Injection of bovine parathyroid poly(A)+ RNA into xenopus oocytes confers sensitivity to high extracellular calcium

Abstract

Parathyroid cells detect increments in the extracellular [Ca²⁺], which lead to substantial increases in intracellular free Ca²⁺ ([Ca²⁺]_i) and, ultimately, to suppression of parathyroid hormone (PTH) secretion. To determine whether mRNA from parathyroid tissue could confer sensitivity to high extracellular Ca²⁺, we isolated and injected total bovine parathyroid poly(A)⁺ RNA into Xenopus laevis oocytes. To assess translational activity of the RNA, PTH released into the media was measured. Intact PTH was detected in the medium for ≤48 h, and injection of increasing amounts of RNA (∼0.5–50 ng/oocyte) led to the release of greater quantities of PTH. We screened for the expression of a putative Ca²⁺ sensor molecule by measuring ⁴⁵Ca efflux from preloaded oocytes, in response to raising extracellular [Ca²⁺] from 0.7 to 5.7 mM. This increment in [Ca²⁺] stimulated ⁴⁵Ca efflux by 249 ± 52 cpm over 20 min from eggs injected with parathyroid poly(A)⁺ RNA (n = 22). This response was significantly greater than ⁴⁵Ca efflux from any group of controls exposed to the same change in extracellular Ca²⁺ (p < 0.02), including oocytes injected with either water, cRNA for the platelet-derived growth factor (PDGF) BB receptor, or T cell poly(A)⁺ RNA. Size-fractionation of poly(A)⁺ RNA over sucrose gradients demonstrated that mRNA, which induced responsiveness to high extracellular Ca²⁺, was present in fractions with transcripts of ˜5–9 kB. Injection of these fractions also conferred sensitivity to the presence of Ba²⁺ or Sr²⁺ (both at 5 mM) in the media. These findings establish that parathyroid cells express mRNA for a molecule capable of detecting changes in the extracellular divalent cation concentration. This molecule may play a role in secretory responses mediated by Ca²⁺ in the parathyroid.