Vitamin D and adaptation to dietary calcium and phosphate deficiencies increase intestinal plasma membrane calcium pump gene expression

Abstract

The effect of vitamin D and other variables on the synthesis of the chicken intestinal plasma membrane calcium pump (PMCA) mRNA was assessed. 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 fragment of the PCR product was cloned into the pBluescript II 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-deficient 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.