| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Biological Sciences and Center for Environmental Signal Transduction, College of Arts and Sciences, Western Michigan University, Kalamazoo, Michigan 49008
 |
ABSTRACT |
|---|
Top
Abstract
Introduction
Methods
Results
Discussion
References
|
|---|
We examined the functional role of
large-conductance Ca2+-activated
K+
(KCa) channels in the hamster
cremasteric microcirculation by intravital videomicroscopy and
characterized the single-channel properties of these channels in
inside-out patches of membrane from enzymatically isolated cremasteric
arteriolar muscle cells. In second-order (39 ± 1 µm,
n = 8) and third-order (19 ± 2 µm, n = 8) cremasteric arterioles
with substantial resting tone, superfusion with the
KCa channel antagonists
tetraethylammonium (TEA, 1 mM) or iberiotoxin (IBTX, 100 nM) had no
significant effect on resting diameters
(P > 0.05). However, TEA potentiated
O2-induced arteriolar constriction
in vivo, and IBTX enhanced norepinephrine-induced contraction of
cremasteric arteriolar muscle cells in vitro. Patch-clamp studies
revealed unitary K+-selective and
IBTX-sensitive currents with a single-channel conductance of 240 ± 2 pS between 
60 and 60 mV (n = 7 patches) in a symmetrical 140 mM
K+ gradient. The free
Ca2+ concentration
([Ca2+]) for
half-maximal channel activation was 44 ± 3, 20 ± 1, 6 ± 0.4, and 3 ± 0.5 µM at membrane potentials of 60,
30, +30, and +60 mV, respectively
(n = 5), with a Hill coefficient of
1.9 ± 0.2. Channel activity increased
e-fold for a 16 ± 1 mV
(n = 6) depolarization. The
plot of log[Ca2+] vs.
voltage for half-maximal activation
(V1/2) was
linear (r2 = 0.9843, n = 6); the change in
V1/2 for a
10-fold change in
[Ca2+] was 84 ± 5 mV, and the [Ca2+] for
half-maximal activation at 0 mV
(Ca0; the
Ca2+ set point) was 9 µM. Thus, in vivo,
KCa channels are silent in cremasteric arterioles at rest but can be recruited during
vasoconstriction. We propose that the high
Ca0 is responsible for the
apparent lack of activity of these channels in resting cremasteric
arterioles, and we suggest that this may result from expression of
unique KCa channels in the
microcirculation.
microcirculation; vasoconstriction; iberiotoxin; tetraethylammonium; skeletal muscle; vascular smooth muscle; calcium ions; oxygen; patch clamp; norepinephrine; hamster; cremaster muscle
| |
INTRODUCTION |
|---|
|
Top
Abstract
Introduction
Methods
Results
Discussion
References
|
|---|
CALCIUM-ACTIVATED potassium (KCa) channels are one of the dominant ion channels found in the plasma membrane of vascular smooth muscle (25). These K+ channels are activated both by elevated concentrations of intracellular Ca2+ ([Ca2+]i) and by membrane depolarization (25). Recent studies have provided evidence that large-conductance KCa channels regulate membrane potential and resting diameter of small arteries that develop myogenic tone (4, 22-25). Furthermore, recent studies by Nelson and colleagues have suggested that Ca2+ sparks, representing focal, subsarcolemmal Ca2+ transients with peak amplitudes of ~300 nM, are sufficient to activate KCa channels at physiological membrane potentials in muscle cells isolated from small cerebral arteries (23). The Ca2+ sensitivity of KCa channels implied from this study, suggesting activation of the channels when [Ca2+]i = 100-300 nM, is consistent with the Ca2+ sensitivity for KCa channels in mesenteric arteries reported by Benham et al. (3). However, other studies have found that KCa channels require micromolar concentrations of Ca2+ to be active at physiological membrane potentials (29; see also 6). Thus there may be regional or species-dependent differences in the properties of KCa channels that may affect their contribution to the regulation of vascular function in different vascular beds. Furthermore, the single-channel properties and functional role played by KCa channels in muscle cells in the walls of arterioles in the microcirculation are not known. Therefore, we examined the contribution of KCa channels to the regulation of resting vascular tone of arterioles in hamster cremasteric muscles in vivo and then characterized the single-channel properties of KCa channels in inside-out patches of membrane from vascular smooth muscle cells isolated from the same arterioles. Our studies indicate that KCa channels do not appear to regulate the resting diameter of cremasteric arterioles in vivo, although they may limit vasoconstriction. This functional profile may reflect a relatively high Ca2+ set point displayed by KCa channels in the microcirculation, suggesting that a distinct KCa channel subtype may be expressed in cremasteric arteriolar muscle cells.
| |
METHODS |
|---|
|
Top
Abstract
Introduction
Methods
Results
Discussion
References
|
|---|
Preparation of cremaster muscles for intravital microscopy. All experiments involving animal use were approved by the Western Michigan University Institutional Animal Care and Use Committee and conform with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Animal Welfare Act. Male golden Syrian hamsters (100-170 g) were anesthetized with pentobarbital sodium (75 mg/kg ip). Cremaster muscles (2) were prepared for intravital video microscopy by standard methods as described previously (10-12). The muscles were exteriorized and pinned out over a Sylgard pedestal and superfused at 5-10 ml/min with a warm, pH 7.4, bicarbonate-buffered physiological salt solution [PSS; composition (in mM): 132 NaCl, 4.7 KCl, 2 CaCl2, 1.2 MgSO4, 0.03 EDTA, and 20 NaHCO3] that was equilibrated with a gas mixture containing 5% CO2-95% N2. The superfusion system allowed the superfusate to be rapidly changed without affecting pH, gas composition, or temperature (34°C) of the superfusion solution. Preparations were viewed with a Leitz Laborlux 12 FS microscope equipped with a 25× long-working distance objective (Zeiss UD 40, NA 0.65). The microscope was coupled to a Sony color video camera, and arterioles in the preparation were observed at a final magnification of ×800 measured at the face of a Sony color video monitor. Continuous records of arteriolar internal diameters were measured with a video caliper system that was accurate to ±1 µm (15).
Protocol for in vivo experiments. Only the central regions of preparations were observed in order to obviate cut-edge effects (27). Arterioles were selected for observation on the basis of their visibility and the presence of resting tone, and only one arteriole was studied per animal. Resting tone was evaluated by observing the dilation of vessels to topical application of 3-10 µl of 1 mM acetyl-
-methylcholine chloride
(methacholine). All vessels reported in this study were either on the
surface of the muscle or only one skeletal muscle cell layer below the surface of the cremaster. In a typical experiment, arteriolar diameter
was measured and the response to methacholine was determined in normal
PSS. After recovery from the methacholine, the superfusate was rapidly
switched to one containing either iberiotoxin (100 nM) or
tetraethylammonium chloride (TEA; 1 mM), which have been shown to
selectively inhibit KCa channels
in other vascular muscle cells (25). The diameter of the arteriole was
then measured continuously for up to 30 min after exposure to a
blocker.
In some experiments, oxygen-induced arteriolar constriction was
assessed before and during exposure to 1 mM TEA by equilibrating the
superfusate with 21% O2-5%
CO2-74%
N2 as described
previously (10-12).
Preparation of single arteriolar muscle
cells. Single arteriolar muscle cells were
enzymatically isolated from second- or third-order cremasteric
arterioles as described previously (14) with only minor modifications.
Hamsters were euthanized with pentobarbital sodium (>150 mg/kg), and
cremaster muscles were removed and placed in
3-(N-morpholino)propanesulfonic acid (MOPS)-buffered PSS
(4°C) containing 100 µM
Ca2+, 10 µM sodium
nitroprusside, and 1 mg/ml bovine serum albumin (BSA, see
Solutions). Single microvessels were
then hand-dissected from these tissues and transferred to 1-2 ml
of dissociation solution (see
Solutions) containing 10 µM sodium
nitroprusside for 10 min at room temperature. This solution was then
replaced by 1 ml of fresh room temperature solution containing 1.5 mg/ml papain and 1 mg/ml dithioerythritol. The solution was warmed to
37°C and incubated for 35 min with occasional agitation. The
papain-containing solution was then replaced with 1 ml of 37°C
solution containing 1.5 mg/ml collagenase, 1 mg/ml elastase, and 1 mg/ml soybean trypsin inhibitor. This solution was incubated at
37°C for 12-15 min, after which time the enzyme-containing
solution was replaced by 2 ml of room temperature dissociation
solution. The vessel fragments were allowed to settle to the bottom of
the tube, and most of this wash solution was removed. For
single-channel studies, 1-3 ml of room temperature
Kraftbrühe (KB) solution (17) was then rapidly added to the tube
to dissociate the vessel. If further dissociation was required, the
solution in the tube was gently triturated using a 1-ml Eppendorf-style
pipette. The cell-containing solution was then placed in
six 35-mm plastic culture dishes along with sufficient
additional KB solution to make 2 ml and was stored at 4°C for up to
4 h. For contraction studies, cells were isolated as described
previously (14).
Single-channel studies. Culture dishes
containing arteriolar muscle cells were placed on the stage of a Leitz
DMIL inverted microscope equipped with contrast enhancement optics and
allowed to warm to room temperature for 10 min. They were then
superfused with 20-30 ml of 100 µM
Ca2+ MOPS-buffered PSS, followed
by 10-20 ml of 2 mM Ca2+
MOPS-buffered PSS. The cells were then allowed to settle and attach to
the bottom of the dishes for 10-20 min. Solutions were delivered
by gravity at 1-1.5 ml/min and siphoned through a U-shaped capillary tube positioned to maintain bath volume between 1.5 and 2 ml.
Single-channel experiments were carried out in the inside-out
configuration (8). Pipettes were pulled from borosilicate glass tubes
(no. 6175, A-M Systems, Everett, WA) and fire polished and showed tip
resistances of 4-7 M
when filled with pipette solution (see
Solutions). Seals of 5-20 G
were established by application of light suction (8), and inside-out
patches were formed by rapidly withdrawing the pipette from the cell.
All experiments were conducted at room temperature (20°C). Currents
were acquired using an Axopatch 200A amplifier controlled by a Digidata
1200 data-acquisition system that was interfaced to a 80486 DX-2 66 personal computer running pCLAMP version 6 software (all from Axon
Instruments, Foster City, CA). Currents were filtered at 1 kHz with the
Axopatch 200 internal four-pole bessel low-pass filter and digitized at
5 kHz. Single-channel records were collected for 15-60 s or until
1,500-2,000 events were recorded per channel in the patch. At
Ca2+ concentrations 
3 µM,
currents were recorded for 5 min. Only data from patches with three or
fewer channels were analyzed and reported.
Single-cell contraction studies.
Contraction of single cells was assessed as described previously (14).
Cells were placed in a 1-ml chamber mounted on the stage of an inverted
microscope and allowed to settle, and the chamber was perfused with
bath solution (see Solutions). After
a 10-min equilibration period, a superfusion pipette (10- to 50-µm
tip ID) filled with a norepinephrine-containing solution (1 µM) was
positioned adjacent to a cell with a micromanipulator, and fluid was
ejected from the pipette onto the cells by pressurizing the back of the
pipette with a water manometer. The response of the cell was then
monitored through the eyepiece of the microscope at ×100
magnification (×10, NA 0.2 objective). A positive response was
defined as a >30% shortening of a cell within 15 s of continuous exposure to norepinephrine as described previously (14). At least 30 cells from several different fields were tested in each aliquot, and
the final response was calculated as the percentage of cells responding
(14). Three such trials were performed for each isolate of cells: two
controls in normal bath solution and one in the presence of 100 nM
iberiotoxin. The order of treatment was randomized and the results from
the two control trials were pooled for final display of the data.
Solutions. MOPS-buffered PSS contained
(in mM) 148 NaCl, 4.7 KCl, 1.2 MgSO4, 0.1 or 2 CaCl2, 0.03 EDTA, 2 MOPS, 5 glucose, and 2 Na pyruvate (pH = 7.4 with NaOH/HCl) at room
temperature. Dissociation solution contained (in mM) 137 NaCl, 5.6 KCl,
1 MgCl2, 0.42 Na2HPO4,
0.44 NaH2PO4,
10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 4.17 NaHCO3, and 0.1 CaCl2 (pH 7.4). KB solution contained (in mM) 85 KCl, 30 KH2PO4,
5 MgSO4, 2 Na2ATP, 0.2 ethylene glycol-bis(-aminoethyl
ether)-N,N,N',N'-tetraacetic
acid (EGTA), 5 Na pyruvate, 5 succinate, 5 creatine, 20 glucose, and 20 taurine (pH = 7.2) and 10 mg/ml BSA. Bath solution for inside-out
patch-clamp studies contained (in mM) 140 KCl, 1 MgSO4, 0.03 Na2EDTA, 10 NaMOPS, 1 EGTA, 1 N-hydroxyethylethylenediaminetriacetic
acid, and 1 nitrilotriacetic acid (pH = 7.2 with NaOH). In
some experiments 135 mM KCl in this solution was replaced by 135 mM
NaCl. CaCl2 was then added from a
1 M stock to yield the appropriate free
Ca2+ concentrations. This was
monitored with a Ca2+-selective
macroelectrode (Orion 93200 electrode, Fisher Acumet pH meter) that was
calibrated between 10 nM and 1 mM with solutions of known free
Ca2+ concentrations (32). All
Ca2+ concentrations reported in
the text refer to the concentration of free
Ca2+. The pipette solution for
inside-out patch-clamp studies contained (in mM) 100 K gluconate, 40 KCl, 1 MgCl2, 2 MOPS, 1 EGTA, and 0.1 CaCl2 (pH = 7.2 with NaOH).
Bath solution for single-cell contraction studies consisted of (in mM)
135 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES, and 10 glucose
(pH = 7.4 with NaOH).
Drugs and chemicals. Iberiotoxin was
purchased from Research Biochemicals and BSA from U.S. Biochemicals.
All other compounds were purchased from Sigma. Iberiotoxin was
dissolved in water to yield a concentration of 100 µM. Aliquots of
this solution were then stored at 20°C until used. On the
day of an experiment an appropriate amount of this solution was thawed
and diluted in the appropriate PSS or pipette solution to yield a final
concentration of 100 nM. Solutions of TEA (1 M) were stored frozen and
then diluted in the appropriate solution on the day of an experiment. All other solutions were made fresh each day.
Data analysis and statistics. Data are
presented as means ± SE or means ± 95% confidence
intervals. Curve fitting was performed with SigmaPlot for Windows
(Jandel Scientific, San Rafael, CA). All statistical comparisons were
performed at the 95% confidence level.
| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Methods
Results
Discussion
References
|
|---|
Effects of KCa channel blockers on resting tone of cremasteric arterioles in vivo. Arterioles in the present study had substantial resting tone (Fig. 1). Topical application of methacholine (3-10 µl of 1 mM solution) caused second-order arterioles to dilate 106 ± 12% from a resting diameter of 39 ± 1 µm (n = 8) and third-order arterioles to increase their diameters by 107 ± 21% from a resting diameter of 19 ± 2 µm (n = 8) (Fig. 1).
|
Effects of KCa channel blockers on arteriolar muscle cell contraction. To test whether KCa channels might be involved in the negative feedback regulation of arteriolar muscle cell tone in the presence of a vasoconstrictor, we assessed the effects of 1 mM TEA on oxygen-induced constriction of arterioles in vivo. We found that TEA significantly potentiated the effects of oxygen on these microvessels (Fig. 2A). Because of the expense of using iberiotoxin in vivo, we were precluded from assessing its effects on oxygen reactivity. However, we did assess the effects of this peptide KCa channel blocker on norepinephrine-induced contraction of single arteriolar muscle cells in vitro (Fig. 2B). As was observed for TEA and oxygen in vivo, iberiotoxin significantly increased the percentage of single arteriolar muscle cells that contracted when exposed to 1 µM norepinephrine (Fig. 2B).
|
Characterization of KCa channels in
inside-out patches of arteriolar muscle cell membrane.
Large-conductance unitary currents were observed in inside-out patches
of membrane from hamster cremasteric arteriolar muscle cells that were
both Ca2+ and voltage dependent
(see Figs. 3 and 5). The channels underlying these events appeared to
be potassium selective (Fig.
3B) and had a single-channel conductance, measured as the slope of the current-voltage relationship between 60 and +60 mV in
symmetrical 140 mM K+, of 240 ± 2 pS (n = 7 patches, Fig.
3B). These channels were observed in
essentially every patch excised from these cells with 
2 channels
present in every patch.
|
|
Ca2+ and
voltage sensitivity of arteriolar muscle KCa
channels.
Figure 5 summarizes the effects of
Ca2+ and voltage on the activity
of KCa channels in inside-out
patches of arteriolar muscle membrane. At negative membrane potentials
that spanned the physiological range (60 to 30 mV), no
channel activity was observed during 5-min recordings until the
Ca2+ concentration bathing the
cytosolic side of inside-out patches was increased from 1 to 3 µM;
at 1 µM, no events were detected in 5-min recordings at 60
or 30 in five separate patches. At membrane potentials of
60, 30, +30, and +60 mV, the
Ca2+ concentrations required for
50% activation (i.e.,
NPo/NPo max = 0.5, where N is the number of
channels in the patch,
Po is the individual channel open probability,
NPo is the
channel activity, and
NPo max is
the maximum
NPo)
were 44 ± 3, 20 ± 1, 6 ± 0.4, and 3 ± 0.5 µM, respectively. Hill coefficients were not significantly different
among the four membrane potentials examined
(P > 0.05) and averaged 1.9 ± 0.2 (n = 4), suggesting
parallel shifts of the Ca2+
response curves induced by changes in voltage.
|
Vm)/K]}, where
Vm is the
membrane potential,
V1/2 is the
voltage for half-maximal activation, and
K is the logarithmic voltage
sensitivity (
V required for e-fold increase in activity) (5); the
resulting V1/2
values were then plotted against
log[Ca2+] as suggested
by Carl et al. (5) (Fig. 5B), where
[Ca2+] is free
Ca2+ concentration.
The voltage sensitivities estimated from the curve fits were similar
for all concentrations of Ca2+
tested and indicated that channel activity increased
e-fold (~2.72 times) for a 16 ± 1 mV (n = 6) depolarization. As can be
seen in Fig. 5B, the semilog plot of
Ca2+ vs.
V1/2 yielded a
linear relationship {slope = 84 ± 5 mV/log[Ca2+] (in M),
intercept = 423 ± 26 mV,
r2 = 0.9843, P < 0.05}. From the equation
for the line fit through these data we estimated that
V1/2, the
change in V1/2 for a 10-fold change in Ca2+, was
84 ± 5 mV and that the
Ca2+-axis intercept
[Ca0; the
Ca2+ set point (5)] was ~9
µM.
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Methods
Results
Discussion
References
|
|---|
KCa channels have been proposed to play a substantial role in the regulation of membrane potential and hence tone of vascular smooth muscle cells in arteries that display myogenic tone (4, 23, 25). Our studies suggest that, under resting conditions, this may not be the case in the microcirculation of skeletal muscle. We found that two blockers of KCa channels, TEA and iberiotoxin, at concentrations that inhibited the activity of these channels in patch-clamp experiments in cells from cremasteric arterioles as well as other types of vascular muscle (4, 23, 25), had either no effect (iberiotoxin) or only a transient effect (TEA) on the resting diameters of arterioles in cremaster muscles in vivo. These data suggest that KCa channels are not very active under native conditions in the cremasteric microcirculation, despite the fact that these arterioles display substantial resting tone. Similar results have been reported in the cerebral (26) and cremasteric (18) microcirculations of rats. These observations are in direct contrast to findings in small, myogenically active arteries studied in vitro, where inhibition of KCa channels by either TEA or iberiotoxin depolarized and constricted these vessels (4, 23, 25). Our patch-clamp studies provide an initial explanation for these observations. We propose that the KCa channels found in cremasteric arteriolar muscle cells have a high Ca2+ set point under resting conditions such that they are not active at normal resting membrane potentials.
In inside-out patches of cremasteric arteriolar muscle membrane, we
found that internal Ca2+
concentrations 3 µM were required to observe unitary
KCa channel current between
60 and 30 mV, voltages that span the normal range of
membrane potential observed in cremasteric arteriolar muscle cells in
vivo (30, 31) and in vitro (14). Consistent with these findings, we
previously observed that iberiotoxin had no effect on macroscopic
K+ currents elicited between
90 and 0 mV in cremasteric arteriolar muscle cells studied using
the whole cell perforated-patch technique (14) or using conventional
whole cell methods with cells dialyzed with pipette solutions
containing Ca2+ concentrations as
high as 300 nM (13).
The lack of activity of KCa channels in arteriolar smooth muscle cells at negative membrane potentials could be caused by a low voltage sensitivity, a low Ca2+ sensitivity, or a high Ca2+ set point (5). We found that a 16-mV depolarization was required to produce an e-fold increase in channel activity in cremasteric arteriolar muscle cells. This voltage sensitivity is well within the range of values estimated in other smooth muscle cells [e-fold increase per 10-20 mV (5, 25)]. Thus arteriolar muscle cells do not appear to display a low voltage sensitivity.
We assessed the Ca2+ sensitivity
of KCa channels in inside-out
patches of cremasteric arteriolar muscle cell membranes by estimating V1/2, as
suggested by Carl et al. (5). We obtained a value of 84 ± 5 mV from
our analysis. This value is also similar to
V1/2 values
observed in other smooth muscles (5) and suggests that the
Ca2+ sensitivity of these channels
is not low. The average Hill coefficient that we measured (1.9 ± 0.2) also was similar to values reported in earlier studies of other
smooth muscles (1, 3, 5, 9, 20), further supporting this conclusion.
On the other hand, the Ca0 value [the Ca2+ set point (5)] was found to be 9 µM. Previous studies of KCa channels in myocytes isolated from guinea pig mesenteric arteries, rat pulmonary arteries, and rabbit portal veins have found Ca0 to be 0.5 µM (3), 1.5 µM (1), and 1 µM (9), respectively, values consistent with the Ca0 estimated in a variety of smooth muscles (5, 20). Our estimate of the Ca2+ set point is 6- to 18-fold higher than that observed in myocytes from larger vessels supplying other tissues. This implies that subsarcolemmal Ca2+ concentrations must be 6- to 18-fold greater in arteriolar muscle cells to initiate channel activity compared with muscle cells in larger vessels, which is consistent with our observations that channel activity is not observed at physiological membrane potentials for Ca2+ concentrations < 3 µM and that blockade of KCa channels in vivo has little effect on resting arteriolar diameter.
The mechanisms responsible for the high Ca2+ set point that we observed remain to be established. We do not think that our method for isolation of the cells, which included the use of sodium nitroprusside, somehow elevated the Ca0 in these cells. We have observed similar behavior in hamster cells isolated using diltiazem instead of sodium nitroprusside (Jackson, unpublished observations) and in rat cremasteric arteriolar muscle cells isolated in the absence of any dilator (W. F. Jackson and N. J. Rusch, unpublished observations). Thus it is unlikely that our data can be explained by simple methodological differences.
We think that it is more likely that cremasteric arteriolar muscle
cells express KCa channels that
inherently have an elevated Ca0.
Recent molecular studies suggest that variations in expression of both
the 
- and -subunits, which are thought to make up these channels,
can influence the response of the channels to changes in
Ca2+ (19). For example, expression
of the -subunit along with the -subunit for the human vascular
smooth muscle KCa channels reduces the Ca0 value for unitary currents
from 30 µM (-subunit alone) to 5 µM (- + -subunits)
(estimated from data in Ref. 19). Tissue-specific expression of spliced
variants of these two subunits could explain the differences in
Ca0 values that have been
reported. It is also possible that the high
Ca0 results from stable covalent modification of the channel proteins (i.e., stable phosphorylation). This should prove to be fertile ground for future investigations.
We propose that this high Ca2+ set point is responsible for the apparent lack of activity of KCa channels that we observed in resting arterioles. [Ca2+]i in rat cremasteric arteriolar muscle cells in vitro measured in cannulated pressurized vessels with substantial myogenic tone has been estimated to be <100 nM (21), although this value may underestimate the near-membrane Ca2+ concentration to which the channels are exposed (7, 23). Focal release of Ca2+ from the sarcoplasmic reticulum (Ca2+ "sparks") have been suggested to elevate local Ca2+ concentrations to ~300 nM (23). Even if the peak concentration of Ca2+ sparks has been underestimated (7), it seems unlikely that concentrations substantially greater than 1 µM would be routinely observed near the membrane of the arteriolar muscle cells except during conditions when Ca2+ release from the sarcoplasmic reticulum is increased or influx of Ca2+ through Ca2+ channels is augmented. However, a final conclusion will require the simultaneous measurement of membrane potential, near-membrane Ca2+ concentrations, and channel activity in the living microcirculation.
We did observe transient effects of TEA on third-order arterioles in vivo. This may be interpreted as evidence in support of the hypothesis that there is some KCa channel activity in the microcirculation that contributes to resting arteriolar tone in this tissue. However, the lack of effect of iberiotoxin argues against this conclusion. Instead, we suggest that the diameter responses observed in the presence of TEA may have resulted from some other effect, such as nonselective blockade of another K+ channel type. In support of this hypothesis, Kleppisch and Nelson (16) have shown that 1 mM TEA is not selective for KCa channels but may also inhibit ATP-sensitive K+ channels in some arterial muscle cells. Thus TEA may not be a completely selective blocker of KCa channels, which may explain its transient effects observed in vivo.
The apparent lack of activity of KCa channels in vivo and whole cell experiments in vitro (13, 14) does not mean that these channels do not play a role in the regulation of arteriolar tone in the microcirculation. On the contrary, we found that iberiotoxin (100 nM) in vitro and TEA (1 mM) in vivo significantly potentiated norepinephrine-induced contraction of single muscle cells and oxygen-induced arteriolar constriction, respectively. Therefore KCa channels may participate in the negative feedback regulation of arteriolar muscle cell contraction during activation by vasoconstrictors. This most likely occurs because of an increase in Ca2+ influx and release and membrane depolarization produced during vasoconstriction. In addition, recent studies have provided evidence that these channels may be activated by vasodilators that act through the adenosine 3',5'-cyclic monophosphate or guanosine 3',5'-cyclic monophosphate second messenger systems (26, 28). Thus, although not active under resting conditions, KCa channels may be recruited and participate in the regulation of arteriolar tone during vasoconstriction and may be activated to induce vasodilation. Hence, these channels may still play a role in the local, neural, and hormonal regulation of blood flow in the microcirculation.
In summary, we propose that the KCa channels expressed in cremasteric arteriolar muscle cells have a high Ca2+ set point that renders them silent under resting conditions even in arterioles with substantial resting tone. Our observations are in conflict with a number of in vitro experiments on cannulated arteries that display myogenic tone (see Ref. 25), and this points to the necessity of examining the physiological function and characteristics of ion channels in the microcirculation. Future studies should be directed toward examining the molecular expression of KCa channels in arterioles to determine if the high Ca2+ set point that we observed reflects expression of unique channel subunits or some other modification of the channels.
| |
ACKNOWLEDGEMENTS |
|---|
We thank James M. Huebner for technical and editorial assistance. We also express our appreciation to Dr. Nancy J. Rusch for helpful comments and discussion.
| |
FOOTNOTES |
|---|
This research was supported by National Heart, Lung, and Blood Institute Grant HL-32469 to W. F. Jackson and a Center for Environmental Signal Transduction postdoctoral fellowship to K. L. Blair.
Address for reprint requests: W. F. Jackson, Dept. of Biological Sciences, Western Michigan Univ., Kalamazoo, MI 49008.
Received 10 June 1997; accepted in final form 25 August 1997.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Methods
Results
Discussion
References
|
|---|
1.
Albarwani, S.,
B. E. Robertson,
P. C. G. Nye,
and
R. Z. Kozlowski.
Biophysical properties of Ca2+- and Mg-ATP-activated K+ channels in pulmonary arterial smooth muscle cells isolated from the rat.
Pflügers Arch.
428:
446-454,
1994[Medline].
2.
Baez, S.
An open cremaster muscle preparation for the study of blood vessels by in vivo microscopy.
Microvasc. Res.
5:
384-394,
1973[Medline].
3.
Benham, C. D.,
T. B. Bolton,
R. J. Lang,
and
T. Takewaki.
Calcium-activated potassium channels in single smooth muscle cells of rabbit jejunum and guinea-pig mesenteric artery.
J. Physiol. (Lond.)
371:
45-67,
1986
4.
Brayden, J. E.,
and
M. T. Nelson.
Regulation of arterial tone by activation of calcium-dependent potassium channels.
Science
256:
532-535,
1992
5.
Carl, A.,
H. K. Lee,
and
K. M. Sanders.
Regulation of ion channels in smooth muscles by calcium.
Am. J. Physiol.
271 (Cell Physiol. 40):
C9-C34,
1996
6.
Fay, F. S.
Calcium sparks in vascular smooth muscle: relaxation regulators.
Science
270:
588-589,
1995
7.
Guerrero, A.,
J. J. Singer,
and
F. S. Fay.
Simultaneous measurement of Ca2+ release and influx into smooth muscle cells in response to caffeine. A novel approach for calculating the fraction of current carried by calcium.
J. Gen. Physiol.
104:
395-422,
1994
8.
Hamill, O. P.,
A. Marty,
E. Neher,
B. Sakmann,
and
F. J. Sigworth.
Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.
Pflügers Arch.
391:
85-100,
1981[Medline].
9.
Inoue, R.,
K. Kitamura,
and
H. Kuriyama.
Two Ca-dependent K-channels classified by the application of tetraethylammonium distribute to smooth muscle membranes of the rabbit portal vein.
Pflügers Arch.
405:
173-179,
1985[Medline].
10.
Jackson, W. F.
Prostaglandins do not mediate arteriolar oxygen reactivity.
Am. J. Physiol.
250 (Heart Circ. Physiol. 19):
H1102-H1108,
1986.
11.
Jackson, W. F.
Arteriolar tone is determined by activity of ATP-sensitive potassium channels.
Am. J. Physiol.
265 (Heart Circ. Physiol. 34):
H1797-H1803,
1993
12.
Jackson, W. F.
Regional differences in mechanism of action of oxygen on hamster arterioles.
Am. J. Physiol.
265 (Heart Circ. Physiol. 34):
H599-H603,
1993
13.
Jackson, W. F.,
K. L. Blair,
and
N. J. Rusch.
Large-conductance, Ca2+-activated K+ channels in arteriolar muscle cells have low Ca2+-sensitivity (Abstract).
Microcirculation
3:
92,
1996.
14.
Jackson, W. F.,
J. M. Huebner,
and
N. J. Rusch.
Enzymatic isolation and characterization of single vascular smooth muscle cells from cremasteric arterioles.
Microcirculation
4:
35-50,
1977.
15.
Jackson, W. F.,
and
J. R. Searle.
An affordable video caliper (Abstract).
FASEB J.
3:
A1390,
1989.
16.
Kleppisch, T.,
and
M. T. Nelson.
ATP-sensitive K+ currents in cerebral arterial smooth muscle: pharmacological and hormonal modulation.
Am. J. Physiol.
269 (Heart Circ. Physiol. 38):
H1634-H1640,
1995
17.
Klöckner, U.,
and
G. Isenberg.
Endothelin depolarizes myocytes from porcine coronary and human mesenteric arteries through a Ca-activated chloride current.
Pflügers Arch.
418:
168-175,
1991[Medline].
18.
Loeb, A. L.,
I. Gödény,
and
D. E. Longnecker.
Functional evidence for inward-rectifier potassium channels in rat cremaster muscle arterioles in vivo (Abstract).
Microcirculation
4:
160,
1997.
19.
McCobb, D. P.,
N. L. Fowler,
T. Featherstone,
C. J. Lingle,
M. Saito,
J. E. Krause,
and
L. Salkoff.
A human calcium-activated potassium channel gene expressed in vascular smooth muscle.
Am. J. Physiol.
269 (Heart Circ. Physiol. 38):
H767-H777,
1995
20.
McManus, O. B.
Calcium-activated potassium channels: regulation by calcium.
J. Bioenerg. Biomembr.
23:
537-560,
1991[Medline].
21.
Meininger, G. A.,
D. C. Zawieja,
J. C. Falcone,
M. A. Hill,
and
J. P. Davey.
Calcium measurement in isolated arterioles during myogenic and agonist stimulation.
Am. J. Physiol.
261 (Heart Circ. Physiol. 30):
H950-H959,
1991
22.
Nelson, M. T. Regulation of arterial tone by
potassium channels. Jpn. J. Pharmacol. 58, Suppl.
2: 238P-242P, 1992.
23.
Nelson, M. T.,
H. Cheng,
M. Rubart,
L. F. Santana,
A. D. Bonev,
H. J. Knot,
and
W. J. Lederer.
Relaxation of arterial smooth muscle by calcium sparks.
Science
270:
633-637,
1995
24.
Nelson, M. T.,
J. B. Patlak,
J. F. Worley,
and
N. B. Standen.
Calcium channels, potassium channels, and voltage dependence of arterial smooth muscle tone.
Am. J. Physiol.
259 (Cell Physiol. 28):
C3-C18,
1990
25.
Nelson, M. T.,
and
J. M. Quayle.
Physiological roles and properties of potassium channels in arterial smooth muscle.
Am. J. Physiol.
268 (Cell Physiol. 37):
C799-C822,
1995
26.
Paterno, R.,
F. M. Faraci,
and
D. D. Heistad.
Role of Ca2+-dependent K+ channels in cerebral vasodilatation induced by increases in cyclic GMP and cyclic AMP in the rat.
Stroke
27:
1603-1607,
1996
27.
Proctor, K. G.,
and
D. W. Busija.
Relationships among arteriolar, regional, and whole organ blood flow in cremaster muscle.
Am. J. Physiol.
249 (Heart Circ. Physiol. 18):
H34-H41,
1985.
28.
Robertson, B. E.,
R. Schubert,
J. Hescheler,
and
M. T. Nelson.
cGMP-dependent protein kinase activates Ca-activated K channels in cerebral artery smooth muscle cells.
Am. J. Physiol.
265 (Cell Physiol. 34):
C299-C303,
1993
29.
Stehno-Bittel, L.,
and
M. Sturek.
Spontaneous sarcoplasmic reticulum calcium release and extrusion from bovine, not porcine, coronary artery smooth muscle.
J. Physiol. (Lond.)
451:
49-78,
1992
30.
Stekiel, W. J.,
S. J. Contney,
and
J. H. Lombard.
Sympathetic neural control of vascular muscle in reduced renal mass hypertension.
Hypertension
17:
1185-1191,
1991
31.
Stekiel, W. J.,
S. J. Contney,
and
N. J. Rusch.
Altered -receptor control of in situ membrane potential in hypertensive rats.
Hypertension
21:
1005-1009,
1993
32.
Tsien, R. Y.,
and
T. J. Rink.
Neutral carrier ion-selective microelectrodes for measurement of intracellular free calcium.
Biochim. Biophys. Acta
599:
623-638,
1980[Medline].
This article has been cited by other articles:
|
|
 |
|
  X. Wu, Y. Yang, P. Gui, Y. Sohma, G. A. Meininger, G. E. Davis, A. P. Braun, and M. J. Davis Potentiation of large conductance, Ca2+-activated K+ (BK) channels by {alpha}5{beta}1 integrin activation in arteriolar smooth muscle J. Physiol., March 15, 2008; 586(6): 1699 - 1713. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. A. Dabisch, J. T. Liles, S. R. Baber, N. H. Golwala, S. N. Murthy, and P. J. Kadowitz Analysis of L-NAME-dependent and -resistant responses to acetylcholine in the rat Am J Physiol Heart Circ Physiol, February 1, 2008; 294(2): H688 - H698. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. J. Marvar, J. R. Falck, and M. A. Boegehold High dietary salt reduces the contribution of 20-HETE to arteriolar oxygen responsiveness in skeletal muscle Am J Physiol Heart Circ Physiol, March 1, 2007; 292(3): H1507 - H1515. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Magnusson, C. M. Sorensen, T. H. Braunstein, N.-H. Holstein-Rathlou, and M. Salomonsson Renovascular BKCa channels are not activated in vivo under resting conditions and during agonist stimulation Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2007; 292(1): R345 - R353. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Li, A. Adebiyi, C. W. Leffler, and J. H. Jaggar KCa channel insensitivity to Ca2+ sparks underlies fractional uncoupling in newborn cerebral artery smooth muscle cells Am J Physiol Heart Circ Physiol, September 1, 2006; 291(3): H1118 - H1125. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. T. Lin, D. A. Hessinger, W. J. Pearce, and L. D. Longo Modulation of BK channel calcium affinity by differential phosphorylation in developing ovine basilar artery myocytes Am J Physiol Heart Circ Physiol, August 1, 2006; 291(2): H732 - H740. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. A. Dora Does arterial myogenic tone determine blood flow distribution in vivo? Am J Physiol Heart Circ Physiol, October 1, 2005; 289(4): H1323 - H1325. [Full Text] [PDF]
|
|
|
|
|
|
N. Kotecha and M. A. Hill Myogenic contraction in rat skeletal muscle arterioles: smooth muscle membrane potential and Ca2+ signaling Am J Physiol Heart Circ Physiol, October 1, 2005; 289(4): H1326 - H1334. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. P. Nase, J. Tuttle, and H. G. Bohlen Reduced perivascular PO2 increases nitric oxide release from endothelial cells Am J Physiol Heart Circ Physiol, July 11, 2003; 285(2): H507 - H515. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. T. Lin, D. A. Hessinger, W. J. Pearce, and L. D. Longo Developmental differences in Ca2+-activated K+ channel activity in ovine basilar artery Am J Physiol Heart Circ Physiol, July 11, 2003; 285(2): H701 - H709. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Yashiro and B. R. Duling Participation of intracellular Ca2+ stores in arteriolar conducted responses Am J Physiol Heart Circ Physiol, June 5, 2003; 285(1): H65 - H73. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-I. Kaide, M.-H. Wang, J.-S. Wang, F. Zhang, V.R. Gopal, J. R. Falck, A. Nasjletti, and M. Laniado-Schwartzman Transfection of CYP4A1 cDNA increases vascular reactivity in renal interlobar arteries Am J Physiol Renal Physiol, January 1, 2003; 284(1): F51 - F56. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Zhang, J.-I. Kaide, Y. Wei, H. Jiang, C. Yu, M. Balazy, N. G. Abraham, W. Wang, and A. Nasjletti Carbon monoxide produced by isolated arterioles attenuates pressure-induced vasoconstriction Am J Physiol Heart Circ Physiol, July 1, 2001; 281(1): H350 - H358. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. W. Fallet, J. P. Bast, K. Fujiwara, N. Ishii, S. C. Sansom, and P. K. Carmines Influence of Ca2+-activated K+ channels on rat renal arteriolar responses to depolarizing agonists Am J Physiol Renal Physiol, April 1, 2001; 280(4): F583 - F591. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Horiuchi, H. H. Dietrich, S. Tsugane, R. G. Dacey Jr, C. G. Sobey, and F. M. Faraci Role of Potassium Channels in Regulation of Brain Arteriolar Tone : Comparison of Cerebrum Versus Brain Stem Editorial Comment: Comparison of Cerebrum Versus Brain Stem Stroke, January 1, 2001; 32(1): 218 - 224. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. R. Halla, J. A. Madden, and J. B. Gordon Mediators of alkalosis-induced relaxation of piglet pulmonary veins Am J Physiol Lung Cell Mol Physiol, May 1, 2000; 278(5): L968 - L973. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 |
