A Ca²⁺-dependent chloride current and Ca²⁺ influx via Ca(v)1.2 ion channels play major roles in P2Y receptor-mediated pulmonary vasoconstriction

Abstract

Background and purpose: ATP, UTP and UDP act at smooth muscle P2X and P2Y receptors to constrict rat intrapulmonary arteries, but the underlying signalling pathways are poorly understood. Here, we determined the roles of the Ca²⁺ -dependent chloride ion current (I(Cl,Ca)), Ca(v)1.2 ion channels and Ca²⁺ influx.

Experimental approach: Isometric tension was recorded from endothelium-denuded rat intrapulmonary artery rings (i.d. 200-500 µm) mounted on a wire myograph.

Key results: The I(Cl,Ca) blockers, niflumic acid and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and the Ca(v)1.2 channel blocker, nifedipine, reduced peak amplitude of contractions evoked by UTP and UDP by ∼45-50% and in a non-additive manner. Ca²⁺-free buffer inhibited responses by ∼70%. Niflumic acid and nifedipine similarly depressed contractions to ATP, but Ca²⁺-free buffer almost abolished the response. After peaking, contractions to UTP and UDP decayed slowly by 50-70% to a sustained plateau, which was rapidly inhibited by niflumic acid and nifedipine. Contractions to ATP, however, reversed rapidly and fully. Tannic acid contracted tissues per se and potentiated nucleotide-evoked contractions.

Conclusions and implications: I (Cl,Ca) and Ca²⁺ influx via Ca(v)1.2 ion channels contribute substantially and equally to contractions of rat intrapulmonary arteries evoked by UTP and UDP, via P2Y receptors. ATP also activates these mechanisms via P2Y receptors, but the greater dependence on extracellular Ca²⁺ most likely reflects additional influx through the P2X1 receptor pore. The lack of a sustained response to ATP is probably due to it acting at P2 receptor subtypes that desensitize rapidly. Thus multiple signalling mechanisms contribute to pulmonary artery vasoconstriction mediated by P2 receptors.

Figure 1

Inhibition of the peak amplitude of contractions evoked by UDP and UTP. (A) The superimposed traces shows typical contractions of endothelium-denuded rat isolated IPA evoked by (A) UDP (300 µM) in the absence of (upper traces) and after incubation with (Ai) niflumic acid (1 µM), (Aii) nifedipine (1 µM) and (Aiii) niflumic acid (1 µM) plus nifedipine (1 µM) for 10 min (lower traces). (B) The superimposed traces show responses evoked by UTP (300 µM) in the absence of (upper traces) and after incubation with (Bi) DIDS (100 µM), (Bii) nifedipine (1 µM) and (Biii) DIDS (100 µM) plus nifedipine (1 µM) for 10 min (lower trace). UDP and UTP were applied as indicated by the solid bar. Each pair of traces was obtained in a separate tissue.

Figure 2

Inhibition of the peak amplitude of contractions evoked by UDP and UTP. The mean peak amplitude of contractions of endothelium-denuded rat isolated IPA evoked by (A) UDP (300 µM) and (B) UTP (300 µM) in the presence of niflumic acid (1 µM) (NFA), nifedipine (1 µM) (nifed), niflumic acid (1 µM) plus nifedipine (1 µM) (NFA + nifed), DIDS (100 µM), DIDS (100 µM) plus nifedipine (1 µM) (DIDS + nifed) and in Ca²⁺-free buffer (0[Ca]_ext), expressed as a percentage of control responses, is shown. The numbers in parentheses show n for each. Vertical lines show SEM. *P < 0.05, **P < 0.01, ***P < 0.001 for responses after treatment compared with control. ^#P < 0.05 for responses in Ca²⁺-free buffer compared with the other treatments.

Figure 3

Inhibition of the peak amplitude of contractions evoked by ATP. (A) The superimposed traces show typical contractions of endothelium-denuded rat isolated IPA evoked by ATP (300 µM) in normal buffer (upper trace) and when bathed in Ca²⁺-free buffer for 10 min (lower trace). ATP was applied as indicated by the solid bar. (B) The mean peak amplitude of contractions evoked by ATP (300 µM) in the presence of niflumic acid (1 µM) (NFA), nifedipine (1 µM) (nifed), niflumic acid (1 µM) plus nifedipine (1 µM) (NFA + nifed) and in Ca²⁺-free buffer (0[Ca]_ext), expressed as a percentage of control responses, is shown. The numbers in parentheses show n for each. Vertical lines show SEM. *P < 0.05, **P < 0.01 for responses after treatment compared with control. ^##P < 0.01 for the response to ATP in Ca²⁺-free buffer compared with the other treatments.

Figure 4

Time-course of contractions to UTP, UDP and ATP. The traces shows typical contractions of endothelium-denuded rat isolated IPA evoked by (A) UTP (300 µM) and (B) ATP (300 µM), applied as indicated by the solid bars. The mean amplitude of contractions evoked by (C) UDP (300 µM, n = 5) and UTP (300 µM, n = 6) and (D) ATP (300 µM, n = 4) at peak and 20, 30 and 40 min after agonist administration is shown. Vertical lines show SEM. ***P < 0.001 for contraction amplitude at 20, 30 or 40 min compared with peak.

Figure 5

Inhibition of the plateau of UTP- and UDP-evoked contractions. (A) The trace show a typical contraction of endothelium-denuded rat isolated IPA evoked by UDP (300 µM) and the effect of adding niflumic acid (1 µM) for 10 min, 20 min after UDP. The drugs were applied as indicated by the solid bars. The mean amplitude of contractions evoked by (B) UDP (300 µM) and (C) UTP (300 µM) 30 min after their addition and expressed as a percentage of the peak amplitude, is shown as follows: control responses in the absence of inhibitor (UDP n = 5, UTP n = 6), responses in the presence of niflumic acid (1 µM) (NFA, UDP n = 6, UTP n = 7), in the presence of nifedipine (1 µM) (nif, UDP n = 6, UTP n = 6) and in the presence of niflumic acid (1 µM) plus nifedipine (1 µM) (NFA + nif, UDP n = 7, UTP n = 5). Vertical bars indicate SEM. **P < 0.01 and ***P < 0.001 for response in the presence of inhibitors compared with the control response in their absence.

Figure 6

The effects of tannic acid. (A) The mean peak amplitude of contractions evoked by UDP (n = 5), UTP (n = 4), ATP (n = 5) (300 µM each), phenylephrine (PE) (1 µM) (n = 4) and KCl (40 mM) (n = 6) in the presence of tannic acid (100 µM), expressed as a percentage of the control response, is shown. Vertical lines show SEM. *P < 0.05 for responses after treatment compared with control. (B) The trace shows a typical contraction of endothelium-denuded rat isolated IPA evoked by UDP (300 µM) and the effect of adding tannic acid (100 µM) 20 min after UDP. Drugs were applied as indicated by the solid bars.