Abstract
During sympathetic neurotransmitter release, there is evidence for differential modulation of cotransmitter release by endothelin (ET)-1. Using nerve growth factor (NGF)-differentiated PC12 cells, the effects of ET-1 on K+-stimulated release of ATP, dopamine (DA), and neuropeptide Y (NPY) were quantified using high-pressure liquid chromatography or radioimmunoassay. ET-1, in a concentration-dependent manner, inhibited the release of ATP, but not DA and NPY. Preincubation with the ETA/B antagonist, PD 142893 (N-acetyl-β-phenyl-d-Phe-Leu-Asp-Ile-Ile-Trp), reversed the inhibitory effect of ET-1 on ATP release, which remained unaffected in the presence of the ETA-specific antagonist BQ123 [cyclo(d-Asp-Pro-d-Val-Leu-d-Trp)]. The ETB agonists, sarafotoxin 6c (Cys-Thr-Cys-Asn-Asp-Met-Thr-Asp-Glu-Glu-Cys-Leu-Asn-Phe-Cys-His-Gln-Asp-Val-Ile-Trp), BQ 3020 (N-acetyl-[Ala11,15]-endothelin 1 fragment 6-21Ac-Leu-Met-Asp-Lys-Glu-Ala-Val-Tyr-Phe-Ala-His-Leu-Asp-IIe-IIe-Trp), and IRL 1620 (N-succinyl-[Glu9, Ala11,15]-endothelin 1 fragment 8-21Suc-Asp-Glu-Glu-Ala-Val-Tyr-Phe-Ala-His-Leu-Asp-Ile-Ile-Trp), decreased K+-stimulated release of ATP in a dose-dependent manner, and this effect was reversed by the ETB antagonists RES 701-1 [cyclic (Gly1-Asp9) (Gly-Asn-Trp-His-Gly-Thr-Ala-Pro-Asp-Trp-Phe-Phe-Asn-Tyr-Tyr-Trp)] and BQ 788 (N-[N-[N-[(2,6-dimethyl-1-piperidinyl)carbonyl]-4-methyl-l-leucyl]-1-(methoxycarbonyl)-d-tryptophyl]-d-norleucine sodium salt). Preincubation of PC12 cells with pertussis toxin reversed the ET-1-induced inhibition of the K+-evoked ATP release. Real-time intracellular calcium level recordings were performed on PC-12 cell suspensions, and ET-1 induced a dose-dependent decrease in the K+-evoked calcium levels. Nifedipine, the L-type voltage-dependent Ca2+ channel antagonist, caused inhibition of the K+-stimulated ATP release, but the N-type Ca2+ channel antagonist, ω-conotoxin GVIA, did not reverse the effect on ATP release. These data suggest that ET-1 modulates the release of ATP via the ETB receptor and its associated Gi/o G-protein through attenuation of the influx of extracellular Ca2+ through L-type channels.
Footnotes
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This work was supported by U.S. Public Health Service Grants NHLBI 61836 and 60260.
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doi:10.1124/jpet.104.081075.
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ABBREVIATIONS: NE, norepinephrine; NPY, neuropeptide Y; ET, endothelin; NGF, nerve growth factor; DMEM, Dulbecco's modified Eagle's medium; ir, immunoreactivity; PTX, pertussis toxin; HPLC, high-pressure liquid chromatography; AM, acetoxymethyl ester; PD 142893, N-acetyl-β-phenyl-d-Phe-Leu-Asp-Ile-Ile-Trp; BQ123, cyclo(d-Asp-Pro-d-Val-Leu-d-Trp); sarafotoxin 6c, Cys-Thr-Cys-Asn-Asp-Met-Thr-Asp-Glu-Glu-Cys-Leu-Asn-Phe-Cys-His-Gln-Asp-Val-Ile-Trp; BQ 3020, N-acetyl-[Ala11,15]-endothelin 1 fragment 6-21Ac-Leu-Met-Asp-Lys-Glu-Ala-Val-Tyr-Phe-Ala-His-Leu-Asp-IIe-IIe-Trp; IRL 1620, N-succinyl-[Glu9, Ala11,15]-endothelin 1 fragment 8-21Suc-Asp-Glu-Glu-Ala-Val-Tyr-Phe-Ala-His-Leu-Asp-Ile-Ile-Trp; BQ 788, N-[N-[N-[(2,6-dimethyl-1-piperidinyl)carbonyl]-4-methyl-l-leucyl]-1-(methoxycarbonyl)-d-tryptophyl]-d-norleucine sodium salt; ANOVA, analysis of variance; CTX, ω-conotoxin; RES 701-1, cyclic (Gly1-Asp9)(Gly-Asn-Trp-His-Gly-Thr-Ala-Pro-Asp-Trp-Phe-Phe-Asn-Tyr-Tyr-Trp); BIBP3226, N2-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]-d-arginine amide; BSS, buffered salt solution.
- Received November 19, 2004.
- Accepted January 25, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
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