Abstract
Large-conductance, calcium- and voltage-activated potassium (BKCa) channels hyperpolarize coronary artery smooth muscle cells, causing vasorelaxation. Dopamine activates BKCa channels by stimulating D1-like receptor-mediated increases in cAMP in porcine coronary artery myocytes. There are two D1-like receptors (R), D1R and D5R. We hypothesize that the specific D1-like receptor involved in BKCa channel activation in human coronary artery smooth muscle cells (HCASMCs) is the D5R and that activation occurs via cAMP cross-activation of cGMP-dependent protein kinase (PKG), rather than cAMP-dependent protein kinase (PKA). The effects of D1-like receptor agonists and antagonists on BKCa channel opening in HCASMCs were examined in the presence and absence of PKG/PKA inhibition by cell-attached patch clamp. In the absence of commercially available ligands specific for D1R or D5R, D1R or D5R protein was down-regulated by transfecting HCASMCs with human D1R or D5R antisense oligonucleotides, respectively: cells transfected with scrambled oligonucleotides and nontransfected HCASMCs served as controls. The predominant ion channel conducting outward currents in nontransfected HCASMCs was identified as the large-conductance, calcium- and voltage-activated potassium (BKCa) channel, which was activated by D1-like receptor agonists despite PKA inhibition with (9R,10S,12S)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid (KT 5720) (300 nM), but was abolished by inhibiting PKG with 9-methoxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b-11a-triazadibenzo(a,g) cycloocta(cde)-trinden-1-one (KT 5823) (300 nM). D1-like receptor agonists activated BKCa channels in all transfected cells except those transfected with D5R antisense oligonucleotides. Thus, the dopamine (D1-like) receptor mediates activation of BKCa channels in HCASMCs by D5R, not D1R, and via PKG, not PKA. This is the first report of differential D1-like receptor regulation of vascular smooth muscle function in human cells.
Footnotes
This work was supported by the National Institutes of Health National Center for Research Resources [Grant RR17613] (Mentored Clinical Research Scholar Program); the Department of Pediatrics, Georgetown University Hospital, Washington, DC (to A.N.); the American Heart Association [Grant AHA00078] (to G.H.); the National Institutes of Health National Heart, Lung, and Blood Institute [Grants R01-HL093429, 073890, HL074940, HL068686, HL092196, R37-HL023081] (to S.C., R.W., P.J., and Y.U.P.); and the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grant DK039308] (to P.J. and Y.U.P.).
Parts of this work were presented previously: Natarajan A, Han G, White RE, and Jose PA (2006) D5 receptor mediates dopamine effects on BK(Ca) channels in human coronary artery smooth muscle cells. FASEB J 20:A301; and Natarajan A, Han G, White R, and Jose PA (2006) The human D5 dopamine receptor mediates big K(Ca) channel activity in human coronary artery smooth muscle cells. Hypertension 48:e80.
This work was part of a Ph.D. dissertation in physiology and biophysics: Natarajan A (November 2008) Mechanism of Dopamine-Mediated Activation of BK Channels in Human Coronary Artery Smooth Muscle Cells, Ph.D. dissertation, Georgetown University, Washington, DC.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.109.159871
The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
-
ABBREVIATIONS:
- R
- receptor
- KATP
- ATP-sensitive
- BKCa
- large conductance, calcium- and voltage-activated potassium
- PKA
- cAMP-dependent protein kinase
- PKG
- cGMP-dependent protein kinase
- PLC
- phospholipase C
- PKC
- protein kinase C
- HCASMC
- human coronary artery smooth muscle cell
- RT-PCR
- reverse transcription-polymerase chain reaction
- PCR
- polymerase chain reaction
- qRT-PCR
- quantitative real-time-polymerase chain reaction
- GAPDH
- glyceraldehyde-3-phosphate dehydrogenase
- Scr
- scrambled; AS; antisense
- I/O
- inside-out
- BAPTA
- 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid
- NPo
- number of channels × single-channel open probability
- SCH 23390
- (R)-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H–3-benzazepine
- KT 5720
- (9R,10S,12S)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid
- KT 5823
- 9-methoxy-9-methoxycarbonyl-8-methyl-2,3,9, 10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b-11a-triazadibenzo(a,g) cycloocta(cde)-trinden-1-one
- SKF 81297
- (R)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine)
- PIP2
- phosphatidylinositol 4,5-bisphosphate
- IP3
- inositol 1,4,5-trisphosphate
- bp
- base pair(s)
- ANOVA
- analysis of variance.
- Received August 4, 2009.
- Accepted October 27, 2009.
- 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.
|