Transmitter-Induced Changes in Cytosolic Ca2+ Activity in HT29 Cells

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Abstract
Cl- secretion in HT29 cells involves regulation of Cl– channels via different transduction pathways. One of these transduction pathways is thought to involve the intracellular Ca2+. To address this question further we measured the intracellular Ca2+ activity ([Ca2+]i) in response to various transmitters and secretagogues. As a measure of [Ca2+]i the cells were loaded with fura-2, and the 510-nm fluorescence emission ratio after 335 and 380 nm excitation was estimated in an inverted fluorescence microscope. [Ca2+]i was calibrated at the end of the experiments with ionomycin. Application of ATP (10–6 mol/l; n = 12) raised the fluorescence ratio with seconds from a resting value of 0.76 ± 0.06 to a peak value of 1.68 ± 0.16, followed by a plateau phase at a lower value of 1.05 ± 0.09 (n = 12). [Ca2+]i returned to the resting value after ATP had been removed. These ratios correspond to calculated [Ca2+]i values of approximately 50, 450 and 140 nmol/l, respectively. Other purine and pyrimidine nucleotides had similar effects on [Ca2+]i with the following half-maximal concentrations (EC50): UTP 4·10–7, ATP 2· 10–6 and ITP 4·10–6 mol/l. GTP and TTP were ineffective up to 10–5 mol/l. The order of potency of adenosine nucleotides and adenosine was ATP > ADP > AMP > adenosine = 0. The ATP analogues βγ-methylene-ATP and 2-methylthio-ATP were not effective up to 10–4 mol/l. Using the known purine (P2) antagonists suramin, basilen blue, cibacron blue and coomassie brilliant blue no further subclassification of the P2 receptor was possible. In a similar fashion to ATP, neurotensin (EC50: 5.10–10 mol/l) and carbachol (CCH EC: 4· 10–6 mol/l) induced a rise in [Ca2+]i. The CCH effect was blocked by pirenzepine (10–6 mol/l). The ATP- and CCH-induced [Ca2+]i increases showed an initial release from intracellular Ca2+ stores. The following sustained Ca2+ levels were totally dependent on the presence of extracellular Ca2+. [Ca2+]i was unaffected by iso-protenerol (10–4 mol/l), suprarenin (10–5 mol/l), bradykinin (10–4 mol/l), vasoactive intestinal peptide (10–6 mol/l), 8-chlorophenylthio-cAMP (5·10–4 mol/l), forskolin (10–5 mol/l) pentagastrin (10–6 mol/l). The present data indicate that HT29 cells express P2, neurotensin and muscarinic receptors which mediate a rise in [Ca2+]i. The same agonists, probably via [Ca2+]i increases, induce a pronounced initial membrane depolarization followed by a hyper-polarization which are caused by Cl- and K+ currents, respectively.

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