Adenosine induces cyclic-AMP formation and inhibits endothelin-1 production/secretion in guinea-pig tracheal epithelial cells through A(2B) adenosine receptors

Abstract

1. The adenosine receptor subtype mediating adenosine 3' : 5'-cyclic monophosphate (cyclic AMP) formation and the effect of its activation on endothelin-1 (ET-1) secretion were studied in primary cultures of tracheal epithelial cells. 2. Adenosine analogues showed the following rank order of potency (pD(2) value) and intrinsic activity on the generation of cyclic AMP by tracheal epithelial cells: 5'-N-ethylcarboxyamidoadenosine (NECA, A(1)/A(2A)/A(2B), pD(2): 5.44+/-0.16)>adenosine (ADO, non selective, pD(2): 4.99+/-0. 09; 71+/-9% of NECA response) >/=2-Cl-adenosine (2CADO, non selective, pD(2): 4.72+/-0.14; 65+/-9% of NECA response)>>>CGS21680 (A(2A); inactive at up to 100 microM). 3. Cyclic AMP formation stimulated by NECA in guinea-pig tracheal epithelial cells was inhibited by adenosine receptor antagonist with the following order of apparent affinity (pA(2) value): Xanthine amine congeners (XAC, A(2A)/A(2B), 7.89+/-0.22)>CGS15943 (A(2A)/A(2B), 7.24+/-0. 26)>ZM241385 (A(2A), 6.69+/-0.14)>DPCPX (A(1), 6.51+/-0. 14)>3n-propylxanthine (weak A(2B), 4.30+/-0.10). This rank order of potency is typical for A(2B)-adenosine receptor. 4. Adenosine decreased basal and LPS-stimulated irET production in a concentration-dependent manner. Moreover, NECA but not CGS21680 inhibited LPS-induced irET production. 5. The inhibitory effect of NECA on LPS-induced irET production was reversed by XAC (pA(2)=8.84+/-0. 12) and DPCPX (pA(2)=8.10+/-0.22). 6. These results suggested that adenosine increased cyclic AMP formation and inhibited irET production/secretion by guinea-pig tracheal epithelial cells through the activation of a functional adenosine receptor that is most likely the A(2B) subtype. This adenosine receptor may be involved in the regulation of the level of ET-1 production/secretion by guinea-pig tracheal epithelial cells in physiological as well as in pathophysiological conditions.

Figure 1

Cyclic AMP accumulation in guinea-pig tracheal epithelial cells in the presence of adenosine and adenosine analogues. Cultured cells were preincubated in the presence of Rolipram (10 μM) for 15 min and thereafter stimulated for 5 min with increasing concentrations of (a) adenosine. Each point represents the mean±s.e.mean of four determinations made with different cell preparations. (b) Cultured cells were preincubated in the presence of Rolipram (10 μM) for 15 min and thereafter stimulated for 5 min with increasing concentrations of NECA, 2CADO, adenosine and CGS21680. Values are the mean±s.e.mean of 4–8 determinations made with different cell preparations, expressed as a percentage of the response to 100 μM adenosine. See Methods for further details.

Figure 2

Effect of selective and non-selective adenosine receptor antagonists on the NECA evoked production of cyclic AMP. Cultured cells were preincubated 15 min in the presence of increasing concentrations of XAC, CGS15943, DPCPX, ZM241385 and 3-n-propylxanthine and thereafter stimulated with NECA (10 μM) for 5 min and the reaction was ended as described in Methods. Each point represents the mean±s.e.mean of 4–8 determinations made in different cell preparations, expressed as a percentage of the response of 10 μM NECA.

Figure 3

Effect of N⁶-cyclopentyladenosine (CPA) on Forskolin-stimulated cyclic AMP accumulation in guinea-pig tracheal epithelial cells. Cells were preincubated 15 min in the presence of Rolipram (10 μM) and adenosine deaminase (1.5 u·ml⁻¹) and thereafter stimulated for 5 min in the absence (Control) or in the presence of 10 μM forskolin alone (Forskolin) or with forskolin and increasing concentrations of CPA (+CPA). Values are the mean±s.e.mean of four determinations made with different cell preparations, expressed as percentage of the response to 10 μM forskolin. ^*P<0.05 and ^**P<0.01.

Figure 4

Effect of adenosine on basal (a) and lipopolysaccharide-induced (b) immunoreactive ET-1 production/secretion by tracheal epithelial cells. Cultured guinea-pig tracheal epithelial cells were pretreated or not (Control) with LPS (10 μg·ml⁻¹) during 30 min and therefore incubated 24 h in the presence or absence (control, LPS) of increasing concentrations of adenosine and the irET was measured as described in Methods. Values are the means±s.e.mean of four determinations made with different cell preparations. (f P<0.05 vs LPS, ^**P<0.01 vs Control).

Figure 5

Effect of adenosine analogues on LPS-induced irET production/secretion by guinea-pig tracheal epithelial cells. Cultured cells were preincubated in the presence or absence of increasing concentrations of NECA (a) or CGS21680 (b) during 30 minutes and then incubated with or without LPS (10 ug·ml⁻¹ LPS) for 24 h and the irET was measured as described in methods. Values are means±s.e.mean of 3–4 determinations made in different cell preparations. (^**P<0.01 vs LPS and f P<0.01 vs Control).

Figure 6

Effects of selective adenosine receptor antagonists on the inhibition of LPS-induced irET production/secretion evoked by NECA. Cultured guinea-pig tracheal epithelial cells were first incubated in the presence of (a) DPCPX (1×10⁻⁸ to 1×10⁻⁶ M) or (b) XAC (1×10⁻⁸ to 1×10⁻⁶ M) for 30 min. Thereafter, the cells were incubated in the presence or absence (Control, LPS) of NECA (1 μM) for another 15 min before being stimulated or not (Control) by LPS (LPS, NECA and −8, −7, −6) during 24 h. IrET was measured as described in Methods. Values are means±s.e.mean of 3–4 determinations made in different cell preparations. (f P<0.01 vs Control, ^**P<0.01 vs LPS treated cells and +P<0.05 and ++P<0.01 vs NECA).