Scintillation Autoradiographic Localization of 1,25 Dihydroxyvitamin D3in Chick Intestine*

Abstract

The intracellular binding site of 1,25-dihydroxy-vitamin D3 [1,25(OH)2D3] was determined via preliminary biochemical analysis of radioactive 1,25-(OH)2D3 association with various chick tissues and then by direct autoradiographic visualization. When vitamin D-deficient chicks were injected intracardially with physiological doses of [3H]-1,25(OH)2D3 and killed 2 h later, 2-3 times more radioactivity was found in the intestinal mucosa than was present in equal weights of pancreas, parathyroid, or liver tissue. Very little 3H was found in muscle tissue. The intestinally localized radioactivity was predominantly associated with the nuclear chromatin fraction and binding of 1,25(OH)2[3H]D3 to the nucleus was maximal 2 h after injection and at a dose of at least 0.52 nmol. Using this dose and time period, autoradiographic studies were done on duodenum and thoracic muscle of rachitic chicks injected with radioactive 1,25(OH)2D3 (11.2 Ci/mol). Thin sections of tissue were prepared for thaw and dry mount scintillation autoradiography as well as simple dip-coating autoradiography. After exposure for 4-6 mo., a preferential concentration and retention of 3H-1,25(OH)2D3 was evident in the nuclei of intestinal villi and in the crypt of Lieberkuehn cells when each of the autoradiographic techniques was utilized. Quantitation of the labeled hormone by counting the number of nuclear and cytoplasmic silver grains in target intestinal tissue per 500 .mu.m2 area confirmed the significant nuclear accumulation in both villi and crypt cells. No such nuclear concentration of Ag grains was observed in thoracic muscle cells and the intestinal localization was abolished when a 100-fold excess of unlabeled 1,25(OH)2D3 was injected simultaneously with the radioactive hormone. 1,25(OH)2D3 appears to be bound in a tissue-selective fashion to a high affinity, low capacity site within the nucleus of its intestinal target organ.