Development of a technique for introduction of an expressed complementary deoxyribonucleic acid into parathyroid cells by direct injection.

PTH is a major mediator of bone and mineral metabolism. However, physiological and pathological investigations of parathyroid cells (PTCs) have been limited because of the lack of available cell lines and because the organ is too small for detailed studies. Here, we describe a novel method for adenovirus-mediated cDNA transfer into PTCs, and we show the accuracy of the method in a rat model of uremia-induced secondary hyperparathyroidism. Rats underwent a 5/6-nephrectomy and were fed with a high-phosphate diet for 8 wk. The parathyroid glands were surgically exposed and adenoviruses containing LacZ or Ca-sensing receptor (CaSR) were directly injected into the glands under a zoom-stereo microscope. The parathyroid glands were analyzed for infection of adenovirus and immunohistochemically for expression of CaSR. The functional activity of exogenous CaSR in PTCs after this treatment was investigated based on changes of the calcium and PTH curve. A virus concentration of more than 10(9) plaque-forming units/ml was required for adequate infection of PTCs within 7 d after treatment. Marked increase of CaSR-positive PTCs by 2.39 +/- 0.72 times relative to control treatment, and significant colocalization of CaSR overexpression and virus labeling, were observed in glands after gene introduction. The calcium and PTH curve was shifted to the left from the basal position (set point, 1.10 +/- 0.09 to 0.76 +/- 0.12 mm; P < 0.0001), indicating successful introduction of a functionally active cDNA into the PTCs. This technique may facilitate an elucidation of biological effects through targeting and identification of specific features of PTCs, which may provide the basis for new clinical approaches.

Moduladores alostéricos del receptor sensible al calcio e hiperparatiroidismo primario / Allosteric modulators of the calcium-sensing receptor and primary hyperparathyroidism

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Regulation of parathyroid hypertensive factor secretion by Ca2+ in spontaneously hypertensive rat parathyroid cells.

BACKGROUND: Elevated parathyroid hypertensive factor (PHF) has been suggested to play a causal role in the pathogenesis of hypertension. Previous studies have indicated that PHF secretion is stimulated by low extracellular (EC) Ca2+. Therefore, we hypothesized that the calcium-sensing receptor (CaR) is involved in regulation of PHF release. METHODS: Parathyroid gland (PTG) organ and cell cultures derived from spontaneously hypertensive rats (SHR) or Wistar-Kyoto (WKY) rats were exposed to low and normal EC Ca2+ and PHF release measured by ELISA. Expression of CaR protein was assessed by Western blot. RESULTS: Low EC Ca2+ stimulated both SHR and WKY PTG organ cultures to secrete more PHF, first observable after 60 min incubation. After 4 h, PHF secretion was stimulated (66-fold v 24-fold stimulation for SHR and WKY, respectively). Cultured SHR and WKY parathyroid cells were also stimulated, but to a lesser extent (2.63-fold v 3.75-fold stimulation for SHR and WKY respectively). After 24 h the stimulation by low EC Ca2+ was no longer apparent. Expression of CaR is elevated in the SHR relative to WKY PTG. In both strains expression is higher under conditions of normal (1.5 mmol/L) EC Ca2+ and it increases with incubation time. The apparent suppression of PHF release by normal (1.5 mmol/L) EC Ca2+ is blocked by pre-exposure of the PTG cells to anti-CaR antibody. CONCLUSIONS: Low EC Ca2+ stimulated rapid PHF release from both SHR and WKY PTG. Changes in CaR expression may account for different sensitivity to EC Ca2+ of the two strains and over time.