Vitamin D receptor mRNA measured in leukocytes with the TaqMan fluorogenic detection system: effect of calcitriol administration.

BACKGROUND: The aim of the present study was to investigate the interactions between the circulating concentrations of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and the mRNA concentration of its specific nuclear receptor in human leukocytes. METHODS: We measured vitamin D receptor (VDR) mRNA extracted from leukocytes by use of TaqMan fluorescence analysis applied to the reverse transcription-PCR (RT-PCR) technique in 16 volunteers before and after calcitriol administration. VDR mRNA was also measured in leukocytes from calcium-stone-formers (37 hypercalciuric and 34 normocalciuric patients). The relationship between VDR mRNA concentrations and genetic VDR polymorphisms was analyzed in these patients. RESULTS: Imprecision (CV) of RT-PCR was 1.3% within assay (n = 10) and 1.7% between assays (n = 4). Oral 1,25(OH)2D3 increased mean (SE) serum 1,25(OH)2D3 1.6 (0.3)-fold and VDR mRNA 1.6 (0.1)-fold 8 h after administration. The maximum VDR mRNA was reached 3.6 (1.3) h after 1,25(OH)2D3 ingestion. No differences in leukocyte VDR mRNA concentrations were found between normocalciuric and hypercalciuric stone-formers in the absence of stimulation. Finally, no association was found between VDR mRNA concentrations and genetic VDR polymorphisms in stone-formers. CONCLUSIONS: The TaqMan RT-PCR assay is a rapid and accurate method to measure VDR mRNA, and leukocytes are a useful model to study VDR and 1,25(OH)2D3 interactions. In humans, VDR mRNA is increased by agonist 1,25(OH)2D3, a finding resembling previously reported results obtained in cellular and animal models.

Intestinal calcium absorption is associated with bone mass in stone-forming women with idiopathic hypercalciuria.

BACKGROUND: Patients with idiopathic hypercalciuria are predisposed to osteoporosis despite their high enteral calcium absorption. Conversely, low calcium absorption has been reported in patients with osteoporosis. Because bone loss occurs earlier in women, this work explores the relationship between bone mineral density (BMD) and calcium absorption in premenopausal and postmenopausal hypercalciuric stone-forming women. METHODS: BMD and intestinal calcium absorption were compared in 64 hypercalciuric and 42 normocalciuric calcium stone-forming women. Calcium absorption was assessed by using strontium as a surrogate marker for calcium. Strontium was administered to patients as an oral load, then measured in blood to calculate absorption after 60 minutes. Femoral and lumbar-spine BMD were measured by dual-energy x-ray absorptiometry. RESULTS: Strontium absorption was significantly increased in hypercalciuric stone formers, whereas BMD z score was decreased in hypercalciuric patients at the lumbar spine, but not the femur. The increase in strontium absorption was detected in both postmenopausal (n = 29) and premenopausal (n = 35) hypercalciuric patients. The decrease in lumbar-spine BMD was confirmed in postmenopausal, but not premenopausal, hypercalciuric patients. Strontium absorption was greater in hypercalciuric patients with a lumbar-spine BMD z score of -2 or less (n = 10) than in those with a score greater than -2 (n = 54). Multiple stepwise regression showed that lumbar-spine BMD was related negatively to intestinal strontium absorption and age in hypercalciuric patients. CONCLUSION: Results of the strontium absorption test suggest that the increase in calcium absorption is associated with a decrease in lumbar-spine BMD in hypercalciuric stone-forming women. Hypercalciuric stone-forming women with high calcium intestinal absorption denote a group of patients predisposed to loss of bone mass.

Influence of calcium-sensing receptor gene on urinary calcium excretion in stone-forming patients.

Calcium-sensing receptor (CaSR) is a plasma membrane protein that regulates tubular reabsorption of Ca. To establish its role in idiopathic hypercalciuria, the association of urinary Ca excretion with the polymorphisms of CASR gene has been studied in healthy subjects and in hypercalciuric and normocalciuric Ca stone formers. CASR exon 7 single nucleotide polymorphisms (SNP), G/T at codon 986, G/A at codon 990, and C/G at codon 1011, were evaluated by PCR amplification and direct sequencing in 97 normocalciuric stone formers, 134 hypercalciuric stone formers, and 101 normocalciuric healthy controls. Four haplotypes were defined on the basis of CASR gene SNP: haplotype 1 was characterized by the most frequent sequence; haplotypes 2, 3, or 4 by the presence of a single polymorphic variant at codon 986, 990, or 1011, respectively. The relative risk of hypercalciuria was calculated with multinomial logistic regression and was significantly increased only in individuals carrying haplotype 3 (Odds ratio, 13.0 [95% confidence interval, 1.7 to 99.4]). Accordingly, Ca excretion was higher in subjects bearing haplotype 3, whereas those bearing haplotype 2 showed a slight increase of plasma Ca concentration. Multiple regression analysis showed that haplotype 3 explained 4.1% of the total variance of Ca excretion and 12.6% of the variance explained by the variables considered in the study. In conclusion, CASR gene could be a component of the complex genetic background regulating Ca excretion. Arg990Gly polymorphism could facilitate activation of CaSR and increase Ca excretion and susceptibility to idiopathic hypercalciuria.

Arachidonic acid increases intracellular calcium in erythrocytes.

Recently, we have measured in erythrocytes a voltage-modulated and dihydropyridine-inhibited calcium influx. Since arachidonic acid and other polyunsaturated fatty acids influence the activities of most ion channels, we studied their effects on the erythrocyte Ca(2+) influx. It was measured on fresh erythrocytes, isolated from healthy donors, using the fluorescent dye Fura 2 as indicator of [Ca(2+)](i). AA (5-50 microM) and EPA (20-30 microM) stimulated a concentration-dependent increase in [Ca(2+)](i), deriving from extracellular calcium (1 mM), without affecting the intra- and extracellular pH and membrane voltage. The Ca(2+) influx rate varied from 0.5 to 3 nM Ca(2+)/s in the presence of AA and from 0.9 to 1.7 nM Ca(2+)/s with EPA. The Ca(2+) influx elicited by AA and EPA was not inhibited by dihydropyridines, while cyclooxygenase inhibitors were effective and PGE1 or PGE2 did not produce any effect. We conclude that AA could activate an erythrocyte voltage-independent Ca(2+) transport via an intermediate product of cyclooxygenase pathway; however, a direct interaction with the membrane lipid-protein cannot be excluded.