title: Hypoxic pulmonary vasoconstriction in the absence of pretone: essential role for intracellular Ca2+ release.
abstract: 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 (IPAs) 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 Ca2+ channel antagonists diltiazem (10 and 30 μm) or nifedipine (3 μm). Application of the store-operated Ca2+ entry (SOCE) blockers BTP2 (10 μm) or SKF96365 (50 μm) attenuated phase 2 but not phase 1, whereas a lengthy (30 min) incubation in Ca2+-free physiological saline solution similarly reduced phase 2 but abolished phase 1. No further effect of inhibition of HPV was observed if the sarco/endoplasmic reticulum Ca2+-ATPase inhibitor cyclopiazonic acid (30 μm) was also applied during the 30 min incubation in Ca2+-free physiological saline solution. Pretreatment with 10 μm ryanodine and 15 mm caffeine abolished both phases, whereas treatment with 100 μm ryanodine attenuated both phases. The two-pore channel blocker NED-19 (1 μm) and the nicotinic acid adenine dinucleotide phosphate (NAADP) antagonist BZ194 (200 μm) had no effect on either phase of HPV. The lysosomal Ca2+-depleting agent concanamycin (1 μm) 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 μm) had no effect on either phase of HPV. Neither the Ca2+-sensing receptor (CaSR) blocker NPS2390 (0.1 and 10 μm) nor FK506 (10 μm), a drug which displaces FKBP12.6 from ryanodine receptor 2 (RyR2), had any effect on HPV. HPV was virtually abolished by the rho kinase blocker Y-27632 (1 μm) and attenuated by the protein kinase C inhibitor Gö6983 (3 μm). Hypoxia for 45 min caused a significant increase in the ratio of oxidised to reduced glutathione (GSSG/GSH). HPV was unaffected by the NADPH oxidase inhibitor VAS2870 (10 μm), whereas phase 2 was inhibited but phase 1 was unaffected by the antioxidants ebselen (100 μm) and TEMPOL (3 mm). We conclude that both phases of HPV in this model are mainly dependent on [Ca2+]i release from the sarcoplasmic reticulum. Neither phase of HPV requires voltage-gated Ca2+ entry, but SOCE contributes to phase 2. We can detect no requirement for cyclic ADP ribose, NAADP-dependent lysosomal Ca2+ release, activation of the CaSR, or displacement of FKBP12.6 from RyR2 for either phase of HPV. Sustained HPV is associated with an oxidising shift in the GSSG/GSH redox potential and is inhibited by the antioxidants ebselen and TEMPOL, consistent with the concept that it requires an oxidising shift in the cell redox state or the generation of reactive oxygen species.
authors.affiliation: P. I. Aaronson: Room 1.19, Henriette Raphael House, Guy's Campus, King's College London, London SE1 9HN, UK. philip.aaronson@kcl.ac.uk.
authors.forename: Michelle J
authors.initials: MJ
authors.lastname: Connolly
authors.forename: Jesus
authors.initials: J
authors.lastname: Prieto-Lloret
authors.forename: Silke
authors.initials: S
authors.lastname: Becker
authors.forename: Jeremy P T
authors.initials: JP
authors.lastname: Ward
authors.forename: Philip I
authors.initials: PI
authors.lastname: Aaronson
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D020013:Calcium Signaling; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D005978:Glutathione / Q000378:metabolism; D000860:Hypoxia / Q000378:metabolism*; D008297:Male; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011651:Pulmonary Artery / Q000166:cytology / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D044169:Receptors, Calcium-Sensing / Q000037:antagonists & inhibitors; D012433:Ryanodine / Q000494:pharmacology; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D014661:Vasoconstriction
chemical_list: D000975:Antioxidants; D002121:Calcium Channel Blockers; D047428:Protein Kinase Inhibitors; D044169:Receptors, Calcium-Sensing; D012433:Ryanodine; D000252:Calcium-Transporting ATPases; D005978:Glutathione; D002118:Calcium