Abstract
The novel endocannabinoid-like lipid N-arachidonoyl l-serine (ARA-S) causes vasodilation through both endothelium-dependent and -independent mechanisms. We have analyzed the vasorelaxant effect of ARA-S in isolated vascular preparations and its effects on Ca2+-activated K+ currents in human embryonic kidney cells stably transfected with the α-subunit of the human, large conductance Ca+-activated K+ (BKCa) channel [human embryonic kidney (HEK) 293hSlo cells]. ARA-S caused relaxation of rat isolated, intact and denuded, small mesenteric arteries preconstricted with (R)-(-)-1-(3-hydroxyphenyl)-2-methylaminoethanol hydrochloride (pEC50, 5.49 and 5.14, respectively), whereas it caused further contraction of vessels preconstricted with KCl (pEC50, 5.48 and 4.82, respectively). Vasorelaxation by ARA-S was inhibited by 100 nM iberiotoxin. In human embryonic kidney cells stably transfected with the α-subunit of the human BKCa channel cells, ARA-S and its enantiomer, N-arachidonoyl-d-serine, enhanced the whole-cell outward K+ current with similar potency (pEC50, 5.63 and 5.32, respectively). The potentiation was not altered by the β1 subunit or mediated by ARA-S metabolites, stimulation of known cannabinoid receptors, G proteins, protein kinases, or Ca2+-dependent processes; it was lost after patch excision or after membrane cholesterol depletion but was restored after cholesterol reconstitution. BKCa currents were also enhanced by N-arachidonoyl ethanolamide (pEC50, 5.27) but inhibited by another endocannabinoid, O-arachidonoyl ethanolamine (pIC50, 6.35), or by the synthetic cannabinoid O-1918 [(-)-1,3-dimethoxy-2-(3-3,4-trans-p-menthadien-(1,8)-yl)-orcinol] (pIC50, 6.59), which blocks ARA-S-induced vasodilation. We conclude the following. 1) ARA-S directly activates BKCa channels. 2) This interaction does not involve cannabinoid receptors or cytosolic factors but is dependent on the presence of membrane cholesterol. 3) Direct BKCa channel activation probably contributes to the endothelium-independent component of ARA-S-induced mesenteric vasorelaxation. 4) O-1918 is a BKCa channel inhibitor.
Footnotes
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This study was supported in part by the Intramural Research Program of the National Institutes of Health, National Institute on Alcohol Abuse and Alcoholism.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.108.144717.
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ABBREVIATIONS: CB, cannabinoid; anandamide, N-arachidonoyl ethanolamide; TRPV, transient receptor potential vanilloid; ARA-S, N-arachidonoyl l-serine; BKCa, large conductance Ca+-activated K+; O-1918, (-)-1,3-dimethoxy-2-(3–3,4-trans-p-menthadien-(1,8)-yl)-orcinol; virodhamine, O-arachidonoyl ethanolamine; HEK, human embryonic kidney; HEK293hSlo, human embryonic kidney cells stably transfected with the α-subunit of the human BKCa channel; DMEM, Dulbecco's modified Eagle's medium; ES, external solution; IS, internal solution; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; 2-AG, 2-arachidonoyl glycerol; 11,12-EET, (±)11(12)-epoxy 5Z,8Z,14Z-eicosatrienoic acid; NS 1619, 1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one; PI(4,5)P2, l-α-phosphatidyl-d-myo-inositol-4,5-bisphosphate triammonium salt; RT, reverse transcriptase; PCR, polymerase chain reaction; rapamycin, 23,27-epoxy-3H-pyrido[2,1-c][1,4]oxaazacyclohentriacontine; URB597, (3′-aminocarbonyl)[1,1′-biphenyl]-3-yl)-cyclohexylcarbamate; 14,15-EEZE, 14,15-epoxyeicosa-5(Z)-enoic acid; indomethacin, 1-(4-chlorobenzoyl)-5-methoxy-2-methyl-3-indoleacetic acid; NDGA, nordihydroguaiaretic acid; phenylephrine, (R)-(-)-1-(3-hydroxyphenyl)-2-methylaminoethanol hydrochloride; GDP-β-S, guanosine 5′-[β-thio]diphosphate; NPo, open probability of n channels; HEK293hSloβ1, HEK293hSlo cells cotransfected with β1 subunit.
- Received August 11, 2008.
- Accepted October 7, 2008.
- U.S. Government work not protected by U.S. copyright
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