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. 2002 Dec 15;545(3):829-36.
doi: 10.1113/jphysiol.2002.029843.

Freshly isolated bovine coronary endothelial cells do not express the BK Ca channel gene

Kathryn M Gauthier  1 , Caiqiong Liu, Aleksandra Popovic, Sulayma Albarwani, Nancy J Rusch
Affiliations

Freshly isolated bovine coronary endothelial cells do not express the BK Ca channel gene

Kathryn M Gauthier et al. J Physiol. .

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.

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Figures

Figure 1
Figure 1. Immunological characterization of α-actin and PECAM expression in smooth muscle cells (SMCs) and endothelial cells (ECs) of bovine coronary arteries
A and B, Nomarski and corresponding fluorescent images of histological sections of coronary arteries probed for either α-actin (red) or PECAM (red). Nuclei were fluorescently labelled with DAPI (blue). α-Actin labelling was limited to the SMCs of the medial layer, whereas PECAM labelling was limited to the EC layer. C and D, Western immunoblot analysis of α-actin (45 kDa) and PECAM (130 kDa) expression in coronary SMC and EC membrane protein. An immunoreactive band corresponding to α-actin (45 kDa) was observed only in the three left lanes loaded with SMC membrane protein, whereas a band corresponding to PECAM (130 kDa) was observed only in the three right lanes loaded with EC membrane protein. Each lane was loaded with 2.5 μg protein. E-H, Nomarski and corresponding fluorescent images of SMCs and ECs probed for either α-actin (red) or PECAM (red). Nuclei were labelled with DAPI (blue). Positive labelling for α-actin was detected only in SMCs, and positive labelling for PECAM was only detected in the ECs.
Figure 2
Figure 2. Immunological analysis of BKCaα-subunit expression in SMCs and ECs from bovine coronary arteries
A, Western immunoblot analysis showed an immunoreactive band corresponding to the BKCaα-subunit (125 kDa) in the three left lanes loaded with SMC membrane protein, but not in the three right lanes loaded with EC membrane protein. B, the immunoreactive bands in SMC protein detected in two left control lanes (-CP) were not evident when the antibody directed against the α-subunit was preabsorbed with its antigenic competing peptide in the two right lanes (+CP). C, Nomarski and corresponding fluorescent images of SMCs and ECs labelled for the BKCaα-subunit (red). Nuclei were labelled with DAPI (blue). Only SMCs showed positive labelling for the BKCaα-subunit. D, the positive labelling for the BKCaα-subunit in SMCs was not present when the antibody was preabsorbed with its antigenic competing peptide (+CP).
Figure 3
Figure 3. The mRNA expression profile of α-actin, the BKCa channel α-subunit, and PECAM in SMCs (lanes 1-3) and ECs (lanes 4-6) from bovine coronary arteries
PCR-amplified products generated by specific primers were individually loaded into separate lanes. The grid at the bottom indicates which specific primers were used to obtain the product in the corresponding lane. Lanes 1-3, bands corresponding to mRNA for α-actin and the BKCaα-subunit were observed in lanes loaded with SMC PCR product, whereas PECAM was absent. Lanes 4-6, bands corresponding to α-actin and the BKCaα-subunit were not detected in lanes loaded with EC product, whereas PECAM was amplified from the same cDNA.
Figure 4
Figure 4. Effect of IBTX (100 nmol l−1) on whole-cell K+ currents in bovine coronary SMCs and Ecs
A and B, original traces of K+ current in a single SMC and EC. Currents were elicited by 8-mV depolarizing steps from -70 to +58 mV. IBTX partially blocked the outward current in the SMC. The low level of K+ current observed in the EC cell was not reduced by IBTX. Cell capacitance values were 24.5 pF (SMC) and 9.5 pF (EC). C, the effect of IBTX on the averaged I-V relationship in freshly isolated SMCs and ECs. The high density of K+ current in SMCs was partially blocked by IBTX (n = 12). ECs showed a low density of outward K+ current that was not affected by IBTX (n = 11). Inset, amplification of the ordinate to enhance the visualization of the averaged EC current density plotted in the main graph. Average cell capacitance values were 20.4 ± 2.3 pF (SMCs) and 10.9 ± 1.5 pF (ECs). •, SMC control; ○, SMC IBTX; ▾, EC control; ▿, EC IBTX. * Current density was significantly different compared to control values at the same voltage. P≤ 0.05.
Figure 5
Figure 5. Effect of NS 1619 (30 μmol l−1) on whole-cell K+ current in bovine coronary SMCs and ECs
A and B, original traces of K+ current in a single SMC and EC. NS 1619 increased the outward current in the SMC. The outward current observed in the EC was not increased by NS 1619. Cell capacitances were 23.4 and 11.5 pF. C, the effect of NS 1619 on the averaged I-V relationship in freshly isolated SMCs and ECs. NS 1619 significantly increased K+ current density in SMCs (n = 9). The K+ current in the ECs was not increased by NS 1619 (n = 9). Inset, amplification of the ordinate to enhance the visualization of the averaged EC current density plotted in the main graph. Average cell capacitance values were 17.7 ± 1.1 pF (SMCs) and 9.4 ± 1.2 pF (ECs). •, SMC control; ○, SMC NS 1619; ▾, EC control; ▿, EC NS 1619. * Current density was significantly different compared to control values at the same voltage. P≤ 0.05.
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