Mechanisms of U46619- and 5-HT-induced contraction of bovine pulmonary arteries: role of chloride ions

Abstract

Background and purpose: Thromboxane A(2) and 5-hydroxytryptamine (5-HT) are implicated in pulmonary hypertension. The involvement of chloride, voltage-operated calcium channels (VOCCs), store-operated calcium channels (SOCCs) and the Rho kinase in the contractile response of bovine pulmonary arteries (BPA) to the thromboxane A(2) mimetic U46619 and 5-HT was investigated.

Experimental approach: Endothelium-intact ring segments of BPA were mounted in Krebs/Henseleit buffer (37 degrees C) under a tension of 2g and gassed with 95%O(2)/5%CO(2).

Key results: Depletion or removal of extracellular chloride, inhibition of chloride and SOCC, Na:K:2Cl, Cl/HCO(3), Rho kinase inhibited contractions to U46619. Combining Rho kinase inhibition and chloride channel blockade (with NPPB) almost abolished the contractions to U46619. In contrast 5-HT-induced contraction was inhibited by verapamil and mibefradil. Depletion of stored calcium with caffeine almost abolished the response to U46619 but not 5-HT. The contraction by the sarco(endo)plasmic reticulum Ca(2+)-ATPase inhibitor CPA was abolished by SOCC and chloride channel blockade (with NPPB) and by chloride depletion.

Conclusions and implications: This study suggests that the contractile response of BPA to U46619 involves Rho kinase together with a chloride-sensitive mechanism, which does not involve VOCC but may have a role in calcium release and calcium entry via SOCC. In contrast contraction of the BPA by 5-HT appears to involve verapamil- and mibefradil-sensitive VOCC. This study may indicate that the use of calcium channel blockers in the management of pulmonary hypertension may not always be effective and that Rho kinase and chloride channels may be targets for the development of new therapies.

Figure 1

Effect of chloride channel blockade and chloride transport inhibition on the concentration response–curve for contraction to 5-HT and U46619. Control responses and the effect of NPPB (50 μM), 9-AC (500 μM) and niflumic acid (NFA, 30 μM) are shown in (a) (5-HT) and (b) (U46619) and bumetanide (100 μM), DIDS (500 μM, 5-HT and 100 μM, U46619) and acetazolamide (1mM) are shown in (c) (5-HT) and (d) (U46619). Results are the means±s.e.m. from 5–16 experiments.

Figure 2

Effect of chloride-depleted and chloride-free PSS on the concentration–response curve for contraction to 5-HT (a) and U46619 (b). Control responses, [Cl⁻]_o=129 mM; Cl⁻-depleted, [Cl⁻]_o=10 mM; chloride-free. Results are the means±s.e.m. from 5 to 6 experiments.

Figure 3

Effect of the Rho kinase inhibitor Y27632 on the concentration–response curve for contraction to 5-HT (a) and U46619 (b). Control responses, Y-27632, 30 μM. Results are the means±s.e.m. from 3 to 7 experiments.

Figure 4

Effect of voltage-operated calcium channels blockade and depletion of stored calcium with caffeine on the concentration–response curve for contractions to 5-HT and U46619. Control responses and the effect of nifedipine (1 μM); verapamil (10 μM); mibefradil (10 μM); are shown in (a) (5-HT) and (b) (U46619). In addition the combined effect verapamil+mibefradil on 5-HT is shown in (a). The effect of caffeine (1 mM) is shown in (c) (5-HT) and (d) (U46619). Results are the means±s.e.m. from 5 to 6 experiments.

Figure 5

The effect of SOCC blockade on the concentration response curve for contraction to U46619. Control response and the effect of 2-APB (100 μM) and SKF96365 (100 μM). Results are the means±s.e.m. from 4 to 5 experiments.

Figure 6

Comparison between the contractile responses of bovine pulmonary arteries to U46619 and 5-HT in CO₂-gassed HCO₃-buffered PSS and air-gassed 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered PSS and the effect of La³⁺ on the response to U46619 in air-gassed HEPES PSS. Results are the means±s.e.m. from 4 to 5 experiments.

Figure 7

The effect of store-operated calcium channels (SOCC) blockade or chloride channel blockade on the concentration–response curve to U46619 in the presence of Rho kinase inhibition. Control responses in the absence and presence of Rho kinase inhibition with Y27632 (30 μM) are shown and the combined effect of Rho kinase inhibition with (a) 2-APB (100 μM) or (b) NPPB (50 μM). Results are the means±s.e.m. from 4 to 6 experiments.

Figure 8

Effect of chloride channel blockade or Rho kinase inhibition on the concentration response curve for contraction to U46619 in nominally Ca²⁺-free PSS. Control responses in normal and Ca²⁺-free PSS are shown and the effect of NPPB (50 μM) or Y27632 (30 μM) on the response in Ca²⁺-free PSS. Results are the means±s.e.m. from 4 to 8 experiments.

Figure 9

Effect of the putative store-operated calcium channels blockers SKF96365 (100 μM) and 2-APB (100 μM), chloride channel blockers NPPB (50 μM) and NFA (30 μM), and chloride depletion on the contraction to the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) inhibitor cyclopiazonic acid (10 μM). Results are the means±s.e.m. from 4 to 8 experiments.