Diacylglycerol regulates acute hypoxic pulmonary vasoconstriction via TRPC6

Abstract

Background: Hypoxic pulmonary vasoconstriction (HPV) is an essential mechanism of the lung that matches blood perfusion to alveolar ventilation to optimize gas exchange. Recently we have demonstrated that acute but not sustained HPV is critically dependent on the classical transient receptor potential 6 (TRPC6) channel. However, the mechanism of TRPC6 activation during acute HPV remains elusive. We hypothesize that a diacylglycerol (DAG)-dependent activation of TRPC6 regulates acute HPV.

Methods: We investigated the effect of the DAG analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) on normoxic vascular tone in isolated perfused and ventilated mouse lungs from TRPC6-deficient and wild-type mice. Moreover, the effects of OAG, the DAG kinase inhibitor R59949 and the phospholipase C inhibitor U73122 on the strength of HPV were investigated compared to those on non-hypoxia-induced vasoconstriction elicited by the thromboxane mimeticum U46619.

Results: OAG increased normoxic vascular tone in lungs from wild-type mice, but not in lungs from TRPC6-deficient mice. Under conditions of repetitive hypoxic ventilation, OAG as well as R59949 dose-dependently attenuated the strength of acute HPV whereas U46619-induced vasoconstrictions were not reduced. Like OAG, R59949 mimicked HPV, since it induced a dose-dependent vasoconstriction during normoxic ventilation. In contrast, U73122, a blocker of DAG synthesis, inhibited acute HPV whereas U73343, the inactive form of U73122, had no effect on HPV.

Conclusion: These findings support the conclusion that the TRPC6-dependency of acute HPV is induced via DAG.

Figure 1

No effect of 1-oleoyl-2-acetyl-sn-glycerol (OAG) on normoxic vascular tone in TRPC6^-/- mouse lungs. The effect of OAG was investigated in isolated wild-type and TRPC6^-/- mouse lungs. The OAG-induced vasoconstriction is represented as the increase in pulmonary artery pressure (ΔPAP) during normoxic ventilation. OAG was applied in increasing doses every 25 min. Data are derived from n = 5 and n = 4 wild-type and TRPC6^-/-mice, respectively. * Significant differences compared to wild-type mice (p < 0.05).

Figure 2

1-oleoyl-2-acetyl-sn-glycerol (OAG) diminished HPV specifically. The effect of OAG as well as its solvent on the normoxic vascular tone, the strength of HPV, and the strength of U46619-induced vasoconstriction was investigated in isolated wild-type mouse lungs. (A) Increase in normoxic pulmonary arterial pressure (ΔPAP), assessed directly before each hypoxic ventilation maneuver. (B) Strength of HPV referenced to the effect of the second hypoxic ventilation maneuver (= 100%). (C) Strength of the U46619-induced vasconstriction, referenced to the effect of the second U46619 application (= 100%). Data are derived from n = 5 isolated lung preparations each. * Significant differences compared to control experiments with application of the solvent only (p < 0.05).

Figure 3

Diacylglycerol kinase inhibitor R59949 diminished HPV specifically. The effect of the diacylglycerol kinase inhibitor R59949 as well as its solvent on the normoxic vascular tone, the strength of HPV, and the strength of U46619-induced vasoconstriction was investigated in isolated wild-type mouse lungs. (A) Increase in normoxic pulmonary arterial pressure (ΔPAP), assessed directly before each hypoxic ventilation maneuver. (B) Strength of HPV referenced to the effect of the second hypoxic ventilation maneuver (= 100%). Absolute values of HPV for the second hypoxia maneuver prior to R59949 application were 0.7 ± 0.1 (n = 5). (C) Strength of the U46619-induced vasoconstriction, referenced to the effect of the second U46619 application (= 100%). Data are derived from n = 5 isolated lung preparations each. * Significant differences compared to control experiments with application of the solvent only (p < 0.05).

Figure 4

Inhibitory effect of phospholipase C inhibitor U73122 on HPV and U46619-induced vasoconstriction. The effect of the active (U73122) and inactive (U73343) form of phospholipase C inhibitor as well as their solvent on the normoxic vascular tone, the strength of HPV, and the strength of U46619-induced vasoconstriction was investigated in isolated wild-type mouse lungs. (A) Increase in normoxic pulmonary arterial pressure (ΔPAP), assessed directly before each hypoxic ventilation maneuver. (B) Strength of HPV referenced to the effect of the second hypoxic ventilation maneuver (= 100%). (C) Strength of U46619-induced vasoconstrictions, referenced to the effect of the second U46619 application (= 100%). Data are derived from n = 5 isolated lung preparations each. * Significant differences compared to control experiments with application of the solvent only (p < 0.05).

Figure 5

Inhibition of acute but not sustained HPV by the diacylglycerol kinase inhibitor R59949. The effect of the diacylglycerol kinase inhibitor R59949 as well as its solvent on normoxic vascular tone and sustained HPV was investigated for a period of 120 min. (A) Increase in normoxic pulmonary arterial pressure (ΔPAP) referenced to the value directly before R59949 application. For comparison changes in PAP are given for normoxic lungs in the absence of R59949. (B) Strength of sustained HPV in the presence and the absence of R59949. Data are derived from n = 8 isolated lung preparations each. * Significant differences compared to experiments in the absence of R59949 (p < 0.05).