An alternative pathway for metabolism of leukotriene D(4): effects on contractions to cysteinyl-leukotrienes in the guinea-pig trachea

Abstract

Contractions of guinea-pig tracheal preparations to cysteinyl-leukotrienes (LTC(4), LTD(4) and LTE(4)) were characterized in organ baths, and cysteinyl-leukotriene metabolism was studied using radiolabelled agonists and RP-HPLC separation. In the presence of S-hexyl GSH (100 microM) the metabolism of [(3)H]-LTC(4) into [(3)H]-LTD(4) was inhibited and the LTC(4)-induced contractions were resistant to CysLT(1) receptor antagonism but inhibited by the dual CysLT(1)/CysLT(2) receptor antagonist BAY u9773 (0.3 - 3 microM) with a pA(2)-value of 6.8+/-0.2. In the presence of L-cysteine (5 mM), the metabolism of [(3)H]-LTD(4) into [(3)H]-LTE(4) was inhibited and the LTD(4)-induced contractions were inhibited by the CysLT(1) receptor antagonist ICI 198,615 (1 - 10 nM) with a pA(2)-value of 9.3+/-0.2. However, at higher concentrations of ICI 198,615 (30 - 300 nM) a residual contraction to LTD(4) was unmasked, and this response was inhibited by BAY u9773 (1 - 3 microM). In the presence of the combination of S-hexyl GSH with L-cysteine, the LTD(4)-induced contractions displayed the characteristics of the LTC(4) contractile responses, i.e. resistant to CysLT(1) receptor antagonism, increased maximal contractions and slower time-course. This qualitative change of the LTD(4)-induced contraction was also observed in the presence of S-decyl GSH (100 microM), GSH (10 mM) and GSSG (10 mM). S-hexyl GSH, S-decyl GSH, GSH and GSSG all stimulated a formation of [(3)H]-LTC(4) from [(3)H]-LTD(4). In conclusion, GSH and GSH-related compounds changed the pharmacology of the LTD(4)-induced contractions by stimulating the conversion of LTD(4) into LTC(4). Moreover, the results indicate that, in addition to the metabolism of LTC(4) into LTD(4) and LTE(4), also the formation of LTC(4) from LTD(4) may regulate cysteinyl-leukotriene function.

Figure 1

Concentration-effect curves for leukotriene C₄ (LTC₄) in guinea-pig tracheal spiral preparations (A, n=7–12) and metabolism of radiolabelled leukotriene C₄ ([³H]-LTC₄, B, n=4–7) in the absence and presence of L-cysteine (5 mM, L-cys) in combination with either L-serine borate (45 mM, L-SB) or S-hexyl GSH (100 μM). In (C, n=4) and (D, n=4–10) preparations were treated with S-hexyl GSH (100 μM) and L-cysteine (5 mM) in the absence and presence of either the CysLT₁ receptor antagonist ICI 198,615 (C) or the CysLT₁/CysLT₂ receptor antagonist BAY u9773 (D). Contractions are presented as per cent of a maximal contraction to histamine (1 mM), acetylcholine (1 mM) and KCl (40 mM) and metabolism as per cent of total radioactivity. Vertical lines represent s.e.mean and (*) indicates a significant difference (P<0.05) compared with control.

Figure 2

Concentration-effect curves for leukotriene D₄ (LTD₄) in guinea-pig tracheal spiral preparations (A, n=6–8) and metabolism of radiolabelled leukotriene D₄ ([³H]-LTD₄, B, n=5). In (C, n=5–14) and (D, n=4) preparations were treated with L-cysteine (5 mM) in the absence and presence of the CysLT₁ receptor antagonist ICI 198,615 (C) or the combination of ICI 198,615 (300 nM) with the CysLT₁/CysLT₂ receptor antagonist BAY u9773 (D). Contractions are presented as per cent of a maximal contraction to histamine (1 mM), acetylcholine (1 mM) and KCl (40 mM) and metabolism as per cent of total radioactivity. Vertical lines represent s.e.mean and (*) indicates a significant difference (P<0.05) compared with control.

Figure 3

Concentration-effect curves for leukotriene D₄ (LTD₄) in guinea-pig tracheal spiral preparations (A, n=4–10) and metabolism of radiolabelled leukotriene D₄ ([³H]-LTD₄, B, n=4–7). In (C, n=5–11) and (D, n=4–14) preparations were treated with S-hexyl GSH (100 μM) and L-cysteine (5 mM) in the absence and presence of either the CysLT₁ receptor antagonist ICI 198.615 or the CysLT₁/CysLT₂ receptor antagonist BAY u9773 (D). Contractions are presented as per cent of a maximal contraction to histamine (1 mM), acetylcholine (1 mM) and KCl (40 mM) and metabolism as per cent of total radioactivity. Vertical lines represent s.e.mean and (*) indicates a significant difference (P<0.05) compared with control.

Figure 4

Concentration-effect curves for leukotriene D₄ (LTD₄) and the metabolism of radiolabelled leukotriene D₄ ([³H]-LTD₄) in the guinea-pig trachea in the presence of the combination of L-cysteine (5 mM, L-cys) with (A) reduced glutathione (10 mM, GSH), (B) oxidized glutathione (10 mM, GSSG), (C) S-decyl GSH (100 μM) or (D) glutamic acid (1 mM). The contractions were studied in the absence and presence of the CysLT₁ receptor antagonist ICI 198,615 (300 nM). The contractions (n=4–7) are presented as per cent of a maximal contraction to histamine (1 mM), acetylcholine (1 mM) and KCl (40 mM) and metabolism (n=4) as per cent of total radioactivity (shown as insets). Vertical lines represent s.e.mean.

Figure 5

Concentration-effect curves for leukotriene E₄ (LTE₄) in guinea-pig tracheal spiral preparations (A and B) and the lack of metabolism of radiolabelled leukotriene E₄ ([³H]LTE₄, C, n=4). The contractions were studied in the absence (A, n=5) and presence (B, n=4) of the combination of S-hexyl GSH (100 μM) with L-cysteine (5 mM, L-cys). Preparations were pretreated with the CysLT₁ receptor antagonist ICI 198,615 (1 and 300 nM). The contractions are presented as per cent of a maximal contraction to histamine (1 mM), acetylcholine (1 mM) and KCl (40 mM) and metabolism as per cent of total radioactivity. Vertical lines represent s.e.mean.