From the Department of Vascular Surgery, Imperial College School of
Medicine at Charing Cross, Charing Cross Hospital, Fulham Palace Road,
London W6 8RF, United Kingdom
Whole-cell patch-clamp electrophysiological
investigation of endothelial cells cultured from human saphenous vein
(HSVECs) has identified a voltage-gated Na+ current
with a mean peak magnitude of
595 ± 49 pA (n = 75). This current was inhibited by tetrodotoxin (TTX) in a
concentration-dependent manner, with an IC50
value of 4.7 µM, suggesting that it was of the
TTX-resistant subtype. An antibody directed against the highly conserved intracellular linker region between domains III and IV of
known Na+ channel
-subunits was able to retard current
inactivation when applied intracellularly. This antibody identified a
245-kDa protein from membrane lysates on Western blotting and
positively immunolabeled both cultured HSVECs and intact venous
endothelium. HSVECs were also shown by reverse transcription-polymerase
chain reaction to contain transcripts of the hH1 sodium
channel gene. The expression of Na+ channels by HSVECs was
shown using electrophysiology and cell-based enzyme-linked
immunosorbent assay to be dependent on the concentration and source of
human serum. Together, these results suggest that TTX-resistant
Na+ channels of the hH1 isoform are expressed in human
saphenous vein endothelium and that the presence of these channels is
controlled by a serum factor.
 |
INTRODUCTION |
Vascular endothelial cells form the primary interface between the
blood and the underlying tissue. These cells not only provide a barrier
of varying permeability between the blood and the smooth muscle of the
vessel wall, but are a major contributor to the processes of vascular
growth and repair, vascular autoregulation, and control of vascular
tone by secretion of both relaxant and contractile factors (1, 2).
Endothelial cells are known to possess a broad spectrum of ion channels
that open in response to a variety of stimuli, including membrane
potential, receptor occupation, elevation of
[Ca2+]i, and mechanical deformation induced by
flow (3). Levels of [Ca2+]i are an important
factor in the control of endothelial cell function (4), and ion
channels, with their ability to allow both Ca2+ entry
either directly or indirectly, via control of membrane potential, are
critical to this process (5).
Definitive data regarding the exact repertoire of ion channels
expressed by endothelial cells are still sparse, particularly in venous
endothelium. In this study, we report the presence of a voltage-gated
Na+ current present in human saphenous vein endothelial
cells (HSVECs).1 This type of
channel is normally only expressed by classically excitable cells that
generate action potentials such as neurons and cardiac and skeletal
muscle. Voltage-gated Na+ channels are characterized by
their kinetics; voltage dependence; and sensitivity to the guanidinium
toxin, tetrodotoxin (TTX). TTX-sensitive Na+ channels are
blocked by nanomolar concentrations of TTX and are found in tissues
such as mature skeletal muscle (6). In contrast, TTX-resistant channels
have a substantially lower affinity for the toxin, requiring 0.1-10
µM for inhibition (7). TTX-resistant channels are found
in a wide variety of tissue types, including cardiac cells (8) and
denervated or developing skeletal muscle (9) and corneal endothelium
(10). A third class of voltage-gated Na+ channels,
expressed by embryonic cardiac cells (11) and dorsal root ganglion
neurons (12), remain unblocked by TTX concentrations in excess of 100 µM and are classified as TTX-insensitive. The voltage-gated sodium current we describe here in HSVECs is
TTX-resistant and appears to result from expression of the cardiac
Na+ channel gene (hH1).
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EXPERIMENTAL PROCEDURES |
Human Saphenous Vein Endothelial Cell Isolation and
Culture--
HSVECs were obtained by enzymatic release from saphenous
vein harvested during high ligation of varicose veins or bypass
surgery. After removal of any residual external connective tissue, the vein was carefully opened along its longitudinal axis with a scalpel blade. HSVECs were obtained by placing the vein luminal face down in a
shallow Petri dish containing Ca2+- and
Mg2+-free phosphate-buffered saline (PBS; 150 mM NaCl, 2 mM NaH2PO4, and 10 mM Na2HPO4) and 1 mg/ml
collagenase (Type II, Sigma) and incubating at room temperature
(20-22 °C) for 30 min. Cells were placed in culture on
fibronectin-coated dishes or flasks as appropriate and grown in M199
medium supplemented with heparin, endothelial cell growth supplement,
antibiotic solution (200 units/ml penicillin and 200 µg/ml
streptomycin), and 10% (v/v) heat-inactivated human serum. Serum was
obtained either from non-diabetic patients with peripheral arterial
disease (>65 years old) or from healthy donors (<30 years old).
Cultures, characterized by positive immunostaining for von Willebrand
factor, were maintained at 37 °C in humidified CO2 in
air atmosphere and used in experiments at passages 0-3.
Electrophysiological Recording--
Experiments were performed
at room temperature (20-22 °C) using the whole-cell configuration
of the patch-clamp technique (13) on subconfluent HSVECs grown in 35-mm
diameter Petri dishes. These were placed on the stage of an inverted
microscope (Diaphot 200, Nikon, Tokyo, Japan), visualized with
phase-contrast optics, and continuously superfused at 2 ml/min with
extracellular solution. The standard pipette and extracellular
solutions were designed to isolate INa (pipette:
120 mM CsCl, 10 mM EGTA, 2 mM
MgCl2, 5 mM NaCl, 5 mM HEPES, 2 mM Na2ATP, and 0.5 mM
Na2GTP; extracellular: 120 mM NaCl, 4 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM TEA-Cl, and 10 mM HEPES (pH 7.3) with CsOH), although all current-clamp
and some preliminary experiments employed "quasiphysiological"
solutions (pipette: 140 mM KCl, 2 mM
MgCl2, 1 mM CaCl2, 0.05 mM EGTA, 20 mM HEPES, 2 mM
Na2ATP, and 0.5 mM Na2GTP;
extracellular: 135 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2,
11 mM glucose, and 10 mM HEPES (pH 7.3) with
NaOH). Patch-clamp pipettes were manufactured from borosilicate glass
(GC150-15TF, Clark Electromedical, Reading, United Kingdom) using a
two-stage puller (PB7, Narashige, Tokyo, Japan) and fire-polished to
give final resistances of 1-3 megaohms when filled with pipette
solution. Whole-cell membrane currents (voltage-clamp) and potentials
(current-clamp) were recorded using an Axopatch 200A patch clamp
amplifier (Axon Instruments Inc., Foster City, CA) with analogue cell
capacitance (mean cell capacitance = 35.1 ± 1.4 picofarads,
n = 131) and series resistance (routinely <5 megaohms)
maximally compensated. Signals were low pass-filtered with an 8-pole
Bessel-type filter at either 2 or 5 kHz prior to digitization at 10 kHz
by a Digidata 1200 interface (Scientific Solutions, Solon, OH) and
storage on a computer hard disk (486DX2, Opus Technology). Analysis was
performed using pClamp 6 software (Axon Instruments Inc.), which was
also employed to generate voltage step protocols. When recording
INa linear leakage, currents were subtracted
using a 4-subpulse (P/4) method (14), and INa
amplitude was measured as the difference between the maximal inward
current and the holding current level. Junction potentials were
measured as described previously (15), although only determinations of the INa reversal potential were corrected.
Reverse Transcription-Polymerase Chain Reaction
(RT-PCR)--
Total cellular RNA was extracted from HSVECs grown to
confluence in T75 culture flasks by incubation with 6 ml of RNAzol
reagent (Cinna/Biotecx Labs Inc.) at 4 °C for 10 min. Contaminating
DNA was removed (Message Clean kit, GenHunter Corp.) prior to reverse transcription (Reverse Transcriptor kit, R&D Systems Ltd., Abingdon, UK), both according to the manufacturers' protocols. Sodium channel cDNA was amplified by PCR using oligonucleotide primers directed against the 3'-untranslated region of the human cardiac hH1
channel (16). The primer sequences used were 5'-GACCTGTGACCTGGTCTGGT-3' and 5'-CCATGTCCATGGAAAAATCC-3' (Perkin-Elmer, Warrington, UK). HSVEC
RNA (1 µg) was used for PCR amplification using 1 µM
primers for 30 cycles (94 °C, 1 min; 50 °C, 1 min; and 72 °C,
1 min) at a MgCl2 concentration of 1 mM. The
resulting PCR fragments were analyzed on a 2.5% (w/v) agarose gel
containing ethidium bromide and sequenced using an ABI 373 automated
sequencer to confirm fragment identity.
Immunohistochemistry--
Immunohistochemical analysis for the
presence of voltage-gated Na+ channels in HSVECs was
performed on intact tissue sections and cultured HSVECs using a
polyclonal antibody raised in rabbits against the highly conserved
cytosolic linker region between domains III and IV (peptide sequence
TEEQKKYYNAMKKLGSKKP, amino acids 1490-1508) of known
Na+ channel
-subunits ("anti-Na
"; TCS
Biologicals, Botolph Claydon, UK). Small lengths of human saphenous
vein were fixed overnight in Zamboni's solution (consisting of 1.7%
(w/v) paraformaldehyde and 15% (v/v) saturated picric acid in PBS) and
washed daily in PBS/sucrose solution (PBS supplemented with 440 mM sucrose) for 7 days prior to mounting in OCT compound
(Miles Inc.). Veins were cryosectioned using a microtome (Bright
Instruments, Huntingdon, UK) to produce 8-10-µm thick transverse
sections, which were transferred to 3-aminopropyltriethoxysilane-coated
slides prior to overnight incubation at 4 °C with primary antibody
(anti-Na
, 7.5 µg/ml in PBS buffer supplemented with 1% (w/v)
bovine serum albumin and 1% (v/v) human serum). Specific binding was
visualized by alkaline phosphatase staining (Vector Red®,
Vector Labs, Peterborough, UK) subsequent to primary antibody detection
using a biotinylated secondary antibody (goat anti-rabbit, 1:200
dilution, 1 h) and a tertiary streptavidin-alkaline phosphatase conjugate incubation (1:200 dilution, 1 h; Dako, High Wycombe, UK). The presence of endothelium on intact vein sections was verified by using a mouse monoclonal antibody directed against thrombomodulin. A
protocol similar to that described above was employed for cultured HSVECs, which were, however, fixed by incubation in 100% methanol at
4 °C for 2 min. Controls for both intact vein sections and HSVECs
were prepared by omitting either the primary or secondary antibodies.
Western Blotting--
HSVECs, grown to confluence in T25 culture
flasks, were lysed for 30 min at 4 °C using 1 ml of lysis buffer
that consisted of 140 mM NaCl, 10 mM Tris, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium
metavanadate, 1 mM EDTA, 0.2 mg/ml aprotinin, 2 µg/ml pepstatin A, 2 µg/ml leupeptin, and 2.5% (v/v) Nonidet P-40. Lysed samples were centrifuged at 6000 × g for 10 min to
separate membrane (particulate) and cytosolic (supernatant) fractions,
which were suspended in loading buffer (40 µl for particulate and 80 µl for cytosolic fractions). Aliquots (16 µl) of sample lysates
were fractionated by SDS-polyacrylamide gel electrophoresis using an 8-25% gradient gel (PhastGel, Pharmacia, St. Albans, UK) and
transferred to polyvinylidene difluoride filter membranes (Immobilon-P,
Millipore, Amersham, UK) for Western blotting. Membranes were incubated
in blocking solution containing 5% (w/v) milk powder, 5% (w/v) bovine serum albumin, and 2% (v/v) human serum in Tris-buffered saline/Tween 20 solution (20 mM Tris base, 137 mM NaCl, and
0.05% (v/v) Tween 20 (pH 7.4) with HCl) for 6 h at room
temperature and washed prior to overnight incubation at 4 °C in
primary antibody solution (5 µg/ml anti-Na
antibody) prepared in
Tris-buffered saline/Tween 20 solution and 1% (w/v) bovine serum
albumin. After washing, membranes were incubated at room temperature
for 30 min with a biotinylated secondary antibody (goat anti-rabbit,
1:1000) and a tertiary streptavidin-horseradish peroxidase conjugate
(1:1000; Dako). Bound antibodies were detected using enhanced
chemiluminescence (ECL, Amersham, Amersham, UK). Using this method, we
could routinely detect protein levels as low as 0.5 pg/sample.
Cell-based Enzyme-linked Immunosorbent Assay--
HSVECs
(105/well) were seeded onto 24-well plates and grown to
confluence over 48 h. HSVECs were fixed by incubation in 100% methanol at 4 °C for 2 min and washed with Tris-buffered saline supplemented with 0.5% (w/v) bovine serum albumin. Cells were incubated with the anti-Na
antibody (2 µg/ml) for 40 min at
37 °C, washed, and subjected to two further incubations (30 min each at 37 °C) with a biotinylated secondary antibody (1:500) and a final
streptavidin-horseradish peroxidase conjugate (1:500). Cells were
thoroughly washed prior to assessment of anti-Na
antibody binding by
colorimetric assay using o-phenylenediamine as the substrate, and the
optical density was measured at 492 nm.
Data Analysis and Curve Fitting--
Data are expressed as
mean ± S.E. (n = number of observations).
Normalized activation curves for INa were
calculated by dividing conductances (gNa),
derived from peak currents divided by the Na+ driving force
(Vm
ENa), by the largest
conductances measured. Steady-state inactivation curves
(h
) and activation curves
(m
) were fitted with a Boltzmann function,
where V0.5 is the midpoint and
KV is the slope factor:
I/Imax = 1/1 +exp((V
V0.5)/KV).
Kinetic analysis of INa was only performed if
the peak current exceeded 500 pA, and time constants for current
activation and inactivation were derived by fitting a Hodgkin-Huxley
model (17) to the data as described elsewhere (18). The 50% inhibitory concentration (IC50) for TTX was calculated by fitting the
concentration inhibition curve to a logistic plot incorporating Hill
coefficients (nH) using MicroCal Origin
(MicroCal Inc., Northampton, MA): bound = [drug]nH/[drug]nH + IC50. Reversal potentials (Erev)
were obtained by fitting a second-order polynomial to the
current-potential (I-V) plots over the
appropriate voltage regions (usually +20 to +80 mV). Where appropriate,
results were tested for significance using Student's unpaired
t test.
Materials--
Culture materials were obtained from Gibco
Laboratories (Paisley, UK), and the thrombomodulin antibody was a gift
from Dr. J. Amiral (Serbio Research, Paris, France). TTX,
purchased from Calbiochem (Nottingham, UK), was dissolved in water
prior to addition to the appropriate solution. Unless indicated, all
other chemicals were obtained from Sigma (Poole, UK).
 |
RESULTS |
Potassium Currents--
Under whole-cell current clamp using
quasiphysiological K+-containing solutions, the resting
membrane potential of single HSVECs was found to be
28 ± 6 mV
(n = 24, range of
3 to
71 mV). Hyperpolarizing voltage-clamp pulses from a holding potential of
50 mV produced small
inward currents in 14 of the 24 cells (58%) investigated (Fig.
1A). These currents showed
marked time-dependent inactivation at strongly negative
potentials, inwardly rectified, conducting little outward current, and
reversed close to the potassium equilibrium potential (Fig.
1B). Currents were fully eliminated by substituting Cs+ for K+ in the pipette solution and by the
addition of 10 mM TEA in the extracellular solution,
suggesting that they were carried by potassium ions.

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Fig. 1.
Inwardly rectifying K+
current in HSVECs. A, currents elicited by 250-ms
voltage-clamp pulses between 190 and 10 mV (20-mV increments) from
a holding potential of 50 mV using K+-containing
quasiphysiological solutions (see "Experimental Procedures"). Data
are from a single HSVEC, representative of 13 similar experiments.
Currents were sampled at 8 kHz and low pass-filtered at 2 kHz.
B, mean current-voltage (I-V)
relationship for inwardly rectifying currents as shown in A.
Symbols represent the mean peak current at each potential,
with the S.E. indicated by the error bars
(n = 14). The zero current level is indicated by the
horizontal arrow.
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Sodium Currents--
Depolarizing voltage steps from a potential
of
120 mV to more positive potentials (
40 to +60 mV) elicited
transient inward currents in HSVECs (Fig.
2A). These currents were
present in 10 of the 24 cells (42%) investigated using
K+-containing intracellular solutions, but unlike the
inward current described above, these currents remained even when
intracellular K+ was substituted with Cs+. All
further electrophysiological experiments performed in this study used
the Cs+/TEA-containing solutions to isolate this transient
inward current and to avoid contamination from K+
currents.

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Fig. 2.
Voltage-gated Na+
channels in HSVECs are tetrodotoxin-resistant. A,
decrease in INa amplitude with increasing
concentrations of extracellular TTX. Currents were elicited by a 20-ms
voltage step from a holding potential of 120 mV to a test potential
of 0 mV in Na+-selective intra- and extracellular solutions
(see "Experimental Procedures"). INa was
almost entirely blocked by 30 µM TTX.
Inset, family of inward Na+ currents
elicited by 20-ms depolarizing voltage-clamp steps to potentials
between 60 and +80 mV (10-mV increments) from a holding potential of
120 mV in the absence of TTX. Vertical bar, 500 pA; horizontal bar, 2 ms. B,
current-voltage relationships for the cell shown in A prior
to and subsequent to addition of 1, 10, and 30 µM TTX.
C, log concentration inhibition curve for the effects of TTX
upon INa in HSVECs. Symbols represent
the mean of six cells, with the S.E. indicated by the error
bars. The line is a logistic plot fitted to the
data (see "Data Analysis and Curve Fitting"), yielding an
IC50 of 4.7 µM with a Hill coefficient of
1.18.
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With the use of the Cs+/TEA solutions, the current was
found to be present in 75 of the 131 cells (57%) investigated, with a
peak inward amplitude that varied between
70 and
1700 pA (mean of
595 ± 49 pA). The current was voltage-gated, activating at
50
mV and reaching a peak near
10 mV (Fig. 2, A
(inset) and B), and had a mean reversal potential
of +68 ± 4.2 mV (n = 36), close to the calculated
Nernst potential for Na+ of +63 mV
(ENa) for these solutions at 22 °C. The fast
activation and inactivation kinetics of the inward current and its
reversal close to ENa suggested that this was
likely to be a Na+ current. To confirm this, extracellular
Na+ was replaced with equimolar choline chloride, which
totally abolished the inward current (n = 6) (data not
shown). These results indicate that the rapidly activating and
inactivating inward current in HSVECs is a voltage-gated
Na+ current, which we have designated as
INa.
Tetrodotoxin Sensitivity of the Sodium Current--
To facilitate
comparison of INa in HSVECs with other known
Na+ currents, we determined the sensitivity of the current
to the guanidinium toxin, TTX. TTX has been shown to block a wide range of Na+ channels of different origins (7), which are
classified as TTX-sensitive, TTX-resistant, or TTX-insensitive based on
the IC50 value for TTX blockade. In HSVECs, as the
concentration of TTX in the extracellular solution was increased,
INa decreased, with 100% blockade occurring at
30 µM (Fig. 2). An equal reduction in the amplitude of
INa was observed across the entire voltage range
of activation, suggesting that TTX binding was not
voltage-dependent (Fig. 2B). A logistic plot
fitted to the concentration inhibition curve yielded an
IC50 value for TTX of 4.7 µM (Fig.
2C), suggesting that INa belongs to
the TTX-resistant classification of Na+ channels (19).
Activation and Inactivation Kinetics of the Sodium
Current--
The steady-state activation and inactivation properties
of INa were assessed by the construction of
normalized m
and h
curves. Fits to the normalized activation curves (m
; Fig. 3), generated by conversion of the
I-V relationship to conductances, gave a mean
half-activation voltage (Vm) of
29.2 ± 1.2 mV (n = 17) and an average K value
(slope factor) of 7.2 ± 0.3 mV. The steady-state inactivation
parameter (h
) of INa
was measured using a standard two-pulse protocol. Cells, clamped at
120 mV, were conditioned with a 1-s prepulse to potentials between
120 and
20 mV prior to a 30-ms test pulse to
10 mV, the potential that routinely evoked the maximal current (Fig. 3A). These
data show that significant inactivation was observed at
80 mV and that INa was inactivated completely at
40 mV.
h
curves were calculated by normalizing the
peak current recorded during the test pulse to the maximum current
measured on stepping from
120 mV to the test potentials and were
plotted as a function of the prepulse level. The averaged data indicate
that INa was half-inactivated at
75.4 ± 1.3 mV (Vh; n = 17), with a slope
factor of 5.7 ± 0.2 mV (Fig. 3B). There were not any
areas of significant overlap between m and h
curves. The time dependence of recovery from inactivation also was
evaluated using a double-pulse protocol. Cells were stepped from a
holding potential of
120 mV to 0 mV for 20 ms to elicit and
inactivate INa. The cells were then clamped at
120,
100, or
80 mV for a variable duration of between 4 and 64 ms
in 4-ms increments, prior to a second test pulse to 0 mV (Fig.
4A). Recovery from
inactivation was found to be strongly voltage-dependent,
with complete recovery requiring potentials more negative than
80 mV
(Fig. 4B). By fitting a single exponential function to the
data, the recovery time constants were calculated to be 3.3 ± 0.3, 9.4 ± 1.2, and 32.2 ± 1.7 ms for cells held at
120,
100, and
80 mV, respectively (n = 4). Time
constants for activation (
m) and inactivation (
h) were obtained by fitting a Hodgkin-Huxley model (see "Experimental Procedures") to the inward currents. Both
m and
h were voltage-dependent, becoming more rapid as
the test potential became increasingly more depolarized (Fig.
5). This was much more marked with
h, which was reduced from 3.77 ms at
30 mV to 0.43 ms at
+50 mV. The hyperpolarized half-maximal inactivation potential
(Vh) and the inactivation time course are consistent with those reported for the cardiac isoform of Na+ channels
(7, 20).

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Fig. 3.
Steady-state activation and inactivation of
INa in HSVECs. A,
INa currents elicited at a test potential of 0 mV following a 500-ms hyperpolarizing conditioning prepulse to
potentials between 120 and 20 mV. B, normalized
activation ( ) and inactivation curves ( ) for
INa in HSVECs. Symbols represent the
mean fractional current or conductance (calculated as detailed under
"Data Analysis and Curve Fitting") at each potential, with the S.E.
indicted by the error bars (n = 22).
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Fig. 4.
Recovery from inactivation of
INa. A, series of current traces
elicited by a double-pulse protocol. The cell was clamped at 120 mV,
and a 20-ms voltage step to 0 mV was used to elicit and inactivate
INa. The cell was then clamped at 100 mV for
4-64 ms in 4-ms increments before a second test pulse to 0 mV.
B, recovery from inactivation occurs as a function of the
holding potential (Vhold). Plots are of the ratio of
the amplitude of the second and first current pulses as a function of
the interval between the two. The lines represent a single
exponential curve fitted to the data. Time constants estimated from
these fits were 3.6 ms at 120 mV, 9.7 ms at 100 mV, and 34.6 ms at
80 mV.
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Fig. 5.
Analysis of the time course of
INa in HSVECs. A,
open circles show the best fit of
INa (continuous line)
obtained by a voltage pulse to 0 mV from a holding potential of 120
mV by the Hodgkin-Huxley equation (see "Data Analysis and Curve
Fitting"). m = 0.43 ms; h = 0.82 ms.
B, averaged activation ( m) and inactivation
( h) time constants for INa across the
voltage range 30 to +50 mV. Error bars indicate
the S.E. (n = 14).
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The addition of an antibody directed against the cytosolic linker
region between domains III and IV of known Na+ channel
-subunits (anti-Na
) to the pipette solution (10 µg/ml) produced
substantial slowing of Na+ current inactivation. In four
cells,
h at 0 mV was increased from 0.71 ms to 1.25 ms
within 15 min of establishing whole-cell configuration without any
effect on peak current amplitude (data not shown).
Sodium Channel hH1 Transcripts--
On the basis of
electrophysiological data, particularly the current kinetics and
sensitivity to TTX, the voltage-gated Na+ current in HSVECs
appeared to closely resemble the human cardiac sodium channel, hH1. To
test this hypothesis, mRNA isolated from HSVECs was
reverse-transcribed, and the resulting cDNA was amplified (RT-PCR)
with specific primers targeted against the 3'-untranslated region of
the hH1 cDNA (16). The expected product of 180 base pairs was produced only in those samples that had been
reverse-transcribed. In the absence of a RT step, no product was
present after PCR (Fig. 6). DNA
sequencing of the RT-PCR product confirmed that it was identical to the
3'-untranslated region of the human hH1 sodium channel.

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Fig. 6.
PCR analysis of mRNA from HSVECs.
RT-PCR of HSVEC mRNA was performed using oligonucleotide primers
designed to amplify a region of the 3'-untranslated region of the human
heart Na+ channel gene, hH1. Lane
1, genomic DNA from whole blood; lanes
2 and 3, RT product from HSVEC mRNA;
lane 4, mRNA without the RT step. The
first lane shows a 1-kilobase ladder.
bp, base pairs.
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Immunochemical Detection of Sodium Channels in Saphenous Vein
Endothelium--
Using Western blotting and employing the anti-Na
antibody, we were able to detect Na+ channel protein in the
membrane (but not the cytosolic) fraction of HSVECs with guinea pig
cardiac myocytes acting as a positive control (Fig.
7). The antibody routinely recognized a
single protein band that had an apparent molecular mass of 242 ± 9 kDa when separated by SDS-polyacrylamide gel electrophoresis
(n = 5). This value is close to the molecular mass of
the human hH1
-subunit of 230 kDa as calculated from the deduced
amino acid sequence.

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Fig. 7.
Western blot analysis of HSVEC membrane
protein. Membrane proteins from whole HSVECs were separated by
SDS-polyacrylamide gel electrophoresis and transferred to a
polyvinylidene difluoride filter membrane. Blots were probed using the
anti-Na antibody and visualized by enhanced chemiluminescence.
Lane 1, HSVEC membrane fraction; lane
2, guinea pig cardiac myocyte membrane fraction. The
positions of molecular mass markers are indicated.
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Subconfluent HSVECs, which were electrophysiologically confirmed to be
expressing INa, also immunostained positively
with the anti-Na
antibody (Fig. 8).
This antibody was used to demonstrate the presence of Na+
channels in the endothelium of freshly excised human saphenous vein. In
intact saphenous vein endothelium, the immunostaining for
Na+ channel
-subunits was intermittent, with not all
endothelial cells being stained (Fig.
9).

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Fig. 8.
Immunohistochemical detection of
voltage-gated sodium channels in cultured HSVECs. A,
negative control (omission of the anti-Na antibody), cells
counterstained with hematoxylin; B, HSVECs from the same
culture as in A, showing positive immunoreactivity to the
anti-Na antibody (pink stain). These cells were
electrophysiologically confirmed to be expressing
INa.
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Fig. 9.
Immunohistochemical detection of
voltage-gated sodium channels in intact human saphenous vein sections.
A, vein section immunostained with thrombomodulin antibody
to confirm the presence of intact endothelium; B, serial
section as in A, showing positive immunoreactivity to the
anti-Na antibody. Note that not all cells staining positive for
thrombomodulin expression are expressing Na+
channels.
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Serum Induction of Sodium Channels in HSVECs--
To assess the
effects of serum on expression of INa by
subconfluent HSVECs, we measured the magnitude and prevalence of
INa in cells that had been incubated for 48 h in growth medium that was serum-free or supplemented with 10% (v/v)
human serum either from peripheral arterial disease patients 65 years
of age and over ("aged") or from healthy donors under 30 years of
age ("young"). Under serum-free conditions and in medium
supplemented with aged serum, INa was of small
magnitude and was found in relatively few cells (Table
I). However, medium supplemented with
young serum was found to increase significantly both the magnitude of INa and the number of HSVECs in which
INa was observed (Table I). The stimulatory
effect of serum upon INa expression was
confirmed by cell-based enzyme-linked immunosorbent assay of confluent
HSVECs using the anti-Na
antibody. Inclusion of 2 or 10% (v/v)
young serum in the incubation medium of HSVECs for 6 h increased
the relative concentration of sodium channel protein 2- and 4-fold, respectively. The absorbance increased from 0.10 ± 0.03 (n = 8) in serum-free medium to 0.19 ± 0.03 (n = 7) and 0.36 ± 0.06 (n = 6)
for HSVECs cultured in 2 and 10% (v/v) young sera, respectively.
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Table I
Effects of growth conditions upon presence of INa in HSVECs as
assessed by electrophysiology
Subconfluent HSVECs were cultured for 48 h under three growth
conditions: M199 medium containing 10% (v/v) human serum obtained from
volunteers 65 years of age or older (aged) or younger than 30 years
(young) and M199 medium containing human insulin, transferrin, and 1%
(w/v) human albumin (serum-free M199). The presence of
INa was ascertained using the whole-cell
configuration of the patch-clamp technique with Cs+/TEA
solutions.
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DISCUSSION |
In this investigation, we have used a range of techniques to
demonstrate the presence of voltage-gated sodium channels in human
saphenous vein endothelium. First, immunohistochemistry, using an
antibody directed against the conserved cytoplasmic region of the
-subunit, showed the presence of sodium channels in both intact
saphenous vein endothelium and cultured HSVECs. Second, this same
antibody recognized a 245-kDa protein in Western blot analysis of HSVEC
membrane lysates. Third, whole-cell patch-clamp electrophysiology of
HSVECs showed the presence of fast inward voltage-gated sodium
currents, which were TTX-resistant and showed similar kinetics to the
human heart hH1 channel isoform. RT-PCR analysis also showed HSVECs to
contain hH1 transcripts. The expression of this sodium
channel in HSVECs was dependent on serum characteristics and
concentration.
The expression of voltage-gated sodium channels in human saphenous vein
endothelium was unexpected since this type of ion channel classically
is associated with action potential generation in excitable cells. This
is the first report of the presence of sodium channels in the
endothelium of intact human vessels. There has been one previous
electrophysiological study suggesting that cultured human endothelium
from umbilical vein expressed sodium channels, but the subtype of
sodium channel was not identified (21). A potential criticism of this
latter study was that the expression of sodium channels was an artifact
of placing the cells into culture since this phenomenon has been
reported for human coronary myocytes (22).
The antibody used for demonstrating the presence of sodium channels in
intact endothelium was also used in electrophysiological studies; when
the antibody was applied intracellularly to HSVECs, there was
substantial slowing of the current inactivation. The current kinetics
and the TTX inhibition studies suggested that INa in HSVECs closely resembles the principal
TTX-resistant, voltage-gated sodium channel found in human heart, hH1
(8, 23). Electrophysiological and immunohistochemical analyses showed
that the sodium channel was not present in every endothelial cell.
However, the prevalence of sodium currents (57%) was similar to that
of inwardly rectifying potassium currents (58%), the most widely
distributed channel in endothelial cells (3). The data from cell-based
enzyme-linked immunosorbent assays and electrophysiology suggest that
the prevalence and expression of sodium channels in HSVECs are
serum-dependent. Serum harvested from young healthy
volunteers increased the magnitude of INa
3-4-fold compared with serum from aged patients with peripheral arterial disease. The 2-3-fold increase in HSVEC sodium channels, when
serum concentration was increased from 2% to 10%, was similar to the
previously reported serum stimulation of the sodium channel in rat
leiomyosarcoma cells (24).
Endothelial cells have never been reported to produce action potentials
and are classed as non-excitable (3). In keeping with this tenet, the
magnitude of INa in HSVECs is small, with a mean
peak current of
595 ± 49 pA, and INa
requires a membrane potential more negative than
80 mV to remove
inactivation completely (Fig. 4B) when the resting membrane
potential (Em) of cultured HSVECs is around
30 mV.
This would imply that INa normally would be
inactivated and dysfunctional. However, in vivo, it is probable that endothelial cells are more hyperpolarized
(Em more negative): the Em of
endothelial cells on intact saphenous vein is nearer to
70
mV.2 Stimuli that are known
to hyperpolarize vascular endothelial cells, such as hemodynamic shear
stress, could produce potentials that are sufficiently negative to lead
to a partial recovery of INa from inactivation.
The inwardly rectifying K+ channel is the predominant
channel open at rest in endothelial cells and tends to hold
Em close to the potassium equilibrium potential
(EK). As these channels do not pass much
repolarizing current due to their poor outward rectification
characteristics, they would permit relatively small inward currents
carried by other ions to depolarize the endothelial cell. Therefore,
even a small magnitude INa may be able to elicit
substantial and rapid membrane depolarization. However, as HSVECs lack
the outward potassium currents (delayed rectifier currents) necessary
to rapidly repolarize the cells, it is unlikely that these cells could
elicit repetitive action potentials. Similar findings have been
reported in glial cells, which are also considered to be inexcitable,
yet express Na+ channels (25).
There are at least two possible physiological functions for
voltage-gated sodium channels in vascular endothelium, given that they
are unlikely to be involved in action potential generation. First,
INa could have a role in the regulation of
intracellular calcium levels ([Ca2+]i). This
could occur by several mechanisms. An increase in Na+
influx would stimulate Na+/Ca2+ exchange and
thus raise [Ca2+]i (26). It also has been
reported that voltage-dependent Na+ channel
gating is involved in depolarization-induced activation of G-proteins,
a process that could lead to Ca2+ mobilization (27). Also,
some capillary endothelial cells have been reported to possess a
voltage-dependent, BAY K8644-sensitive Ca2+
current (28, 29); thus, INa could provide the
depolarizing stimulus leading to opening of these channels. However,
these Ca2+ channels have yet to be described in large
vessel endothelium. Second, the electrical coupling between vascular
endothelial cells, as well as coupling between endothelial cells and
smooth muscle cells (30), raises the possibility that an electrical
message, such as depolarization, could be conveyed electrotonically by the endothelium. This process also may participate in regulating [Ca2+]i, as it has been shown in capillary
endothelium that some cells possess a "pacemaker" function and pass
an undetermined message via gap junctions to other cells to initiate
Ca2+ oscillations (31).
This is the first report of the presence of sodium channels of the hH1
isoform in human vascular endothelium. The regulation and distribution
of this sodium channel are the focus of current investigations to
assess the role of this channel in endothelial homeostasis.
We thank A. H. Davies, Prof. R. M. Greenhalgh, and Prof. K. M. Taylor (Hammersmith Hospitals Trust)
for providing saphenous vein and Dr. S. Harding (National Heart and
Lung Institute) for the guinea pig cardiac myocytes.