© 1999 American Heart Association, Inc.
Original Contribution |
Mechanical Stimulation Regulates Voltage-Gated Potassium Currents in Cardiac Microvascular Endothelial Cells
From the Department of Pharmacology, University of South Carolina, School of Medicine, Columbia, SC.
Correspondence to Kenneth B. Walsh, Department of Pharmacology, University of South Carolina, School of Medicine, Columbia, SC 29208. E-mail walsh{at}med.sc.edu
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Abstract |
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Abstract—Vascular endothelial cells are constantly exposed to mechanical forces resulting from blood flow and transmural pressure. The goal of this study was to determine whether mechanical stimulation alters the properties of endothelial voltage-gated K+ channels. Cardiac microvascular endothelial cells (CMECs) were isolated from rat ventricular muscle and cultured on thin sheets of silastic membranes. Membrane currents were measured with the use of the whole-cell arrangement of the patch-clamp technique in endothelial cells subjected to static stretch for 24 hours and compared with measurements from control, nonstretched cells. Voltage steps positive to -30 mV resulted in the activation of a time-dependent, delayed rectifier K+current (IK) in the endothelial cells. Mechanically induced increases of 97%, 355%, and 106% at +30 mV were measured in the peak amplitude of IK in cells stretched for 24 hours by 5%, 10%, and 15%, respectively. In addition, the half-maximal voltage required for IK activation was shifted from +34 mV in the nonstretched cells to -5 mV in the stretched cells. Although IK in both groups of CMECs was blocked to a similar extent by tetraethylammonium, currents in the stretched endothelial cells displayed an enhanced sensitivity to inhibition by charybdotoxin. Preincubation of the CMECs with either pertussis toxin or phorbol 12-myristate 13-acetate during the 24 hours of cell stretch did not prevent the increase in IK. The application of phorbol 12-myristate 13-acetate and static stretch stimulated the proliferation of CMECs. Stretch-induced regulation of K+ channels may be important to control the resting potential of the endothelium and may contribute to capillary growth during periods of mechanical perturbation.
Key Words: endothelial cell • voltage-gated K+ channel • stretch • protein kinase C
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Introduction |
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Potassium channels represent a large and diverse family of proteins expressed throughout the cardiovascular system. In excitable tissues, such as cardiac and smooth muscle, voltage-gated K+ channels regulate the frequency and duration of action potentials.1 In nonexcitable cells, K+ channels are involved in other physiological functions including volume regulation, electrolyte transport, and cell proliferation.2 3 In the vascular endothelium, both inward rectifier and voltage-gated K+ channels are involved in setting the cell resting membrane potential.4 5 By the control of the resting membrane potential and thus the influx of Ca2+ into endothelial cells, these channels play a critical role in regulating the release of endothelial-derived vasoactive substances.6
Both swelling-induced and mechano-sensitive ion channels have been identified in endothelial cells. Brief periods of cellular swelling, induced by exposing bovine pulmonary artery endothelial cells to hypotonic solutions, causes the activation of an outward-rectifying Cl- channel.7 8 This swelling-induced Cl- current is postulated to function in restoring cell volume by allowing the transport of Cl- and water.5 The application of mechanical stretch to several types of endothelial cells results in the activation of nonselective, Gd3+-sensitive, cation channels.9 10 In addition, shear stress, simulated though the use of laminar flow, activates a number of different K+ channels including inward rectifier11 12 and Ca2+-activated K+ channels.13
Although mechano-sensitive ion channels have been identified in vascular endothelial cells under conditions that produce relatively small and transient changes in cell shape, little is known concerning the effects of longer periods of mechanical stimulation on endothelial electrical activity. In the present study, cardiac microvascular endothelial cells (CMECs) were plated on silastic membranes and stretched by 5%, 10%, and 15% for 24 hours. We report that stretch induces the up-regulation of a charybdotoxin-sensitive, voltage-gated K+ current in these cells.
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Materials and Methods |
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Isolation and Culture of CMECs
CMECs were isolated and characterized as described previously.14 Briefly, hearts were removed from adult rats (weight, 180 to 200 g), mounted on a Langendorff-type column, and perfused for 5 minutes with Krebs solution composed of (in mmol/L) NaCl 118, KCl 4.7, CaCl2 1.25, MgSO4 1.2, KH2PO4 1.2, and NaHCO3 25, saturated with 5% CO2/95% O2, at pH 7.4. After perfusion, the outer one-fourth of the left ventricle free wall and septum were dissected away to remove epicardial arteries and larger penetrating vessels. The remaining tissue was minced in 0.2% collagenase (type B, Boehringer-Mannheim Biochemicals) and incubated for 30 minutes at 37°C in a shaking bath. Trypsin (0.02%, Sigma Chemical Co) was then added, and the tissue sheared 10 times over a period of 30 minutes. Dissociated cells were filtered through a 100 µm mesh filter, washed with Ca2+-free solution, and centrifuged at 100g for 5 minutes. CMECs were resuspended in DMEM supplemented with 20% fetal bovine serum and antibiotics (penicillin, streptomycin, and fungiozone). The CMECs were plated on laminin-coated dishes at a density of
2.5x103 cells/cm2.
After 2 hours, attached cells were washed with DMEM to allow
differential adhesion.15 Cultures were maintained in
a humidified atmosphere of 5% CO2 at 37°C and
grown to confluency (6 to 7 days) before use in the stretch
experiments. Differential uptake of acetylated low-density
lipoprotein, determined by fluorescence-activated cell
sorting, indicated that the cultures contained >90%
endothelial cells.14
Application of Static and Cyclic Stretch
For stretch application, cells were treated with trypsin and
plated on silastic membranes.16 Membranes were coated with
1% albumin and fixed with 1% glutaraldehyde.
After allowing 24 hours for cell attachment, the silastic membranes
were subjected to varying degrees of stretch (5%, 10%, and 15%) with
the use of adjustable brackets. When compared with cyclic stretch, the
advantage of applying static stretch was that it required a simple
system that could be precisely set and maintained. In addition, the
static stretch device was small enough to fit into the
recording chambers. This feature allowed stretch to be
maintained during the measurement of membrane currents. In one set of
experiments, silastic membranes were attached to a mechanical
stimulator and subjected to 24 hours of cyclic stretch applied at 0.5
Hz with 10% elongation. To reduce cell proliferation during the 24
hours of static and cyclic stretch, the serum concentration in the
media was reduced to 3%. Nonstretched cells were cultured in an
identical manner, with the exception that force was not applied to the
silastic membranes. Pairs of nonstretched and stretched cultures were
examined on the same experimental day to limit culture-to-culture
variations in ion-channel density.
Recording Procedure and Measurement of K+
Currents
The patch-clamp method17 was used to record
whole-cell CMEC currents with L/M EPC-7 (Adams and List Associates) and
Axopatch 200 (Axon Instruments) amplifiers. Pipettes were made from
Gold Seal Accu-fill 90 Micropets (Clay Adams Inc) and had resistances
of 2 to 4 M
when filled with internal solution. Unless stated
otherwise, all experiments were conducted on isolated, noncoupled CMECs
at room temperature (22°C to 24°C). Cells were placed in an
external solution consisting of (in mmol/L) NaCl 132, KCl 5,
MgCl2 2, CaCl2 1, dextrose
5, and HEPES 5, pH 7.4 (with NaOH) (280 mOsm.) The internal solution
consisted of (in mmol/L) KCl 50, K-glutamate 60,
MgCl2 2, CaCl2 1, EGTA 11,
ATP 3, and HEPES 10, pH 7.3 (with KOH)
([K+]=140 mmol/L) (280 mOsm). The ratio of
EGTA/CaCl2 in this solution sets the free
intracellular Ca2+ concentration to 10
nmol/L.18 A reference electrode made from a Ag-AgCl pellet
was connected to the bath with an agar salt bridge saturated with
external solution. Data were adjusted for liquid junction potentials
that occurred between the pipette solution and bath solution and
between the reference electrode and the bath.19 Offset
potentials, created at the junction of the internal/external solution
interface, were measured at the beginning and end of the experiments
and were between -5 and +5 mV.
Membrane currents were recorded with 12-bit analog/digital converters (Axon Instruments). Data were sampled at 10 kHz, filtered at 2 kHz with a low-pass Bessel filter (Frequency Devices), and stored with the use of personal computers (Northgate and Dell). Series resistance was compensated to provide the fastest possible capacity transient without producing oscillations. With this procedure, >50% of the series resistance could be compensated. Linear leak and capacity transients were removed from test currents with records obtained during 4 hyperpolarizing pulses from the holding potential of –80 to -100 mV. These records were averaged and subtracted from the test currents. Use of this protocol was justified because voltage- and/or time-dependent conductances were not present at these potentials. For a typical set of cultures, the membrane capacity of the nonstretched CMECs ranged from 17 to 40 pF with a mean±SE of 29±1 pF (n=24 cells). The capacity of the stretched cells was 29±2 pF (n=24 cells).
All experiments were conducted at room temperature (22°C to 24°C). Averaged values presented are means±SE. Where appropriate, statistical significance was estimated with the use of Student's t test for unpaired observations.
Materials and Drugs
DMEM, 4-aminopyridine, tetraethlyammonium,
phorbol 12-myristate 13-acetate (PMA), and pertussis toxin
(PTX) were purchased from Sigma Chemical Co.. Charybdotoxin (CTX) was
obtained from Alomone Laboratories LTD (Jerusalem, Israel).
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Results |
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Stretch-Induced Increases in the CMEC IK
The top left panel of Figure 1
shows
an example of membrane currents obtained from a CMEC during voltage
steps applied from a holding potential -80 mV to various potentials.
Voltage steps to potentials more positive than -30 mV resulted in the
activation of outward K+ currents. The
time-dependent and voltage-dependent properties of the CMEC current
were similar to those of a family of delayed rectifier
K+ currents found in a large number of cell
types.3 20 21 The CMEC current was identified as a
K+ current on the basis of 3 criteria. First, the
reversal potential of the tail currents -73±2 mV (n=5 cells),
obtained during deactivation of the current, was close to the
theoretical K+ equilibrium potential (-83 mV)
under the conditions of the experiments
([K+]0=5 mmol/L,
[K+]0=140 mmol/L).
Second, the CMEC current was inhibited by a 500 µmol/L
concentration of the K+ channel blocker
4-aminopyridine (95±1% decrease at 0 mV, n=4 cells)
and during substitution of external NaCl with TEA
chloride(Figure 5). Finally, use of a CsCl internal
solution resulted in a complete elimination of the outward
current.14
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To determine the effect of mechanical stimulation on ion channels,
CMECs were stretched by 5%, 10%, and 15% and compared with control,
nonstretched cells cultured under identical conditions. In initial
experiments, membrane currents were measured during voltage steps to
-100 mV and -60 mV to determine whether stretch was activating
time-independent and voltage-independent background currents that might
occur as a result of the opening of mechano-sensitive ion
channels.9 10 Current densities were -0.9±0.1 pA/pF
(-100 mV) and -0.8±0.1 pA/pF (-60 mV) in the nonstretched CMECs and
-1.1±0.1 pA/pF (-100 mV) and -1.0±0.1 pA/pF (-60 mV) in the
stretched CMECs (n=16 cells). However, when a stretched cell was
compared with a nonstretched cell of similar membrane capacity, 24
hours of stretch resulted in a dramatic increase in the size of the
delayed rectifier K+ current
(IK) (Figure 1
). Figures 2 and 3
summarize the results of IK measurements
obtained from >160 CMECs recorded during stretched and
nonstretched conditions. Stretched-induced increases of 97% and 355%
(at +30 mV) in the peak amplitude IK were
measured in cells stretched at 5% and 10%, respectively (Figure 2, left panel). Increases in IK
were also observed in cultures stretched at 15% for 24 hours (Figure 2, left panel). However, this increase (106%) was significantly
less than that measured during 10% stretch (P<0.05). The
higher incidence of cellular damage in the 15% stretched
cultures may have accounted, at least in part, for this decline in
current amplitude. The mean value of IK
increased after 6 hours of stretch and was significant at 12 hours
(Figure 2, right panel). Measurements were not taken after 24
hours of stretch because of difficulties in finding single, noncoupled
cells at later times. Application of cyclic stretch also increased the
amplitude of IK. The results plotted in
Figure 3 compare the effects of 10% static stretch and 10%
cyclic stretch on IK measured at 0, +30,
and +50 mV. There was no significant difference (P>0.05) at
any voltage in the size of IK recorded
after 24 hours of static and cyclic stretch.
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Voltage Dependence of IK Activation in
Stretched and Nonstretched CMECs
The results displayed in Figures 1 to 3 indicate that
mechanical stimulation produces a strong augmentation in the size of
the CMEC IK. One possible explanation for
these results could be that stretch up-regulates the expression of
K+ channels in the endothelial
cells. With respect to those channels present in the nonstretched
cells, these channels might represent the same or possibly a
different class of K+ channels. For this reason,
it was important to compare the voltage-dependent and pharmacological
properties of IK in the stretched and
nonstretched cells.
The normalized conductance for the currents measured in the stretched
and nonstretched cells is plotted as a function of the test voltage in
Figure 4. The continuous lines
represent the best fits of the data points to the Boltzmann
equation (see Figure 4 legend). The half-maximal voltage
(V1/2) required for activation was +34 mV in the
nonstretched cells and decreased to -5 mV in cells stretched at 10%
for 24 hours. In addition, the parameter k, which reflects
the slope of the fitted curve, decreased from 20 (nonstretched cells)
to 8 (stretched cells). The activation curve for
IK in cells stretched by 5% was
intermediate to that of the 10% stretched and nonstretched cells with
a V1/2 of +10 mV (Figure 4). In this case,
k decreased to 15.
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Pharmacology of IK in Stretched and
Nonstretched CMECs
Both tetraethylammonium (TEA) and
charybdotoxin (CTX) serve as useful pharmacological agents for
characterizing voltage-gated K+
channels.3 20 21 As indicated in the top panel of Figure 5, relatively high concentrations of TEA
were required to block the CMEC IK.
Substitution of the 132 mmol/L NaCl in the external solution with
132 mmol/L TEA chloride reduced IK in
the nonstretched cells by 88±4% (n= 4 cells, 0 mV) (Figure 5, left panel). A similar decrease in IK was
recorded in the presence of TEA for CMECs stretched at 10% (%
change=83±4%, n= 4 cells) (Figure 5, right panel). A smaller
decrease in IK (35% to 40%) was measured
after the addition of 25 mmol/L TEA (results not shown). CTX was
also an effective blocker of IK in the
stretched CMECs. In 5 cells examined, 50 nmol/L CTX inhibited
IK by 88±2% at 0 mV (Figure 6, right panel). Block of
IK occurred in a voltage-dependent manner
with a significantly smaller reduction in
IK recorded at +50 mV. Surprisingly,
IK measured in the nonstretched CMECs
displayed a reduced sensitivity to block by CTX, with an overall
decrease of 21±7% at 0 mV (n= 8 cells) (Figure 6, left panel)
recorded after toxin application. This change was significantly
different than the inhibition recorded in the stretched CMECs
(P<0.05).
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Effect of PTX and PMA on Stretch-Induced Increases in
IK
Previous studies have demonstrated that mechanical stimulation of
endothelial cells effects G protein-coupled, signal
transduction pathways22 and protein kinase C-regulated
proteins.23 As a first step in determining the mechanism
of stretch-induced augmentation of IK,
cell cultures were preincubated with either PTX (1 µg/mL), to inhibit
the Gi and Go proteins, or
PMA (500 nmol/L), to down-regulate protein kinase C. Exposure of the
CMECs to PTX during the 24-hour period of stretch had no effect on the
stretch-induced increase in IK (Figure 7, left panel). Twenty four hours of
treatment with PMA also did not prevent stretch-induced increases in
IK. Surprisingly, in the presence of PMA,
there was a significant augmentation in the size of
IK at all measured voltages (Figure 7, left panel). This effect on IK
was associated with a stimulation of cell proliferation and an increase
in the number of cellular processes projecting from the cell
bodies.
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Effect of Stretch on IK in Culture
Medium Containing 3% and 15% Serum
As shown in the right panel of Figure 7, 24 hours of static
stretch stimulated endothelial cell proliferation.
Unlike the increase in cell proliferation observed during PMA
treatment, this action was not associated with any change in cell
structure. Because stretch induces both an increase in
IK density and a stimulation in
endothelial cell proliferation, it was determined that
changes in K+ channel density might be dependent
on cell growth. Figure 8 summarizes the
results of experiments in which the CMECs were grown in a culture
medium containing 15% serum. Under these conditions, the
endothelial cells proliferated to an extent greater
than that measured in 3% serum, and the application of 10% static
stretch did not significantly change the rate of proliferation (results
not shown). As shown in Figure 8, 24 hours of static stretch
produced a significant increase in the IK
density in cells grown in 15% serum. This increase was not as large as
that measured in cells grown in 3% serum (summarized in Figure 8) due to the augmented basal current level recorded in the
CMECs grown in 15% serum. This suggests that multiple factors
(stretch, serum concentration, proliferative state, etc) can regulate
the IK density.
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Discussion |
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Mechanically Induced Changes in Endothelial Ion Channels
Vascular endothelial cells respond to 2 different types of mechanical force: shear stress that results from blood flow, and circumferential wall stretch that results from transmural pressure. The goal of the present study was to determine the effects of mechanical stretch on K+ currents in CMECs. Although mechano-sensitive ion channels have been identified in endothelial cells under conditions that produce small and transient changes in cell shape,7 9 10 the effect of 24 hours of mechanical stimulation on endothelial electrical activity is not known. Previous studies have demonstrated that both stretch and shear stress regulate endothelial ion channels.9 10 11 12 Lansman et al9 identified a 40 pS ion channel that is activated during deformation of membrane patches in aortic endothelial cells. A similar nonselective cation channel with a permeability for Ca2+, K+, and Na+ is present in cerebral capillary endothelial cells.10 A whole-cell, time-independent, background current corresponding to the opening of these channels could not be measured in CMECs stretched for 6, 12 or 24 hours. Because mechano-sensitive ion channels are known to inactivate during continuous stretch,24 it is not surprising that mechanically induced currents, if present in the CMECs, were not recorded in the chronically stretched cells.
The major finding of the present study is that stretch increases
the size of a voltage-gated K+ current. The
different activation parameters and
charybdotoxin-sensitivity of IK in the
nonstretched and stretched cells suggest that a different type of
K+ channel may be up-regulated by stretch. The
voltage-gated K+ channel (Kv) gene family is
represented by a large and diverse number of 
subunits
that compose the pore of the channel.25 Expression of
individual rKv1.2, rKv1.4, and rKv1.5 subunits results in
time-dependent outward K+ currents with a
V1/2s of -20 to 0 mV, moderate sensitivities to
CTX (Kd=10 to 100 nmol/L), and relative
insensitivities to TEA (Kd>50
mmol/L).25 These properties are quite similar to
IK measured in the stretched cells and may
suggest that one or more of these subunits is preferentially
up-regulated by mechanical stimulation. Olesen et
el11 demonstrated that shear stress activates
a whole-cell inward rectifier K+ current in
bovine aortic endothelial cells. The inward rectifier
was found to vary in size as a function of the amount of shear stress,
desensitize slowly with time, and recover on cessation of
flow.11 The Ca2+-sensitivity of
inward rectifier channel,12 and the requirement of G
proteins in the shear stress activation process,26
indicate that this channel differs from the classic inward rectifier
found in endothelial cells.27 28 The shear
stress–activated inward rectifier also clearly differs from
IK recorded in the CMECs and suggests
that the Kv family of proteins represent a hereto unknown
target of mechanically induced regulation. This idea is supported by
the recent finding that cyclic stretch increases the expression of
Kv1.5 channels in cultured cardiac ventricular
myoctyes.29
Mechanically Induced Changes in Endothelial
Intracellular Signaling
Mechanical forces modulate a number of signal transduction
pathways in endothelial cells including heterotrimer G
proteins,22 cAMP,30 intracellular
Ca,2 31 protein kinase C (PKC),23
mitogen-activated protein (MAP) kinases,32 and
tyrosine kinases.33 34 In addition to the direct
modulatory effects on cellular proteins, stimulation of these pathways
leads to the activation of multiple gene transcription
factors.35 Pretreatment of the CMECs with either (PTX), to
inhibit Gi or Go, or PMA,
to down-regulate PKC,36 was ineffective in preventing
stretch-induced increases in IK. In
actuality, PMA potentiated-stretch induced increases in
IK (Figure 7). However, since PKC
activity was not measured directly, we can only infer that this enzyme
was down-regulated. Previous studies have shown that acute stimulation
of PKC by phorbol esters inhibits Kv channels including the cloned
Kv1.3 and Kv1.4 channels.37 38 39 Thus, by
down-regulating PKC, chronic treatment of the cells with PMA may
relieve a basal inhibition of IK caused
through protein phosphorylation. Alternatively, PMA
treatment may result in the induction of genes encoding for Kv
channels. As observed with the CMECs, phorbol esters induce
morphological changes and proliferation in both macrovascular and
microvascular endothelial cells.40
These actions are associated with changes in the expression of adhesion
molecules including E-selectin, vascular cell adhesion molecule-1, and
intercellular adhesion molecule-1.40 In addition,
treatment of cancer cells with mitogens such as concanavalin A,
lipopolysaccharides, and interleukins cause large increases in
the expression of K+ channels.41
Further experiments with specific inhibitors of PKC and
tyrosine kinases will be necessary to understand the role of these
signaling proteins in the stretch-induced increase in
IK.
Relevance of the Study to Cardiovascular Physiology
The expression of K+ channels varies
greatly between different types of endothelial
cells.4 5 In macrovascular endothelial
cells, the inward rectifier K+ channel is the
main contributor in setting the resting membrane potential (-50 to
-70 mV).42 43 In contrast, Cl-
channels and Kv channels play a more dominant role in setting the
resting potential in microvascular endothelial cells
such as the CMECs, which lack inward rectifier currents at
physiological
[K+]44 (our unpublished results).
This is consistent with the more positive resting potential
(-10 to -40 mV) measured in the microvascular
cells.14 44 By the modulation of the
K+ conductance of the cell and thus the resting
potential, mechanical stimulation should increase
Ca2+ influx and the release of vasoactive
substances such as nitric oxide, endothelin, and
prostaglandins from the cells. In addition, increases in
K+ channel density may be important in mediating
endothelium responses to stimulating agents such as
histamine and thrombin.
Recent evidence suggests that K+ channels are involved in the regulatory steps of cell proliferation.41 Experiments that demonstrate that K+ channel blockers inhibit cell division in neuroblastoma, melanoma, and breast cancer cells,45 46 47 imply that there is a relationship between the internal [K+] and the proliferative activity of the cell. In the cardiovascular system, mechanical forces have been shown to stimulate capillary angiogenesis under conditions that produce mild-to-moderate vessel wall stretch.48 49 Because microvascular endothelial cell proliferation is a critical step in angiogenesis, it will be important to access the role of voltage-gated K+ channels in this process. Stretch-induced increases in CMEC K+ currents may contribute to capillary growth during periods of mechanical perturbation. Future experiments will be required to determine whether stimulation of endothelial cell proliferation or ion channel expression is the primary initiator of these vessel changes.
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Acknowledgments |
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This work was supported by US Public Health Service grant HL-45789 and grants from the South Carolina Affiliate of the American Heart Association and the South Carolina Consortium for Cardiovascular Diseases and Stroke.
Received April 22, 1998; accepted December 2, 1998.
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