CysLT1 leukotriene receptor antagonists inhibit the effects of nucleotides acting at P2Y receptors

Abstract

Montelukast and pranlukast are orally active leukotriene receptor antagonists selective for the CysLT1 receptor. Conversely, the hP2Y(1,2,4,6,11,12,13,14) receptors represent a large family of GPCRs responding to either adenine or uracil nucleotides, or to sugar-nucleotides. Montelukast and pranlukast were found to inhibit nucleotide-induced calcium mobilization in a human monocyte-macrophage like cell line, DMSO-differentiated U937 (dU937). Montelukast and pranlukast inhibited the effects of UTP with IC50 values of 7.7 and 4.3 microM, respectively, and inhibited the effects of UDP with IC50 values of 4.5 and 1.6 microM, respectively, in an insurmountable manner. Furthermore, ligand binding studies using [3H]LTD4 excluded the possibility of orthosteric nucleotide binding to the CysLT1 receptor. dU937 cells were shown to express P2Y2, P2Y4, P2Y6, P2Y11, P2Y13 and P2Y14 receptors. Therefore, these antagonists were studied functionally in a heterologous expression system for the human P2Y receptors. In 1321N1 astrocytoma cells stably expressing human P2Y(1,2,4,6) receptors, CysLT1 antagonists inhibited both the P2Y agonist-induced activation of phospholipase C and intracellular Ca2+ mobilization. IC50 values at P2Y1 and P2Y6 receptors were <1 microM. In control astrocytoma cells expressing an endogenous M3 muscarinic receptor, 10 microM montelukast had no effect on the carbachol-induced rise in intracellular Ca2+. These data demonstrated that CysLT1 receptor antagonists interact functionally with signaling pathways of P2Y receptors, and this should foster the study of possible implications for the clinical use of these compounds in asthma or in other inflammatory conditions.

Fig. 1

Expression of P2Y_{2,4,6,11,13,14} in dU937 cells. Total RNA was subjected to RT and the resulting cDNA was amplified with primers specifically designed for human P2Y_{1,2,4,6,11,12,13,14} receptors. PCR products were separated on 2% ethidium bromide-stained agarose gel and visualized under UV light. Identification of the PCR products was made on the basis of their predicted sizes (see Section 3).

Fig. 2

Concentration–response curves of the [Ca²⁺]_i transient induced by nucleotides in dU937 cells. Fluo3/AM loaded cells were challenged with increasing concentrations of ATP (A), UDP (B), and UTP (C). When hexokinase was present in the UDP experiment, the response curve was unchanged (EC₅₀ 69 μM), indicating that this effect was not the result of contamination by UTP. Results are expressed as the ratio of stimulated over basal (S/B) ± S.E.M. and represent the mean from three independent experiments analyzed simultaneously.

Fig. 3

Effect of pranlukast and montelukast on nucleotide-induced [Ca²⁺]_i transients in dU937 cells. Fluo3/AM loaded cells were challenged with 300 μM UDP (A and C) and 30 μM UTP (B and D) in the presence of increasing concentrations of montelukast and pranlukast (5 min pretreatment). Results are expressed as the ratio of stimulated over basal (S/B) ± S.E.M. and represent the mean from at least three independent experiments analyzed simultaneously.

Fig. 4

Effect of pranlukast and montelukast on the concentration–response curve for the [Ca²⁺]_i transients induced by UDP in dU937 cells. Fluo3/AM loaded cells were challenged with increasing concentrations of UDP in the absence and presence of the indicated concentrations of montelukast (A) and pranlukast (B) (5 min pretreatment). Results are expressed as the ratio of stimulated over basal (S/B) ± S.E.M. and represent the mean from two independent experiments analyzed simultaneously. (C) Typical trace of UDP-induced [Ca²⁺]_i transients before and after treatment with 6 μM montelukast.

Fig. 5

Equilibrium binding of [³H]LTD₄ in intact dU937 cells. Data are presented as percent total [³H]LTD4 binding displaced by the indicated concentrations of unlabeled LTD₄, UTP or UDP. Data shown are mean ± S.E.M. from two independent experiments performed in triplicates.

Fig. 6

Functional effects of leukotriene antagonists on 2-MeSADP-induced signaling in stably transfected astrocytoma cells. (A) Effect of montelukast on concentration–response curves for intracellular Ca²⁺ changes induced by 2-MeSADP acting at P2Y₁ receptors expressed in 1321N1 astrocytoma cells. The cells were pre-treated for 20 min at room temperature with antagonist before application of the agonist 2-MeSADP. The fluorescence emission at 525 nm with excitation at 485 nm is proportional to [Ca²⁺]_i. Increases in intracellular calcium are reported as the maximum fluorescence value after exposure minus the basal fluorescence value before exposure. (B) Concentration-dependence of inhibition by montelukast and pranlukast of inositol phosphate production induced by 30 nM 2-MeSADP in human P2Y₁ transfected 1321N1 human astrocytoma cells. (C) Effect of montelukast and pranlukast on the binding of [³H]MRS2279 to membranes from astrocytoma cells stably expressing the human P2Y₁ receptor. Data shown are mean ± S.E.M. from three independent experiments performed in duplicate.

Fig. 7

Functional effects of leukotriene antagonists on UDP-induced signaling in stably transfected P2Y₆ receptor-expressing astrocytoma cells. (A) Effect of montelukast on concentration–response curves for intracellular Ca²⁺ changes induced by UDP acting at P2Y₆ receptors expressed in 1321N1 astrocytoma cells. The cells were pre-treated for 20 min at room temperature with antagonist before application of the agonist UDP. The fluorescence emission at 525 nm with excitation at 485 nm is proportional to [Ca²⁺]_i. Increases in intracellular calcium are reported as the maximum fluorescence value after exposure minus the basal fluorescence value before exposure. Data shown are mean ± S.E.M. from three independent experiments performed in duplicate. (B) Inositol phosphate production in human P2Y₆ transfected 1321N1 human astrocytes. After labeling with myo-[³H]inositol (1 μCi/10⁶ cells) for 24 h, the cells were treated for 20 min at 37 °C with montelukast (10, 3 and 1 μM concentration) in the presence of LiCl, followed by addition of the agonist, UDP, for another 30 min. The quantity of inositol phosphates was analyzed after the extraction though Dowex AG 1-X8 columns (see Section 2). Data shown are mean ± S.E.M. from two independent experiments performed in duplicate. (C) Concentration dependence of inhibition by montelukast and pranlukast of inositol phosphate production induced by 300 nM UDP in human P2Y₆ transfected 1321N1 human astrocytes. Data shown are mean ± S.E.M. from three independent experiments performed in duplicate.

Fig. 8

Functional effects of leukotriene antagonists on signaling in astrocytoma cells (either in control cells or in cells stably expressing P2Y receptors). (A) Inositol phosphate production in human P2Y₂-receptor transfected 1321N1 human astrocytes. After labeling with myo-[³H]inositol (1 μCi/10⁶ cells) for 24 h, the cells were treated for 30 min at 37 °C with montelukast (10 μM concentration) in the presence of LiCl, followed by addition of the agonist, UTP, for another 30 min. The quantity of inositol phosphates was analyzed after the extraction though Dowex AG 1-X8 columns (see Section 2). Data shown are mean ± S.E.M. from three independent experiments performed in duplicate. (B) Inositol phosphate production induced by UTP in human P2Y₄-receptor transfected 1321N1 human astrocytes. (C) Effect of montelukast on concentration–response curves for intracellular Ca²⁺ changes induced by UTP acting at P2Y₄ receptors stably expressed in 1321N1 astrocytoma cells. The fluorescence emission at 525 nm with excitation at 485 nm is proportional to [Ca²⁺]_i. Increases in intracellular calcium are reported as the maximum fluorescence value after exposure minus the basal fluorescence value before exposure. (D) Lack of effect of montelukast on concentration–response curves for intracellular Ca²⁺ changes induced by carbachol acting at endogenous M3 muscarinic acetylcholine receptors in control 1321N1 astrocytoma cells. The cells were pre-treated for 20 min at room temperature with antagonist before application of the agonist carbachol. Data shown are mean ± S.E.M. from two independent experiments performed in triplicate.