Bradykinin increases Na(+)-K(+) pump activity in cultured guinea-pig tracheal smooth muscle cells

Abstract

1. The effect of bradykinin on the Na+-K+ pump of airway smooth muscle was investigated by measuring ouabain-sensitive (86)Rb(+) uptake in cultured guinea-pig tracheal smooth muscle cells. 2. Bradykinin induced a concentration-dependent increase in ouabain-sensitive (86)Rb(+) uptake, with an EC(50) of 3 nM (pD(2) = 8.50+/-0.10). Stimulation was not affected by indomethacin (1 microM) suggesting that it is not mediated by cycloxygenase products of arachidonic acid. 3. The B(1) receptor agonists Lys-des-Arg(9)-bradykinin and des-Arg(9)-bradykinin had no effect on ouabain-sensitive (86)Rb(+) uptake. In contrast, the B(1) and B(2) receptor agonist Lys-bradykinin induced a concentration-dependent increase in ouabain-sensitive (86)Rb(+) uptake with an EC(50) of 6 nM (pD(2) = 8.21 +/- 0.20). 4. The B(1) receptor antagonist des-Arg(10)-HOE 140 (1 microM) had no effect on bradykinin-stimulated ouabain-sensitive (86)Rb(+) uptake. The B(2) receptor antagonists HOE 140 and WIN 64338 antagonized bradykinin-stimulated ouabain-sensitive (86)Rb(+) uptake with pK(B) values (-log M) of 8.20 +/- 0.08 and 8.11 +/- 0.20 respectively. 5. Reducing extracellular Na+ from 146 mM to 11 mM caused a 53.5% decrease in basal ouabain-sensitive (86)Rb+ uptake and abolished bradykinin-induced uptake. Two inhibitors of the Na(+)-H(+) exchanger, methylisobutyl-amiloride (MIA; 1 - 100 microM) and ethylisopropyl-amiloride (EIPA; 0.1 - 10 microM), inhibited bradykinin-stimulated ouabain-sensitive (86)Rb(+) uptake without affecting basal uptake. 6. These results suggest that bradykinin increases Na+-K+ pump activity of guinea-pig tracheal smooth muscle via stimulation of B(2) receptors and activation of the Na+-H+ exchanger.

Figure 1

Effect of bradykinin on ⁸⁶Rb⁺ uptake into cultured guinea-pig tracheal smooth muscle cells. ⁸⁶Rb⁺ uptake is expressed as total uptake in the absence or presence of 100 μM ouabain (A) or as the increase in ouabain-sensitive uptake induced by bradykinin (B). Points represent mean±s.e.mean (n=17).

Figure 2

Effect of the bradykinin receptor agonists des-Arg⁹-bradykinin, Lys-des-Arg⁹-bradykinin and Lys-bradykinin on ouabain-sensitive ⁸⁶Rb⁺ uptake into cultured guinea-pig tracheal smooth muscle cells. Points represent mean±s.e.mean (n=6–8).

Figure 3

Effect of the bradykinin B₁ receptor antagonist des-Arg¹⁰-HOE 140 (1 μM) on bradykinin-stimulated ouabain-sensitive ⁸⁶Rb⁺ uptake into cultured guinea-pig tracheal smooth muscle cells. Points represent mean±s.e.mean (n=8).

Figure 4

Effect of the bradykinin B₂ antagonists HOE 140 (A) and WIN 64338 (B) on bradykinin-stimulated ouabain-sensitive ⁸⁶Rb⁺ uptake into cultured guinea-pig tracheal smooth muscle cells. Uptake was measured in the presence of 0, 10, 30, 100 or 300 nM antagonist. Points represent mean±s.e.mean (n=8 for HOE 140 and n=9 for WIN 64338).

Figure 5

Effect of monensin on ouabain-sensitive ⁸⁶Rb⁺ uptake into cultured guinea-pig tracheal smooth muscle cells. ^*P<0.05, ^**P<0.01 vs no monensin, ANOVA with Bonferroni post-test. Points represent mean±s.e.mean (n=8–10).

Figure 6

Effect of extracellular Na⁺ concentration on the stimulation of ouabain-sensitive ⁸⁶Rb⁺ uptake into cultured guinea-pig tracheal smooth muscle cells by monensin (0.3 μM) and bradykinin (1 μM). Uptake was measured in BSS containing 146 mM Na⁺ or 11 mM Na⁺. ^*P<0.05 vs control in 146 mM Na⁺, ANOVA with Bonferroni post-test. Points represent mean±s.e.mean (n=8).

Figure 7

Effect of the Na⁺-H⁺ exchanger inhibitors MIA (A) and EIPA (B) on the stimulation of ouabain-sensitive ⁸⁶Rb⁺ uptake into cultured guinea-pig tracheal smooth muscle cells by bradykinin (1 μM). Uptake was measured in the presence of 1–100 μM MIA or 0.1–10 μM EIPA. ^**P<0.001, ^*P<0.005 vs no bradykinin, unpaired Student's t-test. Points represent mean±s.e.mean (n=8).