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Am J Physiol Heart Circ Physiol 287: H22-H28, 2004. First published February 12, 2004; doi:10.1152/ajpheart.00880.2003
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Flow-dependent increase of ICAM-1 on saphenous vein endothelium is sensitive to apamin

Sabena Sultan, Martin Gosling, Shadi Abu-Hayyeh, Nessa Carey, and Janet T. Powell

Department of Vascular Surgery, Imperial College at Charing Cross, London W6 8RP, United Kingdom

Submitted 7 October 2003 ; accepted in final form 9 February 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
The potassium channel blocker tetraethylammonium blocks the flow-induced increase in endothelial ICAM-1. We have investigated the subtype of potassium channel that modulates flow-induced increased expression of ICAM-1 on saphenous vein endothelium. Cultured human saphenous vein endothelial cells (HSVECs) or intact saphenous veins were perfused at fixed low and high flows in a laminar shear chamber or flow rig, respectively, in the presence or absence of potassium channel blockers. Expression of K+ channels and endothelial ICAM-1 was measured by real-time polymerase chain reaction and/or immunoassays. In HSVECs, the application of 0.8 N/m2 (8 dyn/cm2) shear stress resulted in a two- to fourfold increase in cellular ICAM-1 within 6 h (P < 0.001). In intact vein a similar shear stress, with pulsatile arterial pressure, resulted in a twofold increase in endothelial ICAM-1/CD31 staining area within 1.5 h (P < 0.001). Both increases in ICAM-1 were blocked by inclusion of 100 nM apamin in the vein perfusate, whereas other K+ channel blockers were less effective. Two subtypes of small conductance Ca2+-activated K+ channel (selectively blocked by apamin) were expressed in HSVECs and vein endothelium (SK3>SK2). Apamin blocked the upregulation of ICAM-1 on saphenous vein endothelium in response to increased flow to implicate small conductance Ca2+-activated K+ channels in shear stress/flow-mediated signaling pathways.

cardiovascular surgery; endothelial function; hemodynamics; K+ channel

THE REMODELING OF VESSELS accompanies all vascular pathology. One of the driving forces for vascular remodeling is hemodynamic stress, ranging from hypertensive remodeling of the aorta, the intimal hyperplasia that follows balloon angioplasty, or vein grafting to the bulbous, fibrotic changes in varicose veins. The endothelium is considered to be the primary sensor of shear stress (25), but the endothelium also responds to stretch or mechanical deformation (9). These events have been studied in cultured endothelial cells, usually human umbilical vein endothelial cells or bovine aortic endothelial cells. Some of the key early cellular changes, in response to shear stress, relate to alterations in intracellular calcium and other cation concentrations, a range of different ion channels being implicated (10). Recently, a stretch-activated cation channel, which also is shear sensitive, has been described to link the effects of differing hemodynamic stresses (18, 24). Downstream of these changes in cation concentrations, there is activation of kinase-dependent signaling cascades with subsequent activation or inactivation of transcription factors.

Previously, Alevriadou et al. (1) have shown that intact saphenous vein endothelium responds to arterial flow with a very rapid upregulation of ICAM-1. The upregulation of ICAM-1 is likely to lead to increased leukocyte adhesion, and leukocyte infiltration has been associated with the failure of vein grafts (23). This upregulation of ICAM-1 can be abolished in the presence of nonselective K+ channel blocker, 3 mM tetraethylammonium (TEA), within the perfusate. Nifedipine (20 µM), a calcium channel blocker, did not affect flow-induced ICAM-1 upregulation. We extended our earlier work with the intact saphenous vein to identify the subtype of endothelial cell potassium channel involved in flow-mediated responses. However, many of the drugs used to inhibit potassium channels (e.g., charybdotoxin) are costly for other than confirmatory experiments in the large volume, intact vein flow circuit experiments. Therefore, we elected initially to pharmacologically dissect the channel(s) involved in shear stress responses using human saphenous vein endothelial cells (HSVECs) cultured in parallel plate flow chambers and then extend the work to the intact vein. Here we report that apamin, a blocker of small conductance calcium-activated K+ channels, prevents the upregulation of endothelial ICAM-1 in response to flow in both cultured cells and intact vein.

Previously, we had calculated the shear stress in the saphenous vein exposed to arterial pressures and flow in vitro to be 0.8 N/m2 (8 dyn/cm2) (1). This is in general agreement with Casey et al. (6), who estimated that the pulsatile shear stress in vein first exposed to the arterial circulation was between 4.8 and 9.2 dyn/cm2 versus 0.1 dyn/cm2 in the venous circulation. Veins increase in diameter when first exposed to arterial pressure to lower the shear stress. Later, inward remodeling with intimal hyperplasia reduces the lumen area, and at 3 mo, shear stresses in vein grafts have increased to between 13.1 and 28.6 dyn/cm2 (8). Therefore, in this study, we used shear stresses of 8 dyn/cm2 to mimic the situation in new vein grafts versus 0.8 dyn/cm2 for simulated venous flow.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
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 DISCLOSURES
 REFERENCES
 
All drugs were purchased from Sigma-Aldrich (Poole, UK) unless otherwise stated. Krebs solution was made fresh each day. TEA (0.3 and 3 mM), barium chloride (1 mM), BAPTA-AM (100 µM), and charybdotoxin (10 nM) were diluted with deionized water. Apamin (100 nM) was diluted in Krebs solution for intact vein experiments and in deionized water for shear stress experiments. TNF-{alpha} and IL-1{beta} (5 ng/ml) were diluted in serum-free media from stock of 10 µg/ml (diluted as advised by manufacturer's instructions, R&D Systems; Minneapolis, MN). Monoclonal mouse anti-ICAM-1 (Serotec; Kidlington, UK) and CD31 (Dako Diagnostics; Cambridge, UK) antibodies were used at 1/800 and 1/60 dilutions, respectively. Rabbit anti-SK2 or anti-SK3 antibodies were used at 1/1,500 dilution (Chemicon International; Harlow, UK). Secondary antibodies were from Dako Diagnostics.

Cell culture. Primary cultured HSVECs were harvested from normal saphenous vein samples obtained from patients undergoing coronary artery bypass grafting (with local ethical committee consent for tissue harvesting). HSVECs were cultured in medium 199 supplemented with 10% human serum on fibronectin-coated tissue culture ware (10).

Static experiments. HSVECs were grown to confluence at passage 3 in 96-well plates. Subsequently, cells were stimulated with various reagents/drugs for 6 h in serum-free media under static conditions (at 37°C in a 95% O2-5% CO2 humidified environment). Cells were fixed for 2 min with ice-cold methanol and 0.03% (vol/vol) H2O2 at room temperature and incubated overnight in PBS containing 1% (wt/vol) BSA at 4°C. Subsequently, ICAM-1 and DNA were measured by ELISA and picogreen assays, respectively.

Shear stress experiments. HSVECs were grown to confluence (passage 3) on chamber slides 75 mm x 25 mm (Nunc; Paisley, UK), which were subjected to shear stress for various times in a parallel plate chamber (Fig. 1). The chamber consists of a baseplate made of brass, which was designed with precisely machined recesses to allow slides (with cultured endothelial cells) to be inserted level with the plate and a poly(methyl)methylacrylate cover, the two were separated through two spacers (91 cm length, 0.5 cm width, and 0.254 mm thickness). A silicone gasket (96 mm length, 50 mm width, and 0.508 mm thickness, Osteotec; Dorset, UK) was sandwiched between the upper and lower plates, which provided a watertight seal. Washers and nuts held the plates and gasket together. After closure of the chamber with a silicone gasket and upper brass plate, with nuts torqued at 1 N/m2 (10 dyn/cm2), the height of the chamber was 0.56 mm. The upper plate contained inlet and exit flow channels, permitting an even flow across the width of the chamber slide, the ports being connected to silicon tubing with 3.2-mm bore and 1.6-mm wall. Cells were washed twice with warm sterile PBS, and the medium was replaced with serum-free medium 199 30 min before application of shear stress. Drugs and/or reagents were added 10 min before the application of shear stress and were included in the perfusate (30-ml reservoir, gassed with 95% O2-5% CO2 to maintain pH 7.4): the entire system was maintained in an enclosure at 37°C. Flow, driven by a peristaltic pump (Watson-Marlow; Cornwall, UK), was altered to generate a different shear stress: venous shear stress [7.6 ml/min, 0.08 N/m2 (0.8 dyn/cm2)] and arterial shear stress [76 ml/min, 0.79 N/m2 (7.9 dyn/cm2)] (24). After exposure to shear stress (0–6 h), the parallel plate chamber was disassembled, and slides were removed. Cells were fixed for 2 min with ice-cold methanol and 0.03% (vol/vol) H2O2 at room temperature before overnight incubation in PBS containing 1% (wt/vol) BSA at 4°C. Cellular ICAM-1 and DNA were measured by ELISA (12) and picogreen assays, respectively.



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  		Fig. 1. Detailed view of the parallel plate flow chamber. Human saphenous vein endothelial cells (HSVECs) were grown to confluence on 1-well chamber slides, which were then placed in the parallel plate chamber flow system, which consisted of a baseplate, cover, gasket, and spacers. Washers and nuts held the plates, gasket, and spacers together.

 
Time-matched stationary controls (static ± drug) and TNF-{alpha} (5 ng/ml)-stimulated ICAM-1 expression (±drug) served as negative and positive controls, respectively. ICAM-1 was measured as described previously (12) and presented as absorbance per cell number.

BAPTA-AM experiments. HSVECs were incubated for 30 min with or without 100 µM BAPTA-AM under static conditions (37°C, 95% O2-5% CO2). BAPTA-AM-containing medium was removed, and cells were exposed to 6 h of arterial shear stress.

PicoGreen assay for cellular DNA and cell number. The cell number was determined from measurement of double-stranded DNA with the use of a PicoGreen double-stranded DNA quantitation kit (Molecular Probes; Leiden, The Netherlands). Fluorescence was measured (excitation at 485 nm, emission at 530 nm) using a Series 4000 multiwell plate reader-Cytofluor (Perseptive Biosystems; Hertford, UK), which had been adapted to read chamber slides as well as 96-well plates. The assay was calibrated with HSVECs and was linear in the range of 4,000–20,000 cells.

Isolation and measurement of mRNA. mRNA was isolated from HSVECs using a mRNA microisolation kit (Sigma-Aldrich). All reagents and solutions were made up according to the manufacturer instructions, prepared using nuclease-free pipettes, and brought to room temperature before use. RNA was quantified using a RiboGreen RNA quantitation reagent (Molecular Probes), according to the manufacturer's instructions.

Intact vein experiments. Freshly excised human saphenous vein was subjected to simulated flow: venous flow [20 ml/min, 20 mmHg, 0.02 ± 0.01 N/m2 (0.21 ± 0.11 dyn/cm2)] or arterial flow [200 ml/min, 120/80 mmHg, 60 circuits/s, 0.26 ± 0.09 N/m2 (2.6 ± 0.9 dyn/cm2)] for 90 min and processed for immunostaining as described previously (9). The area and length of immunostained endothelium (ICAM-1 to CD31) was analyzed by computer-assisted image analysis (Analysis Imaging C; Münster, Germany). To minimize inconsistencies, all samples were analyzed simultaneously in at least four different areas. The intima (endothelial cells stained with ICAM-1 or CD31) was selected for quantification. The area of staining and length of staining (in µm) for ICAM-1 was compared directly with the serial section stained for CD31. The length of staining was also measured and each section was expressed as staining per micrometer (by dividing area of staining by length of staining). Sections of vein also were used for immunostaining for SK2 and SK3.

RT-PCR and real time quantitative RT-PCR. HSVECs were exposed to shear stress in parallel plate chambers. Approximately 0.1–0.7 µg of mRNA was extracted from each slide. cDNA was synthesized with the use of the cDNA Cycle kit (Invitrogen; Paisley, UK) with random primers, according to the manufacturer's protocols. The polymerase chain reaction conditions were the same for both SK2 and SK3 genes (1 cycle of 95°C for 5 min, 35 cycles of 95°C for 1 min, 60°C for 1 min, and 72°C for 1 min; final extension of 1 cycle at 72°C for 5 min), and the primers (10 pmol) used are shown in Table 1. The total volume was 50 µl with 1.5 mM MgCl2, 200 µM dNTPs, and 1 unit of Taq polymerase (Qiagen; Crawley, UK). All primers and probes were cross-exonic and designed using the Applied Biosystems Primer Express program. PCR products (10 µl) were analyzed on a 3% (wt/vol) agarose gel. For real-time quantitative RT-PCR, HSVEC cDNA was diluted 1/15 and the TaqMan reaction mix was composed of 12.5 µl two times PCR master mix (Applied Biosystems; Warrington, UK), forward and reverse primer, probe (each 10 pmol), water, and 5-µl template DNA for a total reaction mix volume of 25 µl. The bulk reaction mix was made up and aliquoted into the appropriate wells of a MicroAmp optical 96-well reaction plate. A minimum of three no-template controls reactions per plate (5 µl of sterile water) were included as a negative control. Three measurements per sample were carried out from four independent experiments on an Applied Biosystems Prism 7700, and raw data were analyzed using Sequence Detector Software version 1.6.3. The expression levels of the SK2 and SK3 genes in each sample were normalized to {beta}-actin (primer sequences also shown in Table 1). Standard curve reactions were set up in quadruplet, {beta}-actin 50 fg-500 pg, and 50 ag-500 fg for SK2 and SK3. Initially, the reaction mix was held at 50°C for 2 min, stepped to 95°C for 10 min before 45 cycles of 95°C (1 s) and 60°C (1 s).


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  		Table 1. Primer sequences

 
Data analysis. All statistical calculations were performed using Stata version 6. ICAM-1 expression, reported as the mean ICAM-1 expression per 10,000 cells ± SE, was compared by Student's paired t-test or ANOVA as appropriate. Nonparametric data were log-transformed before analysis and for limited-number experiments, replicate data were considered as separate points.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
ICAM-1 expression in HSVECs in response to shear stress, in the presence and absence of potassium channel blockers. Low perfusion rates [shear stress 0.08 N/m2 (0.8 dyn/cm2) venous] for periods of up to 6 h did not alter the expression of ICAM-1 on cultured HSVECs. At high perfusion rates [shear stress 0.8 N/m2 (8 dyn/cm2), arterial] ICAM-1 immunoreactivity increased from 0.05 ± 0.01 (A492/104 cells) at the start of the experiment to 0.07 ± 0.02, 0.07 ± 0.01, and 0.16 ± 0.01 after 2, 4, and 6 h, respectively (a significant increase at 6 h ANOVA; P = 0.002, n = 4). The doubling of endothelial ICAM-1 after 6 h required arterial shear stress to be applied for a minimum of 10 min, followed by venous shear stress for the remaining period. HSVECs were preincubated with the potassium channel blockers, 3 mM TEA, 10 nM charybdotoxin, or 100 nM apamin for 10 min before and during application of arterial shear stress for 6 h. Inclusion of TEA and apamin effected a large reduction (by >65%) of cellular ICAM-1 at 6 h, both P < 0.02 (Fig. 2).Inclusion of charybdotoxin had a more limited effect, reducing endothelial ICAM-1 at 6 h by 30%. Incubation (6 h) of HSVECs in the absence of flow, with any of the drugs used in this study, did not alter the basal expression of cellular ICAM-1 and had a minimal effect on the positive control (TNF-{alpha} stimulated cellular expression of ICAM-1) ( Table 2).



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  		Fig. 2. The effect of shear stress and/or K+ channel blockers on ICAM-1 expression. HSVECs were pretreated with serum-free media supplemented with or without ion channel blockers under static conditions. Subsequently, cells were exposed to static, venous [0.08 N/m2 (0.8 dyn/cm2)], or arterial shear stress [0.79 N/m2 (7.9 dyn/cm2)] in the absence or presence of tetraethylammonium (TEA; 3 mM), charybdotoxin (10 nM), and/or apamin (100 nM) within the perfusate for a period of 6 h. ICAM-1 expression was then quantified using an ICAM-1 ELISA, which was then normalized using a picogreen assay. A significant increase in ICAM-1 expression was observed after exposure to arterial shear stress (107.3 ± 187%) for 6 h compared with static levels (26.1 ± 2.1%) (P = 0.036). This upregulation was not seen in HSVECs exposed to venous shear stress (48.2 ± 6.1%) conditions (P = 0.258). The inclusion of TEA (40.0 ± 7.1%) and apamin (57.1 ± 18.2%) significantly inhibited the 6-h shear-induced upregulation of ICAM-1 (P = 0.01). The inclusion of charybdotoxin (69.6 ± 8.2%) reduced the 6-h arterial shear-induced upregulation of ICAM-1 but not significantly (P = 0.172). TEA (32.4 ± 3.2%), charybdotoxin (55.8 ± 3.4%), and apamin (32.9 ± 6.8%) did not affect the expression of ICAM-1 in response to venous shear stress (P = 0.27, 0.18, and 0.07 for TEA, charybdotoxin, and apamin, respectively). Values are means ± SE from 4 separate experiments. *P = 0.01, compared with 6-h arterial flow.

 

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  		Table 2. ICAM-1 expression and K+ channel blockers

 
Intracellular calcium chelation and shear stress-induced ICAM-1 expression. Preincubation of HSVECs with 100 µM BAPTA-AM before exposure to shear stress [0.8 N/m2(8 dyn/cm2)], reduced (by 60%) the elevation of ICAM-1 after 6 h (6 h flow increased ICAM-1 expression from 0.03 ± 0.002 to 0.083 ± 0.01 and in the presence of BAPTA-AM from 0.022 ± 0.004 to 0.045 ± 0.01 measured as A492/104 cells) and (P < 0.01, n = 4).

Expression of SKCa channel subtypes SK2 and SK3 in HSVEC. Apamin is a selective inhibitor of small conductance Ca2+-activated K+ channels (SKCa), of which there are three subtypes. The expression of both SK2 and SK3 mRNA was detected by RT-PCR, yielding bands at 84 and 109 bp, respectively (Fig. 3). Real-time quantitative RT-PCR showed that the copy number (with respect to 1,000 copies of {beta}-actin) was lower for SK2 than for SK3 [0.14 ± 0.03 and 0.87 ± 0.13; P < 0.01, respectively (n = 4)]. The exposure of HSVECs to shear stress [0.8 N/m2(8 dyn/cm2)] for 6 h did not increase the expression of SK3, but the copy number for SK2 increased to 0.64 ± 0.16 (P = 0.05; n = 4).



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  		Fig. 3. Human small-conductance K+ channel subtype (SK2 and SK3) expression in HSVECs as detected by RT-PCR. HSVECs were exposed to static or arterial shear stress conditions for 6 h, and mRNA was extracted and reverse transcribed. Samples were run on a 3% (wt/vol) agarose gel. Lane 1, standard, molecular sizes are indicated in kb; lanes 2–5, cDNA of HSVECs exposed to static conditions (n = 4); lanes 6–9, cDNA of HSVECs exposed to arterial shear stress; Lane 10, negative control, distilled water, no sample cDNA. Paired samples, n = 4, were amplified. RT-PCR analysis revealed the presence of SK2 (A) and SK3 (B) subtypes of small-conductance Ca2+-activated K+ (SKCa) channels in static and/or sheared HSVECs.

 
Effect of K+ channel blockade on flow-induced endothelial ICAM-1 expression in intact saphenous vein. After 90 min of simulated venous flow, the ICAM-1/CD31 staining ratio was 33 ± 5% compared with 62 ± 5% after simulated arterial flow. Inclusion of 1 mM BaCl2 in the Krebs solution used to perfuse saphenous vein segments had no effect on the ICAM-1/CD31 staining ratio, which after 90 min increased from 32 ± 8% with venous flow to 58 ± 4% with arterial flow (n = 6). Perfusion of saphenous vein segments with 100 nM apamin abolished the increase in endothelial ICAM-1 after exposure of the vein to 90 min of simulated arterial flow (Fig. 4, A and B). The staining area ratio ICAM-1/CD31 was 34 ± 3% and 67 ± 9% after 90 min of venous and arterial flow, respectively (n = 6; P = 0.035). After arterial perfusion for 90 min in the presence of 100 nM apamin, the ICAM-1/CD31 staining area was 40 ± 8% (P = 0.009 compared with absence of apamin; n = 6). When vein segments were immunostained for SK2 and SK3 subtypes of SKCa channels, there was strong staining for the SK3 subtype but scant and intermittent staining for SK2 subtype of SKCa channels (Fig. 5).



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  		Fig. 4. A: immunostaining for ICAM-1 on saphenous vein endothelium is reduced after exposure to arterial flow in the presence of apamin. Saphenous vein was exposed to venous flow or arterial flow conditions in an in vitro circuit. Vein segments were then fixed and immunostained for CD31 (a) and ICAM-1 (b). 1, freshly excised vein; 2, vein exposed to venous flow conditions for 90 min; 3, vein exposed to venous flow in the presence of 100 nm of apamin; 4, vein exposed to arterial flow conditions for 90 min; 5, vein exposed to arterial flow in the presence of 100 nm apamin. Arrows indicate positive immunoreactivity of monoclonal CD31 antibody (top) and monoclonal ICAM-1 antibody (bottom). Bar represents 50 µm. B: ICAM-1 staining on saphenous vein endothelium in response to arterial flow in the presence of K+ channel inhibitors, barium chloride, and apamin. Saphenous vein was exposed to venous or arterial flow for 90 min in in vitro circuit in the absence or presence of BaCl2 or apamin. ICAM-1 expression was assessed by immunostaining (as a ratio of ICAM-1 to CD31). The exposure of saphenous vein to arterial flow significantly increased ICAM-1 staining (P = 0.035) compared with static levels (from 33.1 ± 12.0% to 67 ± 9%). The inclusion of 1 mM BaCl2 within the perfusate did not affect ICAM-1/CD31 staining ratio in response to exposure to venous or arterial shear stress. The inclusion of apamin within the perfusate significantly (P = 0.009) reduced ICAM-1 staining in response to arterial flow (from 67 ± 9% to 40.2 ± 7.9%). Values are means ± SE of staining area of ICAM-1/CD31 (as a percentage) from at least four separate experiments.

 


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  		Fig. 5. Immunostaining for SK2 and SK3 subtype of SKCa channels on saphenous vein endothelium. Vein segments were fixed and immunostained for SK2 (A) and SK3 (B). There was scant and intermittent staining for SK2 subtype of SKCa channels, whereas the staining pattern for SK3 subtype of SKCa channels was much more continuous and pronounced. Arrows indicate positive immunoreactivity of polyclonal SK2 antibody (A) and polyclonal SK3 antibody (B). Antibodies against SK2 and SK3 subtype of SKCa channels were used at 1/1,500 dilution. Bar in A represents 50 µm.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 DISCLOSURES
 REFERENCES
 
Alterations in flow and shear stress stimulate changes in cell morphology, gene expression, and cell surface remodeling. We have used ICAM-1 as a reporter to indicate, for the first time, a potential role in these events for SKCa, the only cation channels reported to be blocked by apamin. Apamin prevented the flow-induced upregulation of endothelial ICAM-1 in the intact saphenous vein to confirm the role of apamin-sensitive channels in both primary endothelium and cultured endothelium, both of which expressed the SKCa subtypes SK3 more abundantly than SK2.

Both shear stress and mechanical stretch cause elevations in intracellular calcium concentrations in endothelial cells (2, 19). The increase in intracellular calcium in response to application of shear stress occurs within milliseconds (2) and is dependent on the release of intracellular calcium and influx of extracellular calcium (19). In HSVECs, the chelation of intracellular calcium with BAPTA-AM inhibited the shear stress-induced increase in ICAM-1 to implicate the release of intracellular calcium in the shear stress-induced increase in endothelial ICAM-1. Because the increase in endothelial ICAM-1 required the application of shear stress for at least 10 min, similar experiments to evaluate the role of extracellular calcium could not be performed because of significant endothelial cell detachment.

Several calcium-activated potassium channels have been described and are categorized by their conductance of K+. Of these, it is the SKCa channels that are blocked by apamin. These SKCa channels, activated by the elevation of cystolic Ca2+, are composed of an heteromeric complex of SK subunits and calmodulin. Activation of SKCa channels occurs by Ca2+ binding to calmodulin, which induces conformational changes and channel opening. There are three subtypes of small conductance calcium-activated potassium channels SK1–3. Of these only SK2 and SK3 are consistently blocked by apamin. The presence of SKCa channels in endothelial cells has been identified in cultured human umbilical vein endothelial cells and porcine coronary artery endothelium (17, 5). These channels also appear to have significance for the generation of endothelium-derived hyperpolarizing factor (5, 7). We also have preliminary findings to suggest that in HSVECs apamin inhibits a small conductance potassium current, consistent with the presence of SKCa (M. Gosling, unpublished observations). A possible physiological role for the SKCa channel identified in HSVECs is to repolarize the endothelial cell, after the depolarization consequent on shear stress-stimulated Ca2+ influx (20). The opening of channels and hyperpolarization of endothelial cells would maintain an electrochemical gradient for Ca2+ entry, with continued activation of Ca2+-dependent kinases and other downstream signaling pathways. The shear stress-stimulated Ca2+ influx is likely to alter the activity of several cations.

Although we have no evidence to suggest that SKCa channels are primary sensors of shear stress, these channels have been implicated in mechanotransduction in human articular chondrocytes and human hepatocyte cell lines (15, 27). Apamin blocks the hyperpolarization response of chondrocytes to mechanical stimulation (15). In the human liver, where calcium-dependent changes in K+ permeability coordinate membrane transport, metabolism, and cell volume, apamin-sensitive SK2 channels have been associated with the volume-dependent changes in K+ permeability (27).

Apamin (100 nM) abolished the rapid upregulation of ICAM-1, in response to arterial flow, observed in the intact vein, whereas 1 mM Ba2+, a blocker of inwardly rectifying and other potassium channels, did not. Higher concentrations of Ba2+ cannot be used in physiological solutions. Immunohistochemical studies suggested that SK3 was more strongly expressed than SK2 in saphenous vein endothelium. For cultured cells, although transcripts for both SK2 and SK3 subtypes were present, SK3 was more abundant in the absence of flow. In addition, there was some evidence that SK2 mRNA expression was increased by shear stress, although we did not extend our investigations to protein quantification. Apamin also blocked the slower upregulation of ICAM-1 by shear stress in cultured cells, whereas charybdotoxin (10 µM), a blocker of big- and intermediate-conductance KCa channels had little effect. These big- and intermediate-conductance KCa channels have been shown to be effectively blocked at 10 µM charybdotoxin (3). Together these data support a pivotal role for SKCa channels in the signaling pathway(s) activated by hemodynamic stress, leading to elevation of endothelial ICAM-1. A recent study (28) described selective SK2 and SK3 blockers, but these blockers were not available for our studies to enable discrimination between the effects of SK2 and SK3 in flow-mediated responses.

In the future, it would be useful to extend our studies to include more selective SK channel blockers, functional assays (leukocyte adhesion), and monitoring of other shear stress responses. Despite these limitations, we have identified the probability that SKCa channels have an important role in mechanotransduction events at venous endothelium. This suggests new pharmacological pathways to modulate the adaptation of human venous endothelium after the use of vein for bypass grafts or dialysis fistulas.


   GRANTS
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This study was supported by the Charing Cross Trustees and the British Heart Foundation.


   DISCLOSURES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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REFERENCES
 
Present address for M. Gosling: Novartis, Ion Channel Pharmacology, Novartis Horsham Research Centre, Wimblehurst Rd., Horsham, West Sussex RH12 5AB, UK.

Present address for N. Carey: Vernalis Research Ltd, 613 Reading Rd., Winnersh, Wokingham, Berks RG41 5UA, UK.

Present address for J. T. Powell: University Hospital of Coventry and Warwickshire, Clifford Bridge Rd., Coventry CV2 2DX, UK.


   ACKNOWLEDGMENTS
 
We thank all of the cardiac and vascular surgeons who have provided us with saphenous vein.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Sultan, Centre for Cardiovascular Biology and Medicine, BHF Laboratories, Dept. of Medicine, Univ. College London, 5 University St., London, WC1E 6JJ, UK (E-mail: s.sultan{at}ucl.ac.uk).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 

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