Extracellular pyrimidines have been demonstrated as being responsible for mediating physiological effects by binding to specific pyrimidinoceptors of the plasma membrane (Ralevic & Burnstock, 1998). We have previously reported a role for extracellular uridine 5-diphosphate (UDP) in stimulating Cl^– secretion across the murine intestinal epithelium (Browne & Harvey, 2000). In this study we have examined the changes in intracellular Ca²⁺ (Ca²⁺_i) resulting from the addition of extracellular UDP to T84 cells.

Changes in [Ca²⁺]_i were assayed by spectrofluorescence microscopy using the dye fura-2 AM in isolated T84 cells, a human colonic carcinoma cell line (Dharmsathaphorn et al. 1984). Changes in secretion were monitored using the short-circuit current (I_sc) method, both I_sc and [Ca²⁺]_i were recorded simultaneously in polarized T84 cell monolayers. Statistical significance was monitored using Student’s unpaired t test.

UDP (10 µM) when repetitively added to isolated T84 cells initiated a transient and ever-decreasing increase in [Ca²⁺]_i. This response was blocked with suramin (100 µM) pre-treatment (▓Dgr│Ca²⁺ ± S.E.M. (nM) = 10 ± 3 compared with the control response of 91 ± 15, n = 6, P < 0.05). The involvement of [Ca²⁺]_i stores in the UDP response (10 µM) was tested in the presence of thapsigargin (1 µM): following thapsigargin addition, a significant increase in [Ca²⁺]_i was detected (▓Dgr│Ca²⁺ ± S.E.M. (nM) = 251 ± 41, n = 6, P < 0.0005); with subsequent UDP addition, no [Ca²⁺]_i changes were detected.

As the control [Ca²⁺]_i to UDP was transient, the regulation of the Ca²⁺ signal was investigated using an inhibitor of the plasma membrane Ca²⁺-ATPase pump, sodium orthovanadate (1 mM). Following vanadate pre-treatment, UDP produced a sustained increase in [Ca²⁺]_i (▓Dgr│Ca²⁺ ± S.E.M. (nM) = 152 ± 38, n = 6, P < 0.05).

In polarized monolayers of T84 cells, the superfusion of UDP (10 µM) on the apical side elicited a sustained increase in both I_sc and [Ca²⁺]_i (▓Dgr│I_sc = 52 ± 3 µA cm^-2, ▓Dgr│Ca²⁺ = 116 ± 6 nM, n = 6, P < 0.005).

This study has demonstrated Cl^– secretion across polarized T84 cell monolayers in response to extracellular UDP at the apical membrane. The secretory response involves increases in [Ca²⁺]_i that are dependent on calcium ion release from intracellular stores. The calcium signal is terminated by extrusion of Ca²⁺ out of the cell via a vanadate-sensitive plasma membrane Ca²⁺-ATPase pump.

Browne, G.P. & Harvey, B.J. (2000). J. Physiol. 526.P, 5-6P.

Dharmsathaphorn, K., McRoberts, J.A., Mandel, K.G., Tisdale, L.D. & Masui, H. (1984). Am. J. Physiol. 246, G204-208.

Ralevic, V. & Burnstock, G. (1998). Pharmacol. Rev. 50 (3), 413-492.