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. 2002 Nov 1;545(Pt 3):829–836. doi: 10.1113/jphysiol.2002.029843

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

Kathryn M Gauthier *, Caiqiong Liu *, Aleksandra Popovic *, Sulayma Albarwani *, Nancy J Rusch *
PMCID: PMC2290710  PMID: 12482889

Abstract

Recent reports have suggested that different types of Ca2+-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 Ca2+-activated K+ (BKCa) 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 α-actin and platelet/endothelium cell adhesion molecule (PECAM) proteins, respectively. Subsequently, studies using a sequence-specific antibody directed against the pore-forming α-subunit of the BKCa channel only detected its expression in the SMCs, whereas PECAM-positive ECs were devoid of the α-subunit protein. Additionally, multicell RT-PCR performed using cDNA derived from either SMCs or ECs only detected mRNA encoding the BKCa α-subunit in the SMCs. Finally, whole-cell recordings of outward K+ current detected a prominent iberiotoxin-sensitive BKCa current in SMCs that was absent in ECs, and the BKCa channel opener NS 1619 only enhanced K+ current in the SMCs. Thus, bovine coronary SMCs densely express BKCa channels whereas adjacent ECs in the same artery appear to lack the expression of the BKCa channel gene. These findings indicate a cell-specific distribution of Ca2+-activated K+ channels in SMCs and ECs from a single arterial site.

Functional, molecular and electrophysiological assays indicate that high-conductance, Ca2+-activated K+ (BKCa) channels of the Slo gene family contribute to the regulation of coronary vascular tone. For example, pharmacological block of BKCa channels with iberiotoxin constricts isolated rat and human coronary arteries, and reduces endothelium-dependent dilation in canine epicardial arteries (Wellman et al. 1996; Node et al. 1997; Nishikawa et al. 1999; Marijic et al. 2001; Koch et al. 2001). BKCa channels of arterial smooth muscle cells (SMCs) are thought to primarily mediate the changes in vascular tone. These channels have been intensively studied in coronary SMCs, and the pore-forming α-subunit and ancillary β-subunit have been cloned and characterized (Tanaka et al. 1997).

In contrast, it is unclear if coronary endothelial cells (ECs) also express BKCa channels, although there is evidence for their existence. A voltage- and Ca2+-sensitive K+ channel showing a unitary conductance of 285 pS has been described in primary cultures of porcine coronary ECs (Baron et al. 1996). Its activation partially mediates the hyperpolarizing response to bradykinin, indicating a possible role in regulating the membrane potential of these cells. In contrast, whole-cell current attributable to the BKCa channel was not detected in second or third passages of ECs derived from human coronary macro- or microvessels (Zunkler et al. 1995), and also was not observed in freshly isolated ECs of guinea-pig coronary capillaries (Dittrich & Daut, 1999). In these cells, the regulation of membrane potential apparently relies on a strong component of inwardly rectifying K+ current (von Beckerath et al. 1996).

Notably, the detection of BKCa current in cultured ECs may reflect an upregulation of the channel by culture conditions. A recent report has demonstrated that mRNA encoding the BKCa channel α-subunit is absent in freshly isolated human capillary ECs, whereas conditioning the same ECs with culture media or plating them at high density induced its expression (Jow et al. 1999). This report implies that cultured ECs may not express a normal complement of K+ channels, and emphasizes the importance of examining the profile of K+ channel expression in intact or fresh ECs. Notably, the question of whether BKCa channels in ECs contribute to the regulation of coronary tone cannot be resolved by vascular reactivity studies. In vascular preparations with intact endothelium, pharmacological inhibitors simultaneously block both EC and SMC K+ channels, whereas only the functional contribution of SMC K+ channels to vascular tone can be evaluated in endothelium-denuded preparations.

To clarify the location of BKCa channels in coronary arteries, the present study compared the expression levels of BKCa channel mRNA, protein and iberiotoxin-sensitive current between freshly isolated bovine coronary ECs and SMCs. Small bovine coronary arteries were used as the source of ECs and SMCs, because BKCa channels within the arterial wall are known to mediate vascular tone (Campbell et al. 1996, 2002; Li et al. 1997). Also, the arteries were large enough to provide adequate ECs for a multifaceted analysis of BKCa channel expression. Finally, because SMCs of bovine coronary arteries densely express BKCa channels (Li et al. 1997; Campbell et al. 2002), the SMCs provided a positive control for antibody and molecular probes used to determine the level of BKCa channel expression in the adjacent ECs.

Methods

Coronary artery preparation

Fresh bovine hearts were obtained from a local slaughterhouse and immediately placed on ice. Epicardial coronary arteries were dissected from the left ventricle, cleaned of adhering fat and connective tissue and placed in iced physiological salt solution (PSS) of the following composition (mmol l−1): 119 NaCl, 4.7 KCl, 1.6 CaCl2, 1.17 MgSO4, 5.5 glucose, 24 NaHCO3, 1.18 NaH2PO4, 5.8 Hepes and 0.026 EDTA.

Immunohistochemistry

Coronary arteries (i.d. = 800-1000 μm) were fixed in PSS containing 4 % paraformaldehyde for 1 h, filled with PSS containing 2 % agar, and embedded in an agarose-containing cartridge on dry ice. Using a cryostat, the arteries were cut into 6 μm sections and placed on glass slides etched with 8 % nitric acid. Immunolabelling was performed using methods modified from Song et al. (1999). Briefly, the sections were refixed in 4 % paraformaldehyde for 30 min, and permeabilized by incubating with 0.2 % Triton X-100 for 15 min. Sections were incubated with either anti-α-actin (Sigma), anti-PECAM (kindly provided by Dr Peter Newman), or anti-α913-926 (kindly provided by Dr Hans-Guenther Knaus). The latter antibody is directed against amino acids 913-926 in the carboxyl terminus of the pore-forming α-subunit of the BKCa channel (Liu et al. 1997, 1998). Primary antibodies were diluted 1 : 100 in 0.2 % Triton X-100 containing 1 % normal goat serum, and the sections were incubated overnight at 4 °C, rinsed, and incubated with the appropriately labelled secondary antibody (1 : 200 anti-mouse or anti-rabbit, Alexa-Fluor 594, Molecular Probes) for 1 h at 25 °C. The slides were then rinsed and incubated with 1 % 4,6-diamidino-2-phenylindole (DAPI, Sigma) for 5 min. After a final rinse, the slides were edged with mounting media (Immuno Fluor, ICN) and protected by a glass coverslip. Nomarski and fluorescent images were captured on the same day (× 400 magnification, Nikon Eclipse E600 microscope, Spot Advanced software).

Enzymatic isolation of ECs and SMCs

Bovine coronary ECs were isolated using a modification of Wohlfeil & Campbell (1997). Briefly, the left coronary artery and its branches were dissected from four bovine hearts, injected with PSS containing 2.5 mg ml−1 collagenase, and incubated for 30 min at 37 °C. The arteries were gently ‘milked’ by manual compression and flushed with 6-10 ml of buffer. The EC-containing solution was collected and centrifuged for 3 min at 1200 r.p.m. (450 g). The supernatant was removed and the cell pellet was used for protein isolation, or the ECs were resuspended in buffer for immunocytochemistry. SMCs were isolated from epicardial branches of the left coronary artery. The arteries were cut open laterally and the lumen gently scraped with a rubber spatula to remove the EC layer. The arterial sections were rinsed with PSS, and single SMCs were enzymatically isolated using techniques described in detail earlier (Jackson et al. 1997). A similar isolation method was used to obtain ECs and SMCs from a single artery for RT-PCR and patch-clamp studies. In this case, short arterial segments were exposed to mild enzyme treatment, and ECs were released by gentle trituration. The remaining SMCs were released by further incubation. The dissociated cells were maintained in PSS at 4 °C and used the same day.

Immunocytochemistry

Isolated ECs and SMCs were repeatedly centrifuged (450 g for 3 min) and rinsed with fresh PSS. Single drops of the cell suspensions were placed on glass slides, air-dried and fixed with 95 % ethanol. Immunocytological staining was performed using methods modified from Song et al. (1999). The fixed cells were rinsed with 0.2 % bovine serum albumin (BSA) in PBS, and incubated with one of three antibodies: anti-α913-926 (1 : 200), anti-α-actin (1 : 400), or anti-PECAM (1 : 400) for 45 min at 37 °C. Subsequently, the slides were rinsed, incubated with 5 % goat serum in 0.2 % BSA/PBS for 30 min at 37 °C, drained, and incubated with the appropriately labelled secondary antibody (1 : 100 goat anti-mouse, or 1 : 400 goat anti-rabbit) for an additional 30 min at 37 °C. The slides were rinsed and incubated with 1 % DAPI for 5 min at 37 °C. After a final rinse, the slides were edged in mounting media and protected with a glass coverslip. Cells incubated without primary antibody, without secondary antibody, with mouse ascites fluid or rabbit IgG were included as negative controls. Nomarski and fluorescent images were captured from the same cell preparations.

Western immunoblotting

The isolation of membrane protein and Western immunoblotting for the detection of the BKCa channels were performed as previously described, using anti-α913-926 as the primary antibody (Liu et al. 1997, 1998). Potential cross-contamination between EC and SMC proteins was evaluated using a monoclonal mouse antibody raised against α-actin (Sigma) that served as a SMC-specific marker, and a rabbit polyclonal antibody raised against PECAM that served as an EC-specific marker (Goldberger et al. 1994). Bound antibodies were detected by chemiluminescence (ECL, Amersham) on radiographic film.

Multi-cell RT-PCR

Single ECs and SMCs were aspirated (10-15 cells per aspiration) into patch pipettes that were placed tip-down in sterile Eppendorf tubes. The pipette tips were broken by gentle pressure to release the cell-containing solution and the samples were immediately frozen in liquid nitrogen. Cell lysis was accomplished by a freeze-thaw cycle, and reverse transcription (RT) was performed on the resulting cell lysates. The RT reaction solution was prepared according to the manufacturer's instructions (You-Prime-First-Strand Beads, Amersham), and 16 μl of this solution was added to each tube containing SMC or EC lysate. The tube was incubated at 37 °C for 2 h to permit reverse transcription, and the resulting cDNA-containing solution was divided into three 5-μl aliquots for PCR amplification of either α-actin, the BKCa channel α-subunit or PECAM from the same lysate. The cDNA was amplified using sequence-specific primer pairs for smooth muscle α-actin (GenBank NM-007392):

graphic file with name tjp0545-0829-mu1.jpg

Samples were incubated for 5 min at 95 °C, followed by 60 cycles of 1 min at 95 ° C, 1 min at 59 °C and 2 min at 72 °C. Amplified cDNA products were separated on a 2 % agarose gel containing ethidium bromide. A 100 bp DNA ladder (Life Technologies) served as a molecular size reference. Amplification of cDNA using specific primer pairs resulted in the following PCR products: α-actin (637 bp), BKCa channel α-subunit (407 bp), and PECAM (262 bp). Negative controls included amplifications performed using specific primers in the absence of cDNA, and amplifications that included cDNA but not specific primers. In addition, amplification was performed on RT product obtained from pipette solution aspirate without cells. Negative controls did not reveal unexpected products.

Patch-clamp recording of K+ current

Whole-cell recordings of K+ currents were obtained in freshly isolated ECs or SMCs using standard methods and a patch-clamp station previously described (Rusch et al. 1992; Jackson et al. 1997). Cells were dialysed with a pipette solution which contained (mmol l−1) 145 K+ glutamate, 1 MgCl2, 10 Hepes, 1 EGTA, 1 Na2ATP and 100 nmol l−1 ionized Ca2+ (pH 7.2), and perfused with a bath solution composed of (mmol l−1) 145 NaCl, 4 KCl, 1 MgCl2, 10 glucose, 10 Hepes and 2 CaCl2 (pH 7.4) at room temperature. Pipettes tip resistances averaged 4-8 MΩ. Briefly, macroscopic K+ currents were generated by progressive 8 mV depolarizing steps (500 ms duration, 5 s intervals) from a constant holding potential of -70 mV. BKCa currents were defined as the outward current inhibited by 100 nmol l−1 iberiotoxin (Sigma), a specific blocker of BKCa channels (Galvez et al. 1990). In some studies, the effect of 30 μmol l−1 NS 1619 (Research Biochemicals International), an activator of BKCa channels (Yamamura et al. 2001), was examined. NS 1619 was mixed as a 10 mmol l−1 stock in 50 % ethanol, and solvent controls were performed to rule out vehicle effects. Trials were performed in triplicate and averaged to estimate K+ current density. The membrane capacitance of each cell was estimated by integrating the capacitive current generated by a 10 mV hyperpolarizing pulse after electronic cancellation of pipette-patch capacitance.

Statistical analysis

All data are expressed as means ± s.e.m. Statistical comparisons between groups were made with one-way, repeated measures, analysis of variance with subsequent Student-Newman-Keuls post hoc analysis test. Significance was accepted at P < 0.05.

Results

Separation and identification of SMCs and ECs

Histological sections of bovine coronary arteries were labelled using either an antibody directed against SMC-specific α-actin or an antibody directed against the platelet and EC-specific adhesion molecule, PECAM. Nuclei were fluorescently labelled with 4,6-diamidino-2-phenylindole (DAPI). Nomarski images revealed characteristic EC and SMC layers in the coronary arterial wall (Fig. 1A and B). Only the SMC layers of the media showed positive staining for α-actin (Fig. 1A). In contrast, only the EC layer was positive for the presence of PECAM (Fig. 1B). Nuclear staining by DAPI (in blue) of the SMCs as well as the ECs was evident.

Figure 1. Immunological characterization of α-actin and PECAM expression in smooth muscle cells (SMCs) and endothelial cells (ECs) of bovine coronary arteries.

Figure 1

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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.

To determine if the enzymatic isolation techniques employed in the present study provided clear separation of SMC and EC proteins, Western immunoblotting was performed using anti-α-actin and anti-PECAM antibodies (Fig. 1C and D). Triplicate lanes loaded equally with either SMC or EC membrane proteins (2.5 μg each lane) and probed with anti-α-actin provided the expected 45 kDa band in SMC lanes, whereas weak immunoreactive bands were detected in EC lanes (Fig. 1C). Conversely, anti-PECAM revealed a 130 kDa band corresponding to the expected size of PECAM only in those lanes loaded with EC protein (Fig. 1D). Anti-α-actin and anti-PECAM also verified the identity of single SMCs and ECs chosen from their respective enzymatic digestions. Isolated SMCs exhibited intense α-actin immunofluorescence but were devoid of PECAM immunofluorescence (Fig. 1E and F). Conversely, α-actin was not detected in cells ‘stripped’ from the arterial lumen after mild enzymatic exposure, and presumed to represent ECs (Fig. 1G). Instead, approximately 90 % of these cells stained positive for PECAM (Fig. 1H). These findings verified the success of the EC and SMC enzymatic separation method, setting the stage to compare BKCa channel expression between the coronary EC and SMC isolations.

Expression of BKCa channel protein in coronary ECs and SMCs

The expression of BKCa channels was compared between freshly isolated coronary ECs and SMCs according to previous methods (Liu et al. 1997, 1998). Triplicate lanes loaded with 2.5 μg of SMC protein and incubated with anti-α913-926 revealed a 125 kDa band consistent with the size of the BKCa channel α-subunit. This band was absent in the adjacent triplicate lanes loaded with 2.5 μg of EC membrane proteins (Fig. 2A). Preabsorption of anti-α913-926 with 1 μmol l−1 of its antigenic competing peptide (+CP) abolished the 125 kDa band in the SMC membranes, indicating specificity of the antibody for its putative epitope (Fig. 2B). Subsequently, the apparent lack of BKCa α-subunit expression in ECs was confirmed at the single-cell level. Fluorescent images of SMCs and ECs labelled with anti-α913-926 detected the BKCa channel protein in SMCs but not ECs (Fig. 2C). Preabsorption of anti-α913-926 with 1 μmol l−1 of its antigenic competing peptide (+CP) eliminated the fluorescent signal corresponding to the BKCa α-subunit in single SMCs (Fig. 2D).

Figure 2. Immunological analysis of BKCaα-subunit expression in SMCs and ECs from bovine coronary arteries.

Figure 2

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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).

Expression of BKCa channel mRNA in SMCs and ECs

To determine if mRNA encoding the BKCa α-subunit was expressed in coronary ECs, total RNA was isolated from ten to fifteen SMCs or ECs aspirated into a patch pipette to ensure clean separation of cell types for multi-cell RT-PCR studies (Fig. 3). The cDNAs obtained by RT were amplified using primers specific for the BKCa α-subunit sequence. Primers specific for α-actin and PECAM sequences were utilized to confirm the purity of SMC and EC cDNA preparations. As expected, amplified products corresponding to mRNAs encoding α-actin (637 bp) and the BKCa α-subunit (407 bp) were identified in SMC preparations (Fig. 3, lanes 1 and 2), whereas PECAM (262 bp) was not detected (Fig. 3, lane 3). Overall, eight of eleven (73 %) cDNA samples from SMCs that were positive for α-actin also were positive for the BKCa α-subunit transcript. In contrast, amplification of cDNA derived from eleven EC lysates, which were positive for PECAM and negative for α-actin, were negative for the BKCa α-subunit in each instance (Fig. 3, lanes 4-6).

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.

Figure 3

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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.

Patch-clamp evaluation of BKCa current in SMCs and ECs

Patch-clamp studies were performed to verify that the apparent absence of mRNA and protein for the BKCa α-subunit in coronary ECs corresponded to an absence of BKCa current. Whole-cell K+ currents were elicited by 8 mV depolarizing steps from -70 to +58 mV to compare the density of BKCa current between freshly isolated bovine coronary SMCs and ECs. Preliminary studies confirmed that equimolar replacement of K+ with Na+ in the pipette solution nearly eliminated outward current in ECs (n = 4) and SMCs (n = 3), identifying K+ as the charge carrier (data not shown). Membrane capacitance also was significantly higher in SMCs than in ECs, averaging 19.1 ± 1.4 (n = 21) and 10.2 ± 1.0 pF (n = 20), respectively.

The families of K+ currents generated in SMCs were partially blocked by 100 nmol l−1 iberiotoxin (IBTX), indicating a contribution of BKCa channels to macroscopic current (Fig. 4A). In contrast, ECs showed only low levels of K+ current that were insensitive to IBTX-induced block (Fig. 4B). The current-voltage (I-V) relationships from 12 SMCs and 11 ECs indicated that IBTX significantly reduced K+ current density in SMCs from 26.9 ± 4.2 to 9.6 ± 1.3 pA pF−1, resulting in a 64 % reduction (Fig. 4C). In contrast, EC outward current density averaged 3.1 ± 0.7 pA pF−1, which was 8.7-fold less than SMCs, and the EC current density was not significantly altered by IBTX (Fig. 4C, rescaled in inset).

Figure 4. Effect of IBTX (100 nmol l−1) on whole-cell K+ currents in bovine coronary SMCs and Ecs.

Figure 4

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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.

The BKCa activator NS 1619 (30 μmol l−1) was added to the patch-clamp chamber as a strategy to amplify BKCa current in SMCs and ECs. Outward K+ current increased in SMCs in response to NS 1619, whereas K+ current in freshly isolated ECs was unchanged (Fig. 5A and B, respectively). The I-V relationships from nine SMCs and nine ECs show that NS 1619 significantly increased SMC maximal current density by 70 % from 19.7 ± 2.7 to 33.4 ± 4.0 pA pF−1, whereas EC K+ current was insensitive to the drug (Fig. 5C graph, rescaled in inset). The addition of drug-free solvent did not alter the outward K+ current in SMCs or ECs (n = 5, 4; data not shown).

Figure 5. Effect of NS 1619 (30 μmol l−1) on whole-cell K+ current in bovine coronary SMCs and ECs.

Figure 5

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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.

Discussion

To our knowledge, the present study provides the first detailed comparison of the expression of BKCa channel mRNA, protein and current between freshly isolated ECs and SMCs from the same artery. The main finding is that BKCa channels are selectively expressed in freshly isolated SMCs of bovine coronary arteries but are not detected in ECs from the same preparation. A dense expression of BKCa channels in bovine coronary SMCs has been demonstrated previously (Campbell et al. 1996; Li et al. 1997), and in the present study, this expression served as a positive control to compare BKCa expression between coronary SMCs and ECs. Because the ECs and SMCs were subjected to similar dissection methods and isolation enzymes, the apparent lack of BKCa channels in ECs may be less likely to reflect damage from these interventions than ECs prepared by disparate methods.

Several studies have used immunocytochemistry to detect the BKCa α-subunit in vascular SMCs (Tanaka et al. 1997; Marijic et al. 2001). Using this method, the present study detected α-subunit expression in freshly isolated coronary SMCs but failed to detect this protein in the adjacent ECs. However, because false negative findings can result from limited access of the antibody to its epitope target, Western blots also were performed using proteins isolated from fresh SMCs and ECs. These studies identified a 125 kDa immunoreactive band corresponding to the BKCa α-subunit only in the coronary SMCs. Multicell RT-PCR studies also failed to detect mRNA encoding the BKCa α-subunit in ECs, although the same cDNA preparations demonstrated positive amplification of PECAM. In parallel, an amplified product corresponding to the BKCa α-subunit was verified in SMCs as a positive control. Although negative results in RT-PCR studies using small cell numbers must be viewed with caution (Dixon et al. 2000), the apparent absence of BKCa α-subunit mRNA in the coronary ECs of the present study supports the Western blot results indicating a lack of BKCa α-subunit protein in these cells.

Finally, whole-cell K+ currents were recorded from freshly isolated ECs and SMCs to compare the membrane density of BKCa current. BKCa current was defined as the outward K+ current blocked by IBTX, a peptide inhibitor of BKCa channels (Galvez et al. 1990) and activated by NS 1619, an activator of BKCa channels (Oleson et al. 1994; Yamamura et al. 2001). The coronary SMCs displayed large outward K+ currents that were inhibited by IBTX, whereas IBTX did not affect the small outward K+ currents observed in ECs. Similarly, NS 1619 only amplified outward K+ currents in bovine coronary SMCs but not in ECs. Walker et al. (2001) have also reported that NS 1619 increased outward current in freshly isolated rat mesenteric SMCs, but had no effect on fresh ECs from the same artery. In contrast, Papassotiriou et al. (2000) identified BKCa current blocked by IBTX and activated by NS 1619 in freshly isolated porcine aortic ECs. In the latter preparation, mRNA encoding the BKCa α-subunit also was detected in ECs by RT-PCR. Thus, BKCa channels may be expressed in ECs from some arteries, although their presence was not detected in the bovine coronary ECs of the present study.

Instead, our results suggest that EC and SMC membranes from the same arterial site may express different profiles of Ca2+-sensitive K+ channels, although the influences that govern this cell-specific expression are not readily apparent. Recent evidence demonstrating that BKCa channel expression is a function of cell culture conditions (Jow et al. 1999) implies that factors localized within the arterial wall could induce the expression of BKCa channels in different cell types. BKCa channel activation in SMCs also is linked to the local release of Ca2+ from the sarcoplasmic reticulum (Standen, 2000), a phenomenon that has not been observed in ECs but could influence the necessity for channel expression. Finally, Burnham et al. (2002) recently reported that small conductance Ca2+-activated K+ channels are expressed in EC but not SMC plasma membrane of porcine coronary arteries. This finding supports the existence of a cell-specific distribution of K+ channels within the arterial wall, and in the absence of BKCa channels, small conductance Ca2+-activated K+ channels may provide Ca2+-dependent K+ efflux in coronary ECs.

The vascular endothelium acts to modulate arterial tone, and also influences vessel permeability, angiogenesis and vascular repair. The ion channels expressed in the EC plasma membrane are important regulators of these functions (Nilius et al. 1997). Furthermore, K+ efflux mediated by the activation of K+ channels in coronary ECs also may function at the myoendothelial junction as an endothelium-derived hyperpolarizing factor (Beny & Schaad, 2000). Endothelial function is disturbed in diseases such as hypertension and atherosclerosis and is altered by the aging process (Mombouli & Vanhoutte, 1999; Marijic et al. 2001). Thus, the design of future therapies directed to normalizing endothelial function will require knowledge of the ion channels that are expressed in the ECs of different vascular beds. The findings of the present study indicate that BKCa channels may represent SMC-specific proteins in the coronary circulation, whereas other K+ channel types may regulate the excitability of coronary ECs.

Acknowledgments

K.G. was a postdoctoral fellow of the American Heart Association-Northland Affiliate and was supported by a National Institutes of Health Training Grant (HL-07792). S.A. was a Fulbright Exchange Scholar sponsored by the United States State Department. Research was supported in part by R01 HL-59238 from the USA National Institutes of Health. The authors thank Mr Miodrag Pesic for graphics support and Drs Paulo Ferreira and Phillip Pratt for advice on fluorescence microscopy. The gifts of PECAM and BKCa antibody from Drs Peter Newman (Medical College of Wisconsin) and Hans-Guenther Knaus (University of Innsbruck), respectively, were also greatly appreciated.

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