium transport in vitro in a -D ileum. Filipin, at concentrations of 10 µg per ml (approximately 1.7 x 10-5 m), stimulates the calcium flux, Jms, of -D ileal tissue by 150 to 200%, but has little or no stimulatory effect on Jms of +D ileal tissue. The effect of filipin is specific for calcium as compared to Rb+, sulfate, phosphate, serine, thiourea, and water. The incubation of filipin in vitro with -D ileal tissue mimicked many aspects of dietary vitamin D administration. Filipin when placed in the solution bathing only the microvilli side of the intestinal cell (a) induces an active translocation of calcium against an electrochemical gradient; (b) confers the property of cold sensitivity on the increased Jms flux; (c) confers sensitivity to N-ethylmaleimide on the increased Jms flux; (d) interacts with the membrane-bound carrier on the microvilli side of the cell to effect an increased uptake of calcium into the cell; and (e) binds more rapidly to intact ileal segments obtained from -D compared to +D chicks. Filipin was shown not to be an ionophoretic compound for calcium, so that the enhanced calcium translocation cannot be ascribed to a direct interaction of filipin and calcium. The sum of this and other evidence suggests that filipin is apparently able to effect a structural reorganization of the -D microvilli membrane so that a previously inactive calcium transport system becomes active. The consequences of this direct filipin-mediated reorganization are remarkably similar to the indirect actinomycin D-sensitive effects brought about by dietary vitamin D administration. A plausible hypothesis for the mechanism of action of vitamin D is that its administration to a vitamin D-deficient animal may cause a structural reorganization of the microvilli or brush border membrane by a process of induction of the synthesis de novo of proteins which are active in either a catalytic or structural role in the mucosal brush border membrane to effect efficient translocation of calcium.