Hypoxic pulmonary vasoconstriction in the absence of pretone: essential role forintracellular Ca2+ release

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Hypoxic pulmonary vasoconstriction (HPV) maintains blood oxygenation during acute hypoxia but contributes to pulmonary hypertension during chronic hypoxia. The mechanisms of HPV remain controversial, in part because HPV is usually studied in the presence of agonist-induced preconstriction ('pretone'). This potentiates HPV but may obscure and distort its underlying mechanisms. We therefore carried out an extensive assessment of proposed mechanisms contributing to HPV in isolated intrapulmonary arteries (IPA) in the absence of pretone by using a conventional small vessel myograph. Hypoxia elicited a biphasic constriction consisting of a small transient (phase 1) superimposed upon a sustained (phase 2) component. Neither phase was affected by the L-type Ca(2+) channel antagonists diltiazem (10 and 30μmole/L) or nifedipine (3 μmole/L). Removal of extracellular Ca(2+) (with 0.2 mmole/L EGTA present to chelate residual Ca(2+)) abolished phase 1 but only attenuated phase 2. The SERCA inhibitor CPA had a similar effect in the presence of extracellular Ca(2+), and did not affect HPV in its absence. Pretreatment with 10 μmole/L ryanodine and 15 mmole/L caffeine abolished both phases, whereas treatment with 100 μmole/L ryanodine attenuated both phases. The two pore channel blocker NED-19 (1μmole/L) and the NAADP antagonist BZ-194 (200 μmole/L) had no effect on either phase of HPV. The lysosomal Ca(2+) depleting agent concanamycin (1 μmole/L) enhanced HPV if applied during hypoxia, but had no effect on HPV during a subsequent hypoxic challenge. The cyclic ADP ribose antagonist 8-bromo-cyclic ADP ribose (30 μmole/L) had no effect on either phase of HPV. Neither the Ca(2+) sensing receptor (CaSR) blocker NPS 2390 (0.1 and 10 μmole/L) nor FK506 (10 μmole/L), a drug which displaces FKBP12.6 from RyR2, had any effect on HPV. HPV was virtually abolished by the rho kinase blocker Y27632 (1 μmole/L) and attenuated by the PKC inhibitor Gö 6983 (3μm/L). 45 minutes of hypoxia caused a significant increase in the ratio of oxidized to reduced glutathione (GSSG/GSH). HPV was unaffected by the NADPH oxidase (NOX) inhibitor VAS 2870 (10 μmole/L), whereas phase 2 was inhibited but phase 1 was unaffected by the antioxidants ebselen (100 μmole/L) and TEMPOL (3 mmoles/L). We conclude that phase 1 HPV in this model is mainly dependent on store operated Ca(2+) entry (SOCE), whereas phase 2 HPV requires both [Ca(2+)]i released from the sarcoplasmic reticulum, and SOCE. Neither phase of HPV requires voltage-gated Ca(2+) entry. Ca(2+) release is mainly mediated by ryanodine receptors. However, we can detect no requirement for cyclic ADP ribose, NAADP-dependent lysosomal Ca(2+) release, activation of the CaSR, or displacement of FKBP12.6 from RyR2 for either phase of HPV. Sustained HPV is associated with an oxidizing shift in the GSSG/GSH redox potential and is abolished by the antioxidants ebselen and TEMPOL, consistent with the concept that it requires an oxidizing shift in the cell redox state or the generation of reactive oxygen species.
Original languageEnglish
Article numberN/A
Pages (from-to)4473-4498
Number of pages26
JournalThe Journal of Physiology
Issue number18
Early online date17 Jun 2013
Publication statusPublished - 15 Sept 2013



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