Role of maxi-K+ channels in
endothelin-induced vasoconstriction of mesenteric and submucosal
arterioles
Ceredwyn E.
Hill,
Adam
Kirton,
Douglas D.
Wu, and
Stephen J.
Vanner
Gastrointestinal Diseases Research Unit, Hotel Dieu Hospital,
Kingston, Ontario, Canada K7L 5G2
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ABSTRACT |
The action of endothelin in small intestinal
resistance vessels of the guinea pig was studied by examining
submucosal arteriole vasoactivity in vitro and electrical properties of
mesenteric arteriole smooth muscle cells. Endothelin-1 (ET-1)
constricted submucosal arterioles with a half-maximal effective
concentration of 170 pM. ET-3 caused detectable constriction with a
minimum of 20 nM. The ET-1 response was prolonged, with a
time to 90% relaxation of 41 ± 2.8 min after washout. The
ETA antagonist BQ-123 (200 nM)
decreased the sensitivity to ET-1 ~40-fold. Arterioles preconstricted
with prostaglandin F2
did not
relax when superfused with ET-1, ET-3, or an
ETB agonist, IRL-1620, and
pretreatment with the nitric oxide synthase inhibitor
NG-monomethyl-L-arginine
was ineffective in countering ET-1-induced constriction, indicating the
absence of functional ETB
receptors. Resting membrane potential in isolated cells was
characterized by transient hyperpolarizing spikes (THs).
ET-1 (20 nM) increased TH frequency and caused the emergence of a
larger amplitude population. Under voltage clamp, spontaneous transient
outward currents (STOCs) were seen that reversed at the
K+ equilibrium
potential. ET-1 increased STOC frequency and amplitude. Iberiotoxin (IBTX; 200 nM), a
maxi-K+ channel antagonist,
blocked the ET-1-induced THs and reduced STOC activity. IBTX or
tetraethylammonium increased the rate and extent of ET-1-induced
arteriole constriction. We suggest that ET-1-induced vasoactivity of
ileal resistance arterioles involves ETA receptor-mediated early
activation of maxi-K+ channels
that serves to counter strong constriction.
smooth muscle; potassium channel; ileum; patch clamp
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INTRODUCTION |
ENDOTHELINS ARE A GROUP OF 21-amino acid residue
peptides that have powerful vasoconstrictive effects on vascular smooth
muscle (33). Endothelins are significant hemodynamic
regulators in the microvasculature of the rat intestine (16), and they
appear to mediate the decreased arteriolar blood flow and diameter that occurs during bacteremia (32). Endothelin is synthesized within the
lamina propria cells of the gastrointestinal tract (19), and mRNA for
its receptor has been demonstrated in the small and large intestine
(3). However, the pharmacological properties of endothelin and the
cellular effects it has on small resistance vessels within the
gastrointestinal tract have not been characterized, although these
arterioles serve as the primary sites for control of intestinal
perfusion and ultimately the efficiency of mucosal absorption and
secretion.
In contrast to the small resistance vessels of the intestine, the
mechanisms involved in endothelin-dependent vasoactivity in other
vascular tissue are more clearly established. In general, activation of
endothelin-A (ETA) receptors on
vascular smooth muscle cells leads to changes in membrane excitability
and inositol phospholipid metabolism that result in increased cytosolic
Ca2+ concentration and ultimately
contraction (see Ref. 5 for review). Membrane potential of resting
vascular smooth muscle is mainly contributed to by a variety of
K+ channels (see Refs. 5, 23 for
review). Current flow through large-conductance, voltage- and
Ca2+-sensitive
K+ channels or
maxi-K+ channels is thought to
serve as a negative feedback signal to repolarize the membrane and
counter the actions of vasoconstrictors and to regulate myogenic tone
in pressurized resistance vessels (6). Smooth muscle
preparations from larger and conduit arteries depolarize in response to
ET-1 (7, 11, 13, 15, 21, 26), although initial transient
hyperpolarizations have been reported (20, 30). In comparison, there is
a dearth of studies on the effects of ET-1 on membrane electrical
properties of resistance arteriole smooth muscle. The role of
maxi-K+ channels in
endothelin-induced vasoconstriction is not well defined and is
confounded by reports showing increased (12, 26), decreased (18), or
transiently stimulated (11, 15) activity. Early activation of
maxi-K+ channels may be important
in regulating the activity of voltage-gated and other channels that are
modulated by endothelin.
The aims of this study were to establish the functional role of
endothelin in resistance arterioles of the guinea pig ileum and to
define the contribution of maxi-K+
channels in ET-1-induced vasoactivity. An isolated submucosal preparation was used to measure vasomotor activity in the resistance vessels within the intestine, and membrane electrical properties were
monitored in acutely dissociated smooth muscle cells from small
mesenteric resistance arteries. The results show that ET-1 constricts
ileal resistance vessels through
ETA receptors and that early
activation of maxi-K+ channels
supplies a significant buffer countering the full constrictive potential of this peptide.
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METHODS |
Preparation and measurement of submucosal arteriole diameter in
guinea pig ileum.
Segments of ileum (8-10 cm) were removed from male albino guinea
pigs (150-200 g; Charles River Canada, St. Constant, PQ, Canada)
after cervical dislocation and carotid transection. This method was
approved by the Animal Care committees of Queen's University and the
Canadian Council. We isolated
1-cm2 sections of a submucosal
preparation by removing the mucosa and, from the opposite surface, the
circular, longitudinal, and myenteric plexus, as described previously
(31). The submucosal preparations were pinned serosal side up in an
organ bath and perfused at 15 ml/min with a physiological saline
solution containing (in mM) 126 NaCl, 1.2 NaH2PO4,
1.2 MgCl2, 2.5 CaCl2, 5 KCl, 25 NaHCO3, and 11 glucose, gassed
with 95% O2-5% CO2 and
maintained at 35-36°C. Peptides, antagonists, and modified
saline solutions were applied by bath perfusion.
The outside diameter of submucosal arterioles was monitored using a
video-imaging technique involving a video camera and monitor interfaced
with a frame grabber (PC Vision Imaging Technology, Woburn, MA). The
outer diameter of the imaged vessels was measured as the distance
between two cursors using Diamtrack software (25) and displayed on a
chart recorder. The amount of constriction in response to the peptides
was expressed as the percentage of the resting vessel diameter. In some
experiments, the recordings were normalized to the maximum amount of
constriction induced by 2 nM ET-1 and the resting vessel diameter, so
that the time course of constriction could be examined and
statistically analyzed. At least three experiments were conducted for
each condition. Representative traces are shown, and, where
appropriate, mean and SE are reported.
Isolation of mesenteric arteriole smooth muscle cells and
measurement of membrane potential and whole cell currents.
Mesenteric arterioles (6 to 8 1-cm segments) consisting of the vasa
recta were dissected from between the smallest arcades and the ileal
border of the segments of ileum obtained previously and placed into
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered Hanks' saline containing 0.15% (wt/vol) bovine
serum albumin (BSA) and (in mM) 136.9 NaCl, 4.2
, 0.8 MgSO4, 0.16 CaCl2, 5.9 KCl, and 19.8 HEPES, pH
7.4, and incubated for 10 min at 20°C. The vessels
were then minced in fresh saline containing 1.3 mg/ml papain, 1 mM
dithiothreitol, and 0.05% BSA and incubated at 37°C for 30 min at
75 revolutions/min (rpm). The tissue was collected and resuspended in
fresh saline containing collagenase (type I; 2 mg/ml) and 0.05% BSA
and incubated for 15 min at 37°C and 75 rpm. After gentle
trituration and washing, the cells were resuspended in the initial
Hanks' saline and plated onto
poly-L-lysine-coated glass
coverslips and allowed to adhere for at least 1 h at 4°C.
Patch-clamp experiments were carried out at 20°C the same day on
cells 10-20 µm wide and 20-40 µm long.
Whole cell voltage and current clamp measurements were recorded from
single smooth muscle cells dialyzed with a pipette solution containing
(in mM) 145 KCl, 5 HEPES, and 1 MgSO4, pH 7.4. Ca2+ was weakly buffered to 0.1 µM by the further addition of 7.5 µM ethylene
glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA) and 5 µM CaCl2. The
extracellular solution normally consisted of (in mM) 140 NaCl, 5 KCl, 5 HEPES, 1 MgSO4, 1 CaCl2, and 5 glucose, pH 7.4, although in some experiments KCl was increased to 115 mM with NaCl
decreased to 30 mM. Heat-polished patch pipettes of thin-walled
borosilicate glass (Kimax-51) had resistances of 2-3 M
in these
solutions. Membrane potential and whole cell currents were measured
using an Axopatch 200A amplifier under the current- and voltage-clamp
configurations and a Digidata 1200 A/D board (Axon Instruments, Foster
City, CA). Data were filtered (low pass, 3 dB cutoff) and sampled at
0.2 and 1 kHz respectively. Digitized records were analyzed using
pClamp 6.0 (Axon Instruments) and Origin 4.1 (Microcal Software,
Northampton, MA) software.
Materials.
ET-1, ET-3,
NG-monomethyl-L-arginine
(L-NMMA), and
prostaglandin F2
(PGF2
) were purchased from
Calbiochem (San Diego, CA). IRL-1620 was a gift from CIBA Geigy Canada
(Calgary, AB, Canada). Tetraethylammonium (TEA) chloride was from
Research Biochemicals (Natick, MA). Papain and HEPES were purchased
from Boehringer Mannheim, and collagenase was from GIBCO/BRL (Grand
Island, NY). All other reagents were from British Drug Houses (Toronto,
ON, Canada) or Sigma Chemical (St. Louis, MO) and were of the highest grade available.
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RESULTS |
Characterization of response of submucosal arterioles to
endothelins.
Superfusion of 2 nM ET-1 to submucosal preparations of the guinea pig
ileum caused a decrease in arteriole diameter that reached a steady
state in 20 min (Fig.
1A).
The initial rapid phase of constriction lasted ~2 min and was
followed by a second further decrease to a minimum at 20 min of
exposure. At increasingly lower concentrations, the rate and extent of
constriction were greatly reduced and delayed and became monophasic.
For example, constriction in response to 60 pM ET-1 (Fig.
1A,
top
trace) occurred at ~5% of the
rate and took almost three times longer to reach a steady state than
that in reponse to 2 nM ET-1. To construct a concentration-response relationship, diameter was measured at the steady state (above 60 pM)
or at 1 h in the presence of lower ET-1 concentrations, since
vasoconstriction in response to 300 nM
PGF2
or 50 mM KCl was not
consistent after 90 min (data not shown).

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Fig. 1.
Concentration dependence of endothelin-1 (ET-1)-induced
vasoconstriction of ileal submucosal arterioles.
A: vessel diameter recordings of
arterioles superfused with 60 pM (top) and 2 nM ET-1
(bottom). Resting outside diameters were 65 and 58 µm
for top and
bottom, respectively.
B: concentration-response curves
constructed from arterioles exposed to different ET-1 concentrations in
absence or presence of 200 nM BQ-123. Preparations were superfused with
BQ-123 for 5 min before and during ET-1 application. %Constriction was
calculated as [(resting diameter minimum
diameter)/resting diameter] × 100.
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Vascular constriction in response to endothelins is dependent on the
receptor subtypes present and the relative affinities for different
peptide isoforms. The concentration-response curves shown in Fig.
1B demonstrate that submucosal
arterioles have a high affinity for ET-1 [half-maximal effective
concentration (EC50) = 0.17 ± 0.1 nM; slope = 0.79 ± 0.4;
n = 3 at each concentration]. Furthermore, the constrictive activity is likely due to activation of
ETA receptors since superfusion of
200 nM of the ETA receptor antagonist BQ-123 5 min before and during ET-1 exposure displaced the
dose-response curve for ET-1 to the right without significantly affecting the slope or maximum constriction
(EC50 = 7.22 ± 1.4 nM; slope = 1.58 ± 0.4; n = 3 at each
concentration). The relative affinity for the other constrictor isoform
of endothelin (ET-3) compared with ET-1 was at least 10-fold less (Fig.
2A).

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Fig. 2.
Effects of ET-3 on constriction, and endothelin agonists on submucosal
arterioles preconstricted with prostaglandin
F2
(PGF2 ). Representative
recordings of arteriole diameter during superfusion with 0.2, 2, or 20 nM ET-3 (A) or 20 nM ET-3, 2 nM
ET-1, or 200 nM IRL-1620 (B)
preconstricted with 300 nM PGF2
superfused from the arrow. Resting outside diameters of the vessels
were, from left to
right, 62, 58, and 65 µm in
A and 62, 85, and 64 µm in
B.
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The possibility that endothelins could act through
ETB receptors to dilate submucosal
arterioles was tested by preconstricting vessels with 300 nM
PGF2
followed by superfusion
with ET-3, ET-1, or the ETB
agonist IRL-1620 (Fig. 2B). There
was no vasodilatory effect of any of these compounds over the time
course examined. To confirm that nitric oxide (NO) was not being
generated through ETB receptor
activation, the preparation was exposed to the NO synthase inhibitor
L-NMMA (300 µM) for 3 min
before and during perfusion with 2 nM ET-1. The percent constriction in
the presence of ET-1 alone (58 ± 9%;
n = 3) was not significantly different from that recorded in the
L-NMMA-pretreated
tissue (54 ± 4%; n = 3) (data not
shown).
A well-established characteristic of ET-1-induced vasoconstriction is
its prolonged effect, even after extended washout of the peptide (33).
The submucosal preparation also showed long-lasting constriction in
response to ET-1 compared with several other vasoconstrictors. Figure
3A shows
records of two different vessel measurements made in the presence of 10 µM norepinephrine or 0.2 nM ET-1. The time to 90%
relaxation after removal of five different constrictors shows that
ET-1, in contrast to each of the other compounds, took at least eight
times longer (Fig. 3B).

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Fig. 3.
Time dependence of recovery from constriction of submucosal arterioles
from ET-1 and other vasoactive compounds.
A: representative recordings of
arterioles superfused with 200 pM ET-1
(top) or 10 µM norepinephrine (NE;
bottom).
B: histogram shows means ± SE
(n = 3 for each condition)
of time to 90% relaxation calculated from onset of perfusion with
compound-free perfusate (ET-1, 0.2 nM; NE, 10 µM; PGF, 300 nM
PGF2 ; ET-3, 60 nM; VAS, 10 nM
arginine-vasopressin).
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Membrane potential and whole cell currents in mesenteric arteriole
smooth muscle cells.
Freshly isolated smooth muscle cells did not rest at a constant
membrane potential. When dialyzed with a weakly buffered
Ca2+ solution, membrane potential
was characterized by aperiodic, transient hyperpolarizing spikes (THs)
of variable amplitude. A representative recording shows that
unstimulated cells rested at about
41 mV and had THs ranging
from 5 to 40 mV in amplitude (Fig.
4A).
Under voltage clamp, and at a holding potential of 0 mV, the same cell
showed spontaneous transient outward currents (STOCs) of similar
frequency and duration as the potential spikes (Fig.
4B). To demonstrate that these
oscillations were mainly due to outward current through
K+ channels, cells were bathed in
medium containing 115 mM KCl rather than the usual 5 mM KCl. Under
these conditions, the STOCs reversed near 0 mV so that STOCs were
inwardly directed at
40 mV and outward at +40 mV (Fig.
4C).

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Fig. 4.
Whole cell recordings from single isolated mesenteric arteriole smooth
muscle cells under current or voltage clamp. Cells were
dialyzed with a weakly
Ca2+-buffered,
K+-rich solution and bathed in 5 mM (A,
B) or 115 mM KCl
(C). Membrane potential
(A) and currents
(B,
C) were recorded, the latter at a
holding potential of 0 mV (B) or
±40 mV (C).
A and
B are from the same cell and
C is from a separate cell.
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ET-1 stimulates iberiotoxin-sensitive transient hyperpolarizations
and STOCs with little effect on averaged membrane potential.
The effects of ET-1 and the specific
maxi-K+ channel blocker
iberiotoxin (IBTX) on the resting properties of acutely isolated arteriole smooth muscle cells were assessed by continuously recording membrane potential or current before and during exposure to the peptides. Six of nine cells recorded from responded to the compounds. Figure 5A
shows that 20 nM ET-1 increased TH amplitude and decreased baseline
membrane potential by ~2.5 mV. Relatively high concentrations of IBTX
were required to inhibit the THs, although it is apparent that 40 nM
IBTX decreased TH frequency. The THs were almost fully blocked by 200 nM IBTX. To quantitate the effects of ET-1 and IBTX (200 nM) on smooth
muscle cell membrane potential, the TH data from each responding cell
were binned from identical record lengths in the absence or presence of
the peptides and expressed as fractions of the total number of events.
The data were pooled from all cells for each condition, and the
means ± SE (n = 6) were
plotted as a function of TH amplitude (Fig.
5B). The control data fell roughly
within a single Gaussian distribution, with the peak number of events
falling between 5 and 7.5 mV amplitude. ET-1 broadened and shifted the
peak to a midpoint between 15 and 17.5 mV, with some new THs >30 mV.
Subsequently, 200 nM IBTX reduced TH amplitude and frequency to <10
mV and 7% of the total number of events. The fraction of events during
control, ET-1, and 200 nM IBTX intervals was 0.50 ± 0.2, 0.45 ± 0.2, and 0.07 ± 0.1, respectively. Averaged membrane potential of
the pooled data (baseline plus THs) was depolarized by 0.51 ± 3.7 and 1.06 ± 3.7 mV (n = 6) in the
presence of ET-1 and IBTX, respectively, relative to controls. The
averaged potential of the recording shown in Fig. 5A was
43.3,
41.1, and
39.2 mV during the control, ET-1, and 200 nM IBTX intervals,
respectively. One-way analysis of variance demonstrated that each of
these values was significantly different (P < 0.01) from the others.

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Fig. 5.
Effects of ET-1 and iberiotoxin (IBTX) on frequency and amplitude of
transient hyperpolarizations in single smooth muscle
cells. A: cells were
dialyzed with a weakly
Ca2+-buffered,
K+-rich solution and bathed in 5 mM KCl, and at the large bipolar spikes ET-1 and IBTX were applied to
cell surface through a pressure-ejection pipette. Membrane potential
was recorded under current clamp configuration of patch-clamp
technique; arrow indicates 50 mV membrane potential.
B: histogram shows that the fractional
number of events from each of 6 responding cells were pooled over
control, ET-1, and 200 nM IBTX intervals and plotted as a function of
the transient hyperpolarization amplitude; bin width = 2.5 mV.
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The effects of ET-1 and IBTX on the transient outward currents
underlying the STOCs were observed by recording membrane current across
a range of potentials between
40 and +40 mV. Representative traces from one cell are shown in Fig. 6.
The results show that ET-1 increased the frequency and amplitude of
STOCs, whereas subsequent addition of IBTX reduced these parameters to
less than control values. These results indicate that STOCs were
sensitive to IBTX and were enhanced by ET-1.

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Fig. 6.
ET-1 increases and IBTX decreases frequency and amplitude of
spontaneous transient outward currents. Cells were
dialyzed with a weakly
Ca2+-buffered,
K+-rich solution and bathed in 5 mM KCl in absence (control; left) or
presence of 20 nM ET-1 (middle), or
both ET-1 and 200 nM IBTX (right).
Representative records from a single cell show membrane current under
voltage clamp between 40 and +40 mV.
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Contribution of a TEA- and IBTX-sensitive component to ET-1-induced
vasoconstriction.
Although the role of maxi-K+
channels in classic vasoconstrictor responses is fairly well defined,
it has not been widely studied in the ET-1 stimulus-response pathway
using parallel vessel and single cell models. TEA (10 mM) increased the
extent and almost doubled the mean rate of contraction of seven
submucosal arteriole preparations in response to 2 nM ET-1 (Fig.
7A,
top). A similar trend was observed
with IBTX (88 nM), although the smaller sample size
(n = 3) resulted in an increased error
about the mean (Fig. 7A,
bottom). The IBTX- and TEA-sensitive
components, obtained by subtracting the mean of the control records
from those obtained in the presence of the blockers, had rapid onsets
and attained steady states within 1-2 min of the initiation of
constriction (Fig.
7B). Superfusion of 10 mM TEA for 60 min, or 88 nM IBTX for 5 min, in the absence of ET-1 had
no effect on vessel diameter (not shown).

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Fig. 7.
Tetraethylammonium (TEA)- or IBTX-induced increase in rate and extent
of constriction of submucosal arterioles. Arterioles
were superfused with 2 nM ET-1 from 0 min in absence or presence of 10 mM TEA added 5 min earlier and maintained over course of experiment, or
88 nM IBTX added 5 min before and the first 8 min during ET-1 exposure.
Resting vessel diameter ranged from 38 to 87 µm. Vessel diameter was
normalized by plotting resting diameter × maximum constriction + 2 nM ET-1. TEA and IBTX data were normalized using the resting diameter
to determine appropriate scaling factor.
A: pooled recordings (means ± SE)
made in presence of ET-1 with or without TEA
(top,
n = 7) or IBTX
(bottom,
n = 3).
B: TEA- and IBTX-sensitive components
were calculated as ET-1 + TEA or IBTX control from
A. Dashed line indicates a normalized
vessel diameter of 1.0.
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DISCUSSION |
This study shows that ET-1-induced constriction of small intestinal
resistance arterioles involves ETA
receptors linked to early activation of TEA- and IBTX-sensitive
K+ channels. We conclude that
opening of maxi-K+ type channels
serves to attenuate the force of contraction induced by ET-1. Because
neither the averaged and pooled membrane potential in the isolated
cells nor basal tone in the intact vessels was greatly affected by the
maxi-K+ blockers, it is suggested
that these channels do not contribute significantly to resting membrane
potential but are important in modulating the strong vasoconstrictive
effects of ET-1 and that depolarization is not a prerequisite for the
latter.
ETA receptors in resistance arterioles of
the guinea pig ileum.
The functional studies reported here demonstrate that submucosal
arterioles of the guinea pig ileum contract in response to ET-1 through
ETA receptors. This conclusion is
supported by the observations that the specific
ETA receptor antagonist BQ-123 shifted the concentration-dependence curve to higher concentrations of
ET-1, the ETB agonist IRL-1620 did
not cause vasoconstriction, and the relative affinity of the
constriction for ET-1 was roughly 100-fold greater than that for ET-3.
These properties are similar to
ETA receptor-mediated responses in
a variety of other cells and tissues.
ETA receptors in different
vascular tissues vary between 10- and 100-fold higher affinity for ET-1
over ET-3 (1, 3, 8, 10, 14). The
EC50 for high-affinity ET-1
receptor responses falls within 0.18 and 0.62 nM (3, 10, 14, 33), further demonstrating the similar nature of the submucosal receptors characterized here (0.17 nM). Our results with the NO synthase inhibitor and the ETB agonist in
the submucosal preparation do not support the existence of functional
ETB receptors linked to vasodilation, through activation of endothelial cells, although there
is evidence for such a mechanism in arterial tissue (17, 21).
Submucosal and mesenteric arteriole endothelin receptors linked to
maxi-K+ channel
activation and membrane potential stabilization.
Our results show that ET-1-induced constriction of submucosal
arterioles (<100 µm diameter) was enhanced in the presence of 10 mM
TEA or 88 nM IBTX, suggesting that
maxi-K+ channels moderate the
constrictive effect of ET-1. Furthermore, the kinetics of the
blocker-sensitive components were identical to that observed for the
ET-1-stimulated increase in cytosolic Ca2+ concentration in
endothelium-denuded, fura-2-loaded, mesenteric arteriole strips (34).
In both studies, maximum contraction was attained much later than the
plateau of the Ca2+ concentration
or TEA/IBTX-sensitive constriction. Attenuation of constriction may be
due to the hyperpolarizing effect of increased K+ channel open probability and
hence buffering of the depolarization resulting from cation entry or
Cl
release through
nonselective or Ca2+-activated
channels, respectively, and/or decreased open probability of
voltage-gated Ca2+ channels.
We also show that isolated smooth muscle cells from mesenteric
arterioles do not undergo significant depolarization in response to
ET-1. This contrasts with substantial evidence that saturating concentrations of ET-1 depolarize, by 20 to 30 mV, vascular smooth muscle cells from large, conduit arteries or derived cell lines (7, 11,
13, 15, 24, 26, 30). These studies used a variety of techniques to
measure membrane potential, including whole cell and intracellular
electrode recording and potential-sensitive dyes, so it is unlikely
that the experimental approach used here (whole cell dialysis with
weakly buffered Ca2+ solutions)
could account for the differences. Rather, this may be due to the fact
that the cells were derived from resistance arterioles rather than
larger vessels. There is some evidence that responses to ET-1 and other
vasoactive agents may be phenotype dependent since two populations of
cells isolated from cultured thoracic aorta smooth muscle either
depolarize or hyperpolarize when exposed to ET-1 (24). From the data
available, it is not possible to determine the reasons for the quite
different electrical responses to vasoconstrictors in different
vascular smooth muscle, although their potential importance in the
physiology and pharmacology of the microvasculature is
evident.
Our results, that STOCs in mesenteric arteriole smooth muscle cells are
blocked by IBTX, increase in frequency and amplitude with membrane
depolarization, and reverse at the
K+ equilibrium potential, confirm
earlier reports that STOCs are mainly carried by
maxi-K+ channels (2, 22, 28).
Furthermore, STOCs have been shown to be inhibited by TEA in a
concentration-dependent manner, with 10 mM causing more than 90% block
(4, 9, 28). In contrast, low-conductance
K+ channels of smooth muscle are
generally much less sensitive to TEA (29; see Ref. 23 for review). We
therefore suggest that the majority of the TEA-induced constriction of
the submucosal arterioles, as confirmed by the similar pattern of
constriction by IBTX, was due to block of
maxi-K+ channels activated by
endothelin.
A few studies have looked at the effects of ET-1 on vascular smooth
muscle cell STOC activity. Resting coronary, mesenteric, and renal
arterial smooth muscle cells dialyzed with 20 or 30 µM EGTA and no
added Ca2+ exhibited STOCs that,
when exposed to saturating concentrations of ET-1, transiently
increased in both amplitude and frequency and then disappeared (11,
15). In contrast, we found that ET-1 caused a sustained increase in
amplitude and frequency of STOCs and transient hyperpolarizations in
mesenteric arteriole cells. We used a mixture of 5 µM
CaCl2 and 7.5 µM EGTA to weakly buffer internal Ca2+ to 0.1 µM,
to be more representative of the state in vivo. Taken together, these
results suggest that the relative complement of active
maxi-K+ and depolarizing channels
determines the presence of STOCs and the membrane potential response to
ET-1, and that the increase in cytosolic
Ca2+ caused by ET-1 may have been
curtailed in the studies in which cells were dialyzed with EGTA.
Role of
maxi-K+ in the
stimulus-contraction coupling pathway for ET-1 in resistance
arterioles.
ET-1 is thought to stimulate contraction of smooth muscle cells through
a sequence of events involving ETA
receptor activation and inositol 1,4,5-trisphosphate synthesis, leading
to increased cytosolic Ca2+
concentration as a result of sarcoplasmic reticular release and plasma
membrane influx (see Ref. 17 for review). Although some of the
ET-1-regulated channels have been identified (e.g., see Ref. 11 for
review), there is conflicting evidence regarding the modulation, and
hence physiological role, of
maxi-K+ channels by ET-1 in
vascular smooth muscle (11, 18, 26). At saturating concentrations of
ET-1, we observed that submucosal arteriole constriction was biphasic,
and this pattern was accentuated in the presence of
maxi-K+ channel blockers. All of
the endothelins have biphasic pressor effects in anesthetized rats
(14). Similar effects of hypoxia on intrapulmonary artery tension in
vitro are paralleled by biphasic increases in cytosolic
Ca2+ (27). There is some evidence
that the second phase involves Ca2+ sensitization of the
contractile apparatus (27). The prolonged response to endothelins is
also correlated with increased
Ca2+ sensitivity of the myosin
light chain (34). Our maxi-K+
blockade experiments in both the intact arterioles and single cells
suggest that the first phase of ET-1-induced constriction is partially
due to activation of voltage-gated
Ca2+ channels, with the major
complement of cytosolic Ca2+
derived from voltage-insensitive
Ca2+ inflow and release from
intracellular stores. We speculate that the hyperpolarizing influence
of early activation of maxi-K+
channels would provide an increased driving force for current flow
through nonselective channels, a neutralizing
counterion for Ca2+-activated
Cl
release, and/or
a feedback mechanism for countering depolarization caused by the
increased inward current induced by endothelin.
 |
ACKNOWLEDGEMENTS |
We thank Margaret Bolton and Emma Hollingsworth for expert
technical assistance.
 |
FOOTNOTES |
This research was supported by the Medical Research (S. J. Vanner) and
Natural Sciences and Engineering Research (C. E. Hill) Councils of
Canada.
Address for reprint requests: C. E. Hill, GI Research, Hotel Dieu
Hospital, 166 Brock St., Kingston, Ontario, Canada K7L 5G2.
Received 12 December 1996; accepted in final form 4 August 1997.
 |
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