The effect of vitamin D and other variables on the synthesis of the chicken intestinal plasma membrane calcium pump (PMCA) mRNA was assesed. The DNA probe for Northern analysis was obtained by reverse transcription and PCR with intestinal poly(A)+ RNA, using two 20-mer oligonucleotide primers homologous to the 3' coding region of the human teratoma PMCA. An EcoRI restriction fragent of the PCR product was cloned into the pBluescript I KS(-) phagemid vector, and the chimeric plasmid was used to transform Escherichia coli. The amino acid sequence deduced from the nucleotide DNA sequence of the PCR product and the cloned DNA were 96% homologous with the teratoma sequence. Northern blots of intestinal poly(A)+ RNA with 32P-labeled DNA showed the presence of three major species of chicken PMCA mRNAs at about 6.6, 5.4, and 4.5 kb. Northern analysis with the chicken PMCA DNA indicated that repletion of vitamin D-deficient chickens with vitamin D increased PMCA mRNAs in the duodenum, jejunum, ileum, and colon. After injection of 1,25-dihydroxyvitamin D3 intravenously into vitamin D-deflcient chickens, duodenal PMCA mRNA tended to increase by 2 hr, reached a maximum at about 16 hr, and returned to baseline levels at 48 hr. Adaptation of chickens to either a calcium-or phosphorus-deficient diet resulted in a 2-to 3-fold increase in duodenal PMCA mRNA. These results indicate that vitamin D and specific variables that affect calcium absorption through the vitamin D-endocrine system increase intestinal PMCA gene expression.A major factor controlling the efficiency of calcium absorption is the vitamin D hormone, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] (1, 2). 1,25(OH)2D3 elicits both genomic (3) and nongenomic (4-10) effects on the intestinal cell. Among the former effects are the induction of the synthesis of intestinal calbindin-D9k in mammalian species and intestinal calbindin-D28k in avian species (3). In addition, we recently demonstrated another apparent genomic response, a vitamin D-dependent increase in the amount ofcalcium pump units in the basolateral membrane of the avian enterocyte (11). This finding is consistent with previous observations (12)(13)(14)(15)(16) showing that repletion of vitamin D-deficient animals with vitamin D or 1,25(OH)2D3 results in an enhancement of the ATP-dependent uptake of Ca2+ by isolated basolateral membrane vesicles. Further, animals fed diets deficient in calcium or phosphorus adapt to these deficiencies by an increase in the synthesis of 1,25(0H)2D3 (17)(18)(19)(20)(21), resulting in an increase in the efficiency of calcium and phosphorus absorption. These adaptations are associated with an increased synthesis of calbindin-D and its mRNA (17)(18)(19)(20)(22)(23)(24)(25)(26)(27)(28)
The intestinal absorption of calcium has been proposed to occur by the transcellular transfer of Ca2+ through the enterocyte proper and between the cells of the intestinal epithelium, i.e., the paracellular path. Attention in this report is given to the transcellular models of Ca2+ absorption and, more specifically, the Ca2+ extrusion events occurring at the basolateral membrane. These extrusion processes include the operation of an ATP-dependent Ca2+ pump and a Na+/Ca2+ exchanger, as well as exocytosis as the terminal event in a proposed vesicular transport mechanism. Evidence for the presence of an ATP-dependent Ca2+ pump at the basolateral membrane is documented and illustrated with biochemical and immunological data from studies on the avian intestinal basolateral membrane. As shown immunohistochemically, the Ca2+ pump was primarily localized on the enterocyte basolateral membrane. The ATP-dependency and vitamin D enhancement of Ca2+ uptake by isolated basolateral membrane vesicles are shown. Western blot analysis of intestinal mucosa, by using a monoclonal antibody produced against the erythrocyte Ca2+ pump, indicated that the number of pump units is increased by 1,25-dihydroxycholecalciferol. The possible involvement of calbindin-D28K as a direct stimulator of the Ca2+ pump is discussed, and the quantitative relationship between Ca2+ transport rates and Ca2+ pumping activity has been estimated. Information related to the basolateral membrane Na+/Ca2+ exchanger and the vesicular transport model of Ca2+ absorption is also briefly reviewed.
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