Epoxyeicosatrienoic acid relaxing effects involve Ca2+-activated K+ channel activation and CPI-17 dephosphorylation in human bronchi.
The aim of the present study was to provide a mechanistic insight into how 14,15-epoxyeicosatrienoic acid (EET) relaxes organ-cultured human bronchi. Tension measurements, performed on either fresh or 3-d-cultured bronchi, revealed that the contractile responses to 1 microM methacholine and 10 microM arachidonic acid were largely relaxed by the eicosanoid regioisomer in a concentration-dependent manner (0.01-10 microM). Pretreatments with 14,15-epoxyeicosa-5(Z)-enoic acid, a specific 14,15-EET antagonist, prevented the relaxing effect, whereas iberitoxin pretreatments (10 nM) partially abolished EET-induced relaxations. In contrast, pretreatments with 1 microM indomethacin amplified relaxations in explants and membrane hyperpolarizations triggered by 14,15-EET on airway smooth muscle cells. The relaxing responses induced by 14,15-EET were likely related to reduced Ca2+ sensitivity of the myofilaments, because free Ca2+ concentration-response curves performed on beta-escin-permeabilized cultured explants were shifted toward higher [Ca2+] (lower pCa2+ values). 14,15-EET also abolished the tonic responses induced by phorbol-ester-dybutyrate (PDBu) (a protein kinase C [PKC]-sensitizing agent), on both fresh (intact) and beta-escin-permeabilized explants. Western blot analyses, using two specific primary antibodies against CPI-17 and its PKC-dependent phosphorylated isoform (p-CPI-17), confirmed that the eicosanoid interferes with this intracellular process. These data indicate that 14,15-EET hyperpolarizes airway smooth muscle cells and relaxes precontracted human bronchi while reducing Ca2+ sensitivity of fresh and cultured explants. The intracellular effects are related to a PKC-dependent process involving a lower phosphorylation level of CPI-17.
Morin
Caroline
C
Le Bilarium, Department of Physiology and Biophysics, Service of Thoracic Surgery, Faculty of Medicine and Health Sciences, University of Sherbrooke, 3001 12th Avenue North, Sherbrooke, PQ, J1H 5N4 Canada.
Sirois
Marco
M
Echave
Vincent
V
Gomes
Marcio M
MM
Rousseau
Eric
E
D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D000577:Amides / Q000494:pharmacology; D001980:Bronchi / Q000187:drug effects* / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins; D015640:Ion Channel Gating / Q000187:drug effects*; D008564:Membrane Potentials / Q000187:drug effects; D009124:Muscle Proteins; D009126:Muscle Relaxation / Q000187:drug effects*; D009129:Muscle Tonus / Q000187:drug effects; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000187:drug effects; D010703:Phorbol Esters / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011188:Potassium / Q000494:pharmacology; D024681:Potassium Channels, Calcium-Activated / Q000378:metabolism*
D000577:Amides; D047908:Intracellular Signaling Peptides and Proteins; D009124:Muscle Proteins; C121756:N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide; C107507:PPP1R14A protein, human; D010703:Phorbol Esters; D024681:Potassium Channels, Calcium-Activated; C046782:14,15-epoxy-5,8,11-eicosatrienoic acid; C067407:14,15-episulfide eicosatrienoic acid; D010749:Phosphoprotein Phosphatases; D015126:8,11,14-Eicosatrienoic Acid; D011188:Potassium; D002118:Calcium