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Freshly isolated bovine coronary endothelial cells do not express the BK Ca channel gene
Kathryn M Gauthier1, Caiqiong Liu, Aleksandra Popovic
1Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.
Insights
High-conductance calcium-activated potassium (BKCa) channels are expressed in bovine coronary artery smooth muscle cells (SMCs), but not in endothelial cells (ECs). This study confirms cell-specific BKCa channel gene and protein expression in arteries.
Area of Science:
- Cardiovascular Biology
- Ion Channel Physiology
- Cellular Biology
Background:
- Vascular function relies on ion channel activity in endothelial cells (ECs) and smooth muscle cells (SMCs).
- The role of Ca(2+)-activated K(+) channels, specifically the high-conductance BK(Ca) channel, in distinct vascular cell types remains incompletely understood.
- Previous studies suggested cell-specific expression patterns of ion channels within arteries.
Purpose of the Study:
- To directly compare the expression of the BK(Ca) channel in vascular ECs and SMCs from bovine coronary arteries.
- To investigate the mRNA, protein, and functional presence of the BK(Ca) channel in isolated ECs and SMCs.
- To determine if BK(Ca) channels are selectively localized to either ECs or SMCs within the same artery.
Main Methods:
- Isolation and separation of ECs and SMCs from bovine coronary arteries, verified by immunofluorescence (alpha-actin for SMCs, PECAM for ECs).
- Detection of BK(Ca) channel alpha-subunit protein using sequence-specific antibodies.
- Assessment of BK(Ca) channel alpha-subunit mRNA expression via multicell RT-PCR.
- Electrophysiological recordings (whole-cell patch clamp) to measure outward K(+) currents and assess BK(Ca) channel function using iberiotoxin and NS 1619.
Main Results:
- BK(Ca) channel alpha-subunit protein was detected exclusively in SMCs, with ECs lacking the protein.
- mRNA encoding the BK(Ca) alpha-subunit was found only in SMCs, not in ECs.
- A significant iberiotoxin-sensitive BK(Ca) current was observed in SMCs but was absent in ECs.
- The BK(Ca) channel opener NS 1619 enhanced K(+) current specifically in SMCs.
Conclusions:
- Bovine coronary artery SMCs exhibit dense expression of BK(Ca) channels.
- Adjacent ECs in the same artery appear to lack the gene and protein expression of BK(Ca) channels.
- These findings demonstrate a clear cell-specific distribution of BK(Ca) channels in vascular SMCs and ECs.
Abstract:
Recent reports have suggested that different types of Ca(2+)-activated K(+) channels may be selectively expressed either in the vascular endothelial cells (ECs) or smooth muscle cells (SMCs) of a single artery. In this study, we directly compared mRNA, protein and functional expression of the high-conductance Ca(2+)-activated K(+) (BK(Ca)) channel between freshly isolated ECs and SMCs from bovine coronary arteries. Fresh ECs and SMCs were enzymatically isolated, and their separation verified by immunofluorescent detection of alpha-actin and platelet/endothelium cell adhesion molecule (PECAM) proteins, respectively. Subsequently, studies using a sequence-specific antibody directed against the pore-forming alpha-subunit of the BK(Ca) channel only detected its expression in the SMCs, whereas PECAM-positive ECs were devoid of the alpha-subunit protein. Additionally, multicell RT-PCR performed using cDNA derived from either SMCs or ECs only detected mRNA encoding the BK(Ca) alpha-subunit in the SMCs. Finally, whole-cell recordings of outward K(+) current detected a prominent iberiotoxin-sensitive BK(Ca) current in SMCs that was absent in ECs, and the BK(Ca) channel opener NS 1619 only enhanced K(+) current in the SMCs. Thus, bovine coronary SMCs densely express BK(Ca) channels whereas adjacent ECs in the same artery appear to lack the expression of the BK(Ca) channel gene. These findings indicate a cell-specific distribution of Ca(2+)-activated K(+) channels in SMCs and ECs from a single arterial site.