Abstract
Chronic hypoxia (CH)-induced pulmonary hypertension is associated with decreased basal pulmonary artery endothelial cell (EC) Ca2+, which correlates with reduced store-operated Ca2+ (SOC) entry. Protein kinase C (PKC) attenuates SOC entry in ECs. Therefore, we hypothesized that PKC has a greater inhibitory effect on EC SOC and receptor-operated Ca2+ entry after CH. To test this hypothesis, we assessed SOC in the presence or absence of the nonselective PKC inhibitor GF109203X [2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)maleimide] in freshly isolated, Fura-2-loaded ECs obtained from intrapulmonary arteries of control and CH rats (4 weeks at 0.5 atm). We found that SOC entry and 1-oleoyl-2-acetyl-sn-glycerol (OAG)- and ATP-induced Ca2+ influx were attenuated in ECs from CH rats versus controls, and GF109203X restored SOC and OAG responses to the level of controls. In contrast, nonselective PKC inhibition with GF109203X or the selective PKCε inhibitor myristoylated V1-2 attenuated ATP-induced Ca2+ entry in ECs from control but not CH pulmonary arteries. ATP-induced Ca2+ entry was also attenuated by the T-type voltage-gated Ca2+ channel (VGCC) inhibitor mibefradil in control cells. Consistent with the presence of endothelial T-type VGCC, we observed depolarization-induced Ca2+ influx in control cells that was inhibited by mibefradil. This response was largely absent in ECs from CH arteries. We conclude that CH enhances PKC-dependent inhibition of SOC- and OAG-induced Ca2+ entry. Furthermore, these data suggest that CH may reduce the ATP-dependent Ca2+ entry that is mediated, in part, by PKCε and mibefradil-sensitive Ca2+ channels in control cells.
Footnotes
This work is supported by the American Heart Association [Grant 0615551Z]; and the National Institutes of Health National Heart, Lung, and Blood Institute [Grants HL58124, HL63207, HL77876, HL88192].
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.110.165563.
↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
-
ABBREVIATIONS:
- CH
- chronic hypoxia
- PKC
- protein kinase C
- [Ca2+]i
- intracellular calcium concentration
- EC
- endothelial cell
- SOC
- store-operated Ca2+
- CPA
- cyclopiazonic acid
- OAG
- 1-oleoyl-2-acetyl-sn-glycerol
- ROC
- receptor-operated Ca2+
- VGCC
- voltage-gated Ca2+ channel
- VSM
- vascular smooth muscle
- TRP
- transient receptor potential
- DAG
- diacylglycerol
- HBSS
- HEPES-buffered saline solution
- PECAM-1
- platelet/endothelial cell adhesion molecule-1
- IP3
- inositol trisphosphate
- V1-2myr
- myristoylated V1-2
- GF109203X
- 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)maleimide
- Gö6976
- 5,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo[2,3-a]py rrolo[3,4-c]carbazole-12-propanenitrile
- SKF96365
- 1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl]-1H-imidazole hydrochloride
- U73122
- 1-[6-[[(17b)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione
- U73343
- 1-[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-pyrrolidine-2,5-dione.
- Received January 5, 2010.
- Accepted June 23, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
JPET articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|