Abstract

We have used the fluorescent probe fura-2 to perform agonist studies of the receptor(s) that mobilizes Ca²⁺ ions in response to extracellular ATP in human parathyroid cells. Extracellular ATP induced Ca²⁺ responses in both normal and adenomatous parathyroid cells. Activation resulted in an initial small transient response during which Ca²⁺ ions were released from intracellular stores, followed by a prominent plateau response during which Ca²⁺ ions entered the cells from the extracellular fluid. The responses exhibited moderate desensitization upon repeated stimulation with ATP, and the ratio of the plateau to the peak response remained constant for any given group of activated cells. The baseline intracellular calcium concentration was 100 ± 4·3 nm (mean ± s.e.m., n=3). Following maximal activation by extracellular ATP it rose to a peak of 684 ± 45·7 nm (n=3) and a plateau level of 415 ± 9·9 nm (n=3).

We examined the effects of a variety of nucleotide species. The order of potency was: adenosine, AMP<α,β-methylene ATP<ADP<ATP≈UTP. In the concentration range 1–1000 μm, UTP (the concentration of agonist inducing a half-maximal response, EC₅₀=2·4 μm) was slightly more potent than ATP EC₅₀=3·6 μm), and the two nucleotides evoked similar maximal responses. In the concentration range 0·01–1·0 μm, however, there was a clear difference in the behaviour of the two nucleotides. In particular, ATP, but not UTP, evoked responses that suggested the presence of a second receptor of higher potency but markedly lower efficacy. Responses to 2-methylthioATP (2-MeSATP) in the concentration range 0·01–100 μm defined a dose–response curve that matched the lower concentration component of the ATP dose–response curve.

Together, these data suggest that two receptor species are involved in the Ca²⁺ responses. One is a nucleotide/P_2U receptor that responds to ATP and UTP, which are approximately equipotent agonists; 2-MeSATP is only a weak agonist with respect to this receptor. The other belongs to the P_2Y receptor subclass. It responds to 2-MeSATP, a selective agonist at P_2Y receptors, and to ATP, but not to the pyrimidine nucleotide UTP.

Journal of Endocrinology (1994) 142, 277–283

Fura-2-loaded human cytotrophoblasts responded to elevated extracellular Ca2+ concentration ([Ca2+]o) with monophasic or, in the case of large (> 20 microns) extravillous cells, biphasic elevations in intracellular free Ca2+ ion concentration ([Ca2+]i) that returned to baseline levels after restoration of control [Ca2+]o. Large extravillous cytotrophoblasts also responded to elevated [Mg2+]o with transient elevations in [Ca2+]i, consistent with the behaviour of the parathyroid Ca2(+)-sensing receptor. Expression of the parathyroid Ca2(+)-sensing receptor in placental cells was confirmed using Northern blot and reverse transcription (RT)-PCR analysis. However, the major transcript in human placental cells (6.2 kb) differed from that expressed by human parathyroid cells (5.6 kb). RT-PCR analysis and DNA sequencing of key PCR products also revealed the presence of a splice variant in placental and parathyroid cells that lacks exon 3.