Cardiovascular Research 38 Ž1998. 433–440
Pressure-activated cation channel in intact rat endocardial endothelium
)
Ralf Kohler
, Armin Distler, Joachim Hoyer
¨
Department of Endocrinology and Nephrology, UniÕersity Hospital Benjamin Franklin, Free UniÕersity, Berlin, Germany
Received 26 August 1997; accepted 12 January 1998
Abstract
Objective: The endocardial endothelium ŽEE. regulates myocardial performance in response to humoral and mechanical stimuli. In
vascular endothelium mechanosensitive ion channels ŽMSC. act as mechanosensors for hemodynamic changes. In the present study we
examined whether MSC are present in intact EE of rat papillary muscle segments and characteristics of MSC are altered in experimental
hypertension. Methods: MSC were investigated by the use of standard patch-clamp technique. For a comparative study, ion channel
characteristics were determined in EE of two-kidney-one-clip rats and sham-operated controls. Results: We identified a new class of
MSC with a mean conductance of 21.8 " 4.4 Žs.d.. pS for Kq and Naq and of 4.1 " 1.5 pS for Ca2q. Channel activity was initiated by
positive pipette pressure and blocked by negative pipette pressure. Channel open probability Ž Po . was characterized by its pressure
sensitivity. Po increased from 0.06 at 10 mmHg to 0.37 and 0.55 at 20 mmHg and 30 mmHg, respectively. Gadolinium Ž20 mM., a
blocker of MSC, completely inhibited channel activity. In some experiments activation of this pressure-activated channel ŽPAC. was
followed by the opening of a Ca2q-dependent non-selective cation channel ŽNSC.. This indicates that Ca2q influx through PAC may be
sufficient to increase intracellular Ca2q concentration and thereby to activate neighboring NSC. In renovascular hypertension Ž2K1C.,
channel density of PAC was significantly increased compared to sham-operated controls. Channel density of NSC was not changed in
2K1C compared to sham-operated controls. Conclusion: A novel type of Ca2q permeable MSC in intact EE of rat ventricular papillary
muscle was identified, which is regulated by membrane pressure. PAC might be implicated in EE mechanotransduction by inducing an
intracellular Ca2q signal. Up-regulation of PAC density in EE from 2K1C might contribute to an altered mechanotransduction in
hypertension. q 1998 Elsevier Science B.V. All rights reserved.
Keywords: Endocardial endothelium; Mechanosensitive ion channel; Hypertension; Left ventricular hypertrophy
1. Introduction
The endothelium modulates vascular tone and myocardial contractile behavior w1x in similar ways. The functional
role of the endocardial endothelium ŽEE. has been proven
by studies showing that the removal of the endothelium
results in a negative inotropic effect on myocardial contraction w2x. The EE influences performance of the subjacent myocardium by the release of nitric oxide ŽNO.,
prostacyclin, prostaglandin E 2 and of endothelin, which
control contractile duration w1,3–6x.
In vascular endothelium intracellular alterations of calcium concentration seem to play a key role in the media-
tion of humoral and mechanical stimuli. For instance, in
vascular endothelial cells fluid shear stress induces an
increase of intracellular calcium concentrations ŽwCa2q x i .
and thereby stimulates Ca2q-dependent NO and prostacyclin synthesis w7–10x. However, the early mechanism of
endothelial mechanosensing leading to an increase of
wCa2q x i are incompletely understood. Ca2q permeable
stretch-activated cation channels ŽSAC. have been identified in vascular endothelial cells w11,12x and in intact EE
of porcine right atrium w13x, and have been proposed to act
as microscopic mechanotransducers by converting a hemodynamic stimulus into an intracellular Ca2q signal.
Recently we reported a novel pressure-activated channel
ŽPAC. in intact endothelium of rat mesenteric artery and
aorta. In experimental hypertension an up-regulation of
)
Corresponding author. Abteilung fur
¨ Endokrinologie und Nephrologie, Universitatsklinikum
Benjamin Franklin, Hindenburgdamm 30, 12200
¨
Berlin, Germany. Tel.: q49 30 84452398; Fax: q49 30 84452398;
E-mail: [email protected]
0008-6363r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.
PII S 0 0 0 8 - 6 3 6 3 Ž 9 8 . 0 0 0 3 0 - 3
Time for primary review 29 days.
434
R. Kohler
et al.r CardioÕascular Research 38 (1998) 433–440
¨
PAC was observed after the development of an increased
blood pressure w16x. An implication of an altered regulation
of ion channels has also been demonstrated in renal tubule
cells and vascular smooth muscle cells in genetic hypertension w14,15x.
An increased peripheral resistance present in hypertension leads to left ventricular pressure overload and
subsequently to left ventricular hypertrophy. In the present
study we examined whether mechanosensitive ion channels are present in intact EE of left ventricular papillary
muscle and ion channel properties are changed in experimental hypertension with left ventricular hypertrophy.
2. Materials and methods
Animal studies were conducted according to the guidelines set by the Ethics Committee of the Berlin government. Rats were killed by cervical dislocation and exsanguinated. The hearts were removed and cut open. Entire
papillary muscles ŽPM. were carefully excised out of the
left ventricle. For patch-clamp experiments, PM were
placed in a chamber on a stage of an inverted Axiovert
microscope ŽZeiss, Germany. with the EE facing the bath
solution. This enabled a direct approach of the endothelial
cells with the patch pipette.
Fig. 1. Ža. Increase of spontaneous channel activity of pressure-activated cation channel ŽPAC. in endocardial endothelium of rat left ventricular papillary
muscle by positive pipette pressure Ž30 mmHg.. A total of seven channels was activated. Inactivation of PAC by negative pipette pressure Žy30 mmHg..
Activation or inactivation of PAC occurred immediately after changing pipette pressure. Channel openings in the downward direction indicate Kq currents
flowing from the pipette into the cell. c ™, closed state of channels. Channel activity was measured at a holding potential y80 mV in cell-attached
patches. Žb. Gradual increase of channel activity in response to positive pipette pressure. Open probability Ž Po . increased to 0.06, 0.37 and 0.55 at 10, 20
and 30 mmHg, respectively. A total of four channels was simultaneously activated in this cell-attached patch. c ™, closed state of channels.
R. Kohler
et al.r CardioÕascular Research 38 (1998) 433–440
¨
Patch-clamp experiments and data analysis were carried
out as described w17–19x. Membrane currents were recorded
with a HEKA electronics ŽLambrecht, Germany. EPC-9
patch-clamp amplifier. Patch pipettes were prepared from
borosilicate glass with a tip resistance of 5–6 M V. Seal
resistance ranged from 5 to 10 GV. Mechanical stimulation of the cell membrane was performed by application of
negative or positive pressures to the rear of the patch
pipette. The applied pressure was adjusted and controlled
with a water manometer and monitored with a differential
pressure transducer.
Data were low-pass-filtered Žy3 dB, 1000 Hz. at a
sample time of 0.5 ms. The given potential values resemble the clamp potential and the sign of the potential refers
to the cytosolic side. If not otherwise stated, the patch
pipette was filled with a KCl solution containing Žin mM.:
140 KCl, 1.3 CaCl 2 , 1 MgCl 2 , 10 HEPES, adjusted to pH
7.4 with KOH. A NaCl solution was used as bath solution
containing Žin mM.: 140 NaCl, 4.3 KCl, 1.3 CaCl 2 , 1
435
MgCl 2 , adjusted to pH 7.4 with NaOH. High Ca2q solution contained 90 mM CaCl 2 instead of NaCl. For Cly
substitution solutions were prepared with Na-cyclamate.
Experiments were performed at 358C. In multichannel
patches the number of channels was determined by the
maximum number of superimposed channel openings during maximal mechanical activation. Single channel open
probability Ž Po . in multichannel patches was calculated as
described previously w18x.
For a comparative randomized study, male Wistar–
Kyoto rats ŽWKY. were obtained from Møllegard Breeding and Research Center ŽSkensved, Denmark.. For induction of renovascular hypertension a two-kidney one clip
model was used. 8-week old WKY were anesthetized with
pentobarbital Ž30 mg P kgy1 i.v.. and the left renal artery
was exposed and dissected free of the renal vein. A silver
clip with an internal diameter of 0.23 mm was placed
around the artery, causing partial occlusion and the wound
was closed. For a control group, sham operation was
Fig. 2. Ža. Ca2q influx through PACs in cell-attached patches at a holding potential of y80 mV. Channel openings in the downward direction indicate
Ca2q currents flowing from the pipette into the cell. c ™, closed state of channels. Pipette solution contained 90 mM CaCl 2 , 1 mM MgCl 2 and 10 mM
HEPES, pH 7.4. Žb. Current–voltage Ž I – V . relationship of PAC in inside-out patches was determined in the presence of the following pipette and bath
solutions: open trianglessCaCl 2 Žpipette and bath.; filled circlessCaCl 2 Žpipette., NaCl Žbath.; open squaress KCl Žpipette., NaCl Žbath.. Curves were
fitted by the Goldmann-Hogkin-Katz equation.
436
R. Kohler
et al.r CardioÕascular Research 38 (1998) 433–440
¨
carried out in WKY of same age and body weight. Rats
were allowed to recover and after 8 weeks experiments
were conducted as detailed below. Systolic blood pressure
was monitored by tail cuff sphygmomanography ŽHarvard
Apparatus, South Natick, MA, USA..
Differences between groups were calculated by use of
Wilcoxon rank sumrMann–Whitney U-test.
3. Results
3.1. Pressure-actiÕated channel
In cell-attached patches of intact endocardial endothelium from papillary muscle, a mechanosensitive cation
channel was identified. The channel had a low basal
channel activity with a channel open probability Ž Po . of
less than 0.1 in the absence of mechanical stimulation.
Channel activity was strongly enhanced by positive pressure applied to the cell membrane via the patch pipette
ŽFig. 1a.. The simultaneous activation of up to 10 channels
was observed in a single patch. Stimulation of channel
activity strongly depended on the magnitude of the applied
pressure ŽFig. 1b.. The application of a pressure of 10
mmHg was sufficient to increase basal channel activity
from Po 0.01 to 0.06. A further increase of pipette pressure from 10 to 20 and 30 mmHg gradually increased
channel activity to a Po of 0.37 and 0.55, respectively. In
cell-attached patches with basal channel activity, negative
pipette pressure Žy20 mmHg. completely abolished channel activity ŽFig. 1a.. The channel was organized in clusters of 3 to 10 channels per patch. Less than 3 channels per
patch were observed only in 2 out of 126 patches.
At membrane potentials in a range from 0 to y80 mV
and with pipette containing KCl, NaCl or CaCl 2 solution
ŽFig. 2a., respectively, inward-directed currents were observed. Channel conductance at negative membrane potentials was 21.8 " 4.4 Žs.d.. pS Ž n s 26. with a KCl pipette
solution and 4.1 " 1.5 pS Ž n s 6. with a CaCl 2 pipette
solution. Excision of the patch into the bath solution
resulted in a rapid inactivation of the channel within 1 to 5
s. However, in some experiments Ž n s 14., channel activity was preserved in inside-out patches. Ion substitution
protocols revealed a permeability ratio for Kq:Naq:Ca2q
of 1:0.9:0.3 as calculated from reversal potentials of 3.6
mV with a KCl pipette solution and a NaCl bath solution,
and of y20.1 mV with a CaCl 2 pipette solution and a
NaCl bath solution, by the use of the Goldman-HogkinKatz equation ŽFig. 2b.. Cly substitution with cyclamate
Ž n s 3. in the pipette solution had no effect on channel
conductance and reversal potential in inside-out patches,
thus excluding an apparent anion permeability of PAC. In
inside-out patches, channel conductance showed a tendency to an inward rectification. At membrane potentials
in a range from y80 mV to y10 mV and at q10 mV to
q100 mV, channel conductance was 15.9 " 0.7 pS and
8.5 " 0.8 pS, respectively. A voltage dependence of channel open probability was not observed at membrane potentials in a range from q100 to y80 mV. Also, reduction of
cytosolic calcium concentration from 1.3 mM to 0.1 mM
did not modify channel activity. In five experiments with
patch pipettes backfilled with Gd 3q Ž50 mM., a blocker of
MSC w20x, channel activity was completely inhibited in
cell-attached patches. Block of channel activity started
about 20 s after seal formation and was complete after 1–2
min ŽFig. 3.. Outward-directed currents recorded at a
Fig. 3. Inhibition of inward currents through PAC by Gd 3q in cell-attached patches. Patch pipettes were backfilled with standard pipette solution
containing Gd 3q Ž50 mM.. c ™, closed state of channels.
R. Kohler
et al.r CardioÕascular Research 38 (1998) 433–440
¨
pipette potential of y80 mV were not affected. In contrast, addition of flufenamic acid Ž20 mM, n s 5., a blocker
of non-selective cation channels w21x, did not block PAC.
In cell-attached patches, PACs were the predominant class
of ion channels and were observed in about 50% of the
experiments performed.
3.2. Ca
437
trophysiological properties of ion channels in intact endocardial endothelium of left ventricular papillary muscle
resemble the results of experiments performed in papillary
muscle preparation from 25 WKY rats.
3.3. ComparatiÕe study
2q
-dependent cation channel
After excising the patch-clamped membrane into the
bath solution, another type of ion channel was activated in
about 40% of the experiments performed. This channel had
a mean conductance 31 " 4 pS Ž n s 28. for Kq. Ion
substitution protocols and revealed a permeability ratio for
Kq:Naq:Ca2q of 1:1:0.1 as calculated from reversal potentials of y0.7 mV and y39.7 mV with KCl pipette
solution and NaCl bath solution and with CaCl 2 pipette
solution and NaCl Bath solution, respectively. Substitution
of Cly with cyclamate in the pipette solution did not
change reversal potential thus excluding apparent anion
permeability of this non-selective cation channel ŽNSC..
Channel activity of NSC depended on cytosolic calcium
concentration. Half-maximal activation occurred at a calcium concentration of 8.2 " 0.9 mM wCa2q x i Ž n s 5.. In
cell-attached and inside-out patches mechanical stimulation of the cell membrane by positive pressure or pipette
suction failed to influence NSC activity. NSC was not
blocked in the presence of gadolinium Ž50 mM, n s 3.. In
contrast, addition of flufenamic acid Ž20 mM, n s 5. to the
bath solution completely and reversibly blocked NSC Žnot
shown..
In some experiments Ž n s 5. in cell-attached patches
with NaCl solution containing 1 mM Ca2q in the patchpipette activation of PAC was followed by the opening of
the Ca2q-dependent NSC ŽFig. 4.. In cell-attached patches,
NSC activity was only observed after opening of PAC and
remained present during PAC activity. After inactivation
of PAC, NSC activity subsequently ceased. Data on elec-
In a comparative randomized study, ion channel properties of PAC were investigated in experimental hypertension. Eight weeks after surgery, 2K1C rats had a systolic blood pressure of 214 " 9 mmHg Ž n s 6. compared
to 126 " 6 mmHg Ž n s 7. in the sham-operated animals
Ž P - 0.01.. Body weight was not significantly different
between 2K1C Ž320 " 19 g. and sham Ž281 " 15 g.. Wet
heart weight was increased in 2K1C Ž1.63 " 0.02 g. compared to sham-operated controls Ž1.30 " 0.04 g, P - 0.01..
In order to compare ion channel density in the two
groups, 10 tight-seal patch-clamp experiments were performed in intact EE of each rat. Only tight-seal patch-clamp
experiments with seal resistance of more than 4 GV were
included in the statistical analysis. A difference in successful seal formation between 2K1C and sham rats was not
observed. Apparent channel density of PAC was determined as the percentage of patches with PAC activity.
PAC density was significantly increased in 2K1C Ž72.2 "
7.6%, n s 6. compared to sham-operated controls Ž37.2 "
8.4%, n s 7; P s 0.012; Fig. 5.. The number of PAC per
cluster was not changed in 2K1C Ž6.4 " 0.5, n s 27.
compared to controls Ž6.3 " 0.7, n s 16.. Channel conductance of PAC were not different in 2K1C Ž20.3 " 3.9 pS,
n s 22. compared to controls Ž21.3 " 3.5 pS, n s 22..
Density of NSC were not different between 2K1C Ž46.1 "
10.5%. and controls Ž40.0 " 6.8%, not significant.. For
determination of the pressure sensitivity of PAC, the Po at
10, 20 and 30 mmHg was calculated. Pressure sensitivity
was not changed in 2K1C Ž Po s 0.17 " 0.06, 0.33 " 0.06,
0.56 " 0.08 at 10, 20 and 30 mmHg, respectively. com-
Fig. 4. Co-activation of Ca2q dependent NSC in cell-attached patches with PAC activity. NSC activity can be distinguished from PAC activity by the
larger current amplitude and shorter open states of the channel at a holding potential of y80 mV. PAC o 1 ™–o 3 ™ denotes open states of PACs; § o
NSC denotes open state of Ca2q dependent non-selective cation channel; c ™, closed state of channels.
438
R. Kohler
et al.r CardioÕascular Research 38 (1998) 433–440
¨
Fig. 5. Density of PAC in intact endocardial endothelium of left papillary
muscle from 2K1C Ž ns6. and sham-operated controls Ž ns 7.. Channel
density was determined in each rat as percentage of patches with PAC
activity. Data are given as mean"s.e., ) P s 0.012, Mann–Whitney
U-test.
pared to sham-operated controls Ž Po s 0.20 " 0.06, 0.45 "
0.06, 0.62 " 0.03, at 10, 20 and 30 mmHg, respectively..
4. Discussion
Mechanosensitive ion channels have been described in
several cell types from vertebrate and non-vertebrate
species w22x. A well-characterized class of MSC are the
stretch-activated non-selective cation channels ŽSAC.,
which are activated by pipette suction associated with a
stretch of the membrane and membrane coupled cytoskeletal structures. In vascular endothelium, MSC have
been proposed to act as mechanosensors for hemodynamic
forces associated with blood flow and pressure. In porcine
aortic endothelial cells w11x, intact aortic endothelium of
the rat w23x, and in intact tissue preparation of EE from
porcine right atrium w13x cation selective SACs with a
conductance for monovalent cations ranging from 20 to 40
pS have been reported. Stretch-inactivated ion channels
have been described in snail neurons w24x, smooth muscle
cells of toad stomach w25x or mdx myotubes w26x. These
channels did not show any distinct responsiveness to positive pipette pressure. Therefore, the activation mechanism
of PAC seems to be different from other MSC. Under
pipette pressure the patch-clamped membrane becomes
spherical and compresses the underlying cytoskeleton, as
revealed by videomicroscopic studies w27x, and might be
responsible for the activation of PAC.
A mechanosensitive ion channel with an opposite response to negative and positive pressure has not so far
been described in endocardial endothelial cells. The identification of PAC in EE might be due to the fact that we
performed patch-clamp experiments in intact tissue preparations. Patch-clamp experiments in intact tissue slices do
not require an isolation of EE presumably associated with
cytoskeletal changes. Moreover, EE remained in their original shape and surroundings. The PAC in intact EE of
papillary muscle described in this study resembles the
characteristics of PAC with respect to mechanosensitivity,
conductance and selectivity recently described in intact
endothelium of rat aorta and mesenteric arteries w16x. However, apparent channel density was about 2-fold and about
6-fold higher in papillary muscle EE than in intact endothelium preparations from aorta and mesenteric artery,
respectively.
It should be pointed out that the pressures applied to the
patch pipette for mechanical stimulation of the cell membrane and the underlying cytoskeleton are not comparable
to blood pressures occurring in vivo. However, these mechanical manipulations are useful tools for the identification of MSC on the level of single-channel patch-clamp
experiments. The mechanical factor controlling the opening of PAC in vivo might be either changes in wall shear
stress or myocardial shortening during heart cycle.
The functional role of MSC for cardiovascular function
is associated to their Ca2q-permeability w11x. In response to
mechanical stress, endothelial MSC provide a Ca2q influx
triggering Ca2q-dependent synthesis of vasoactive factors.
In the present study pressure activation of the Ca2q-permeable PAC led to a Ca2q influx as concluded from the
co-activation of Ca2q-dependent ion channels present in
the same membrane patch. The latter co-activation indicates that the calcium influx through PAC under physiological wCa2q x o is apparently sufficient to increase at least
locally wCa2q x i and thereby to activate neighboring NSC.
A similar co-activation of Ca2q-dependent ion channels
after mechanical stimulation of MSC has been described
for SAC and Ca2q-dependent maxi Kq in intact EE from
porcine right atrium w13x and in Necturus proximal tubulus
cells w28x, and for PAC and Ca2q-dependent maxi Kq or
Ca2q-dependent NSC in intact rat aortic endothelium w16x.
The NSC described in this study showed similar electrophysiological properties with respect to channel conductance, Ca2q dependence and ion selectivity of those reported earlier for non-selective cation channels in endothelial cells with a conductance in a range of 26–36 pS
w29,30x.
Endothelial NO production has been reported to be
stimulated by an increase of wCa2q x i and cell hyperpolarization in vascular endothelium w31x. The EE seems to
regulate cardiac myocyte function in a similar NO-mediated fashion w4x. Therefore, activation of PAC could stimulate Ca2q-dependent NO-production by a Ca2q influx and
subsequently shortens papillary muscle contraction. However, we cannot exclude the possibility that the depolarizing current would impair endothelial NO production. A
concomitant activation of NSC and additional sodium influx could lead to a further cell depolarization. Therefore,
the functional role of PAC in EE remains to be determined
by the use of a specific blocker.
In human and experimental hypertension, an impaired
endothelial function has been demonstrated w32–34x. In the
spontaneous hypertensive rat and in renovascular hyper-
R. Kohler
et al.r CardioÕascular Research 38 (1998) 433–440
¨
tension, a decreased endothelium-dependent vasodilation
has been observed w35,36x.
Although experimental evidence is limited, it is likely
that an altered EE function may play an important role in
the pathophysiology of left ventricular hypertrophy w37x. A
prolonged action potential of papillary muscle has been
reported in renovascular hypertensive cats with left ventricular hypertrophy w38x. In renovascular hypertensive rats
an EE-dependent decreased maximal tension development
of papillary muscle has been observed, indicating a compensatory function of the EE in left ventricular hypertrophy w39x.
In experimental hypertension ŽSHR and 2K1C., we
observed an up-regulation of PAC density in aortic and
mesenteric endothelium w16x. Since PAC up-regulation was
only observed after induction of hypertension, altered PAC
density appeared to be secondary to hypertension. Ion
channel characteristics in EE of left ventricular papillary
muscle from hypertrophied hearts have not so far been
investigated. In the present study we were interested in
determining whether PAC properties are changed in EE of
renovascular hypertensive rats with left ventricular hypertrophy. Apparent PAC density was increased in EE of
2K1C compared to sham-operated controls whereas channel conductance and mechanosensitivity were unchanged.
The increased PAC density in EE of 2K1C compared to
sham-operated controls indicates that PACs in EE are
regulated in a similar way to those in aortic and mesenteric
endothelium from hypertensive rats.
In conclusion, we identified a novel type of Ca2q-permeable MSC in intact EE of rat ventricular papillary
muscle, which is regulated by membrane pressure. PAC
contributes to EE mechanotransduction by inducing an
intracellular Ca2q signal. Up-regulation of PAC density in
EE from 2K1C might indicate an altered mechanotransduction in hypertension.
Acknowledgements
This work was supported by the Deutsche Forschungsgemeinschaft ŽHo 1103r2-2. and by the European Community ŽBM H4 CT 96 0602..
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