Smooth Muscle Group, Department of Physiology, Queen's University, Belfast BT9 7BL, United Kingdom
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Freshly dispersed sheep mesenteric lymphatic
smooth muscle cells were studied at 37°C using the perforated
patch-clamp technique with Cs+- and K+-filled
pipettes. Depolarizing steps evoked currents that consisted of
L-type Ca2+ [ICa(L)]
current and a slowly developing current. The slow current reversed at
1 ± 1.5 mV with symmetrical Cl concentrations
compared with 23.2 ± 1.2 mV (n = 5) and
34.3 ± 3.5 mV (n = 4) when external
Cl
was substituted with either glutamate (86 mM) or
I
(125 mM). Nifedipine (1 µM) blocked and BAY K 8644 enhanced ICa(L), the slow-developing sustained
current, and the tail current. The Cl
channel blocker
anthracene-9-carboxylic acid (9-AC) reduced only the slowly developing
inward and tail currents. Application of caffeine (10 mM) to
voltage-clamped cells evoked currents that reversed close to the
Cl
equilibrium potential and were sensitive to 9-AC.
Small spontaneous transient depolarizations and larger action
potentials were observed in current clamp, and these were blocked by
9-AC. Evoked action potentials were triphasic and had a prominent
plateau phase that was selectively blocked by 9-AC. Similarly, fluid
output was reduced by 9-AC in doubly cannulated segments of
spontaneously pumping sheep lymphatics, suggesting that the
Ca2+-activated Cl
current plays an important
role in the electrical activity underlying spontaneous activity in this tissue.
lymphatics; pacemaking; action potentials; spontaneous activity
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ALTHOUGH IT IS NOW
well accepted that lymphatic vessels produce regular spontaneous
contractions that serve to propel fluid from the interstitium back to
the general circulation (4, 6, 7, 12, 25, 31), relatively
little is known about the ionic mechanisms underlying this spontaneous
activity. Van Helden (31) demonstrated that segments of
guinea pig mesenteric lymphatic smooth muscle generated small
spontaneous transient depolarizations (STDs) and larger action
potentials. The initial phase of the action potentials exhibited a
similar time course to the rising phase of the STDs, suggesting that
STDs either singularly, or through summation, depolarized the membrane
sufficiently to initiate action potential firing. Thus STDs were
proposed to provide the mechanism for pacemaking in guinea pig
mesenteric lymphatics. Van Helden (31) postulated that
STDs were generated by the transient activation of a Cl
conductance, since reducing extracellular Cl
or chelation
of intracellular Ca2+ with
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM reduced STD amplitude. However, as of yet, such a Cl
conductance has not been demonstrated or characterized under voltage
clamp in lymphatic smooth muscle.
In the present study, we demonstrate for the first time the presence of
a Ca2+-activated Cl conductance in lymphatics
and further suggest that its presence is necessary for normal
spontaneous contractions in these vessels.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell isolation. Mesenteric lymphatic vessels were removed from sheep of either sex at a local abattoir within 15 min of slaughter. These were transported to the laboratory in oxygenated Krebs solution at 37°C where they were dissected free from the mesenteric fat and either used immediately or stored at 4°C for use the following day. Several 2- to 3-cm lengths of lymphatic smooth muscle were cut into small pieces (<1 mm3) and placed in dispersal medium containing [per 5 ml of Ca2+-free Hanks' solution (see Solutions)] 15 mg collagenase (Sigma type 1A), 0.5 mg protease (Sigma type XXIV), 5 mg BSA (Sigma), and 15 mg trypsin inhibitor (Sigma) for 25-35 min at 37°C. Tissue was then transferred to Ca2+-free Hanks' solution and stirred for a further 15-30 min to release single relaxed smooth muscle cells. These cells were plated in petri dishes containing 100 µM Ca2+ Hanks' solution and were stored at 4°C for use within 8 h.
Solutions.
The composition of the solutions used was as follows (in mM):
1) Hanks' solution: 129.8 Na+, 5.8 K+, 135 Cl, 4.17 HCO3
, 0.34 HPO42
, 0.44 H2PO4
, 1.8 Ca2+, 0.9 Mg2+, 0.4 SO42
, 10 glucose, 2.9 sucrose, and 10 HEPES, pH adjusted to 7.4 with NaOH;
2) 49 mM Cl
Hanks' solution: 129.8 Na+, 5.8 K+, 86 glutamate, 49 Cl
,
4.17 HCO3
, 0.34 HPO42
, 0.44 H2PO4
, 1.8 Ca2+, 0.9 Mg2+, 0.4 SO42
, 10 dextrose, 2.9 sucrose,
and 10 HEPES, pH adjusted to 7.4 with NaOH; 3)
I
-substituted Hanks' solution: 129.8 Na+,
5.8 K+, 125 I
, 10 Cl
, 4.17 HCO3
, 0.34 HPO42
, 0.44 H2PO4
, 1.8 Ca2+, 0.9 Mg2+, 0.4 SO42
, 10 dextrose, 2.9 sucrose,
and 10 HEPES, pH adjusted to 7.4 with NaOH; 4)
Cs+ perforated patch pipette solution: 133 Cs+,
135 Cl
, 1.0 Mg2+, 0.5 EGTA, and 10 HEPES, pH
adjusted to 7.2 with CsOH; 5) K+ perforated
patch pipette solution: 133 K+, 135 Cl
, 1.0 Mg2+, 0.5 EGTA, and 10 HEPES, pH adjusted to 7.2 with KOH;
and 6) Krebs solution: 146.2 Na+, 5.9 K+, 133.3 Cl
, 25 HCO3
, 1.2 H2PO4
, 2.5 Ca2+, 1.2 Mg2+, and 11 glucose, pH maintained at 7.4 by bubbling with
95% O2-5% CO2.
Perforated patch voltage clamp of single cells.
Currents were recorded using the perforated patch configuration of the
whole cell patch-clamp technique (15, 22, 30). This
circumvented the problem of current rundown encountered using the
conventional whole cell configuration. The cell membrane was perforated
using the antibiotic amphotericin B (600 µg/ml). Patch pipettes were
initially front filled by being dipped in pipette solution and then
were back filled with the amphotericin B-containing solution. Pipettes
were pulled from borosilicate glass capillary tubing (1.5 mm outer
diameter, 1.17 mm inner diameter; Clark Medical Instruments) to a tip
diameter of ~1-1.5 µm and resistance of 2-4 M.
Isolated doubly cannulated lymphatic vessel recordings. Sections of mesenteric lymphatic vessels 4-7 cm long and 1-3 mm in diameter were cannulated at both ends with polythene tubing, mounted horizontally in an open-topped, water-jacketed organ bath, and perfused with Krebs' solution at 37 ± 1°C as described previously (25). The inflow cannula was connected to a pressure reservoir of variable height, and the outflow cannula was connected to a drop counter. The inflow reservoir and outflow cannulas were maintained at the same level so that when the vessel was not contracting the inflow and outflow pressures were equal and there was no movement of fluid through the vessel. However, when the vessel developed contractile activity, any flow that occurred was due to pumping activity of the vessel. The transmural pressure was set to 2-4 cmH20, and the preparation was allowed to equilibrate for 30 min, during which time spontaneous pumping activity usually developed. Contractions were measured as pressure fluctuations at the outflow end of the lymphatic using a Statham P23H pressure transducer with the output recorded on a Gould 2200 chart recorder. Fluid output was measured by a custom-built drop counter and was recorded simultaneously on the Gould chart recorder. Flow was intermittent, with each contraction resulting in the expulsion of a number of drops. The drop counter was reset at 1-min intervals so that output consisted of a series of ramps, the heights of which were an index of the flow rate. During experiments, the Krebs solution in the organ bath could be completely exchanged for a drug-containing solution within 5 min.
The following drugs were used: anthracene-9-carboxylic acid (9-AC), caffeine (Sigma), BAY K 8644, nifedipine (Bayer), and TTX (Tocris Cookson). Stock solutions of nifedipine, BAY K 8644, and TTX were made up in ethanol (1 mM stock). 9-AC was made up in DMSO (100 mM stock). All other compounds were dissolved directly in the perfusate. ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
L-type Ca2+ current and Ca2+-activated
Cl current.
As noted previously (9), the yield of cells was usually
modest, but enough healthy relaxed smooth muscle cells were obtained to
make experimentation possible. When cells were held at
60 mV and then
stepped to a series of potentials from
80 mV to +50 mV for 500 ms in
10-mV increments, a family of inward, outward, and tail currents
(Itail) were evoked and are shown in Fig.
1A. Similar responses to
voltage steps were found in >70% of cells studied. The inward current
was comprised of two components: an initial fast-activating,
fast-inactivating current and a more slowly developing, sustained
current. After repolarization to
60 mV, a slowly decaying inward
Itail was observed. The fast inward current was
measured within the first 20 ms of the test pulse, as indicated in Fig.
1A, and the slowly developing current was measured at the
end of the 500-ms pulse. This procedure was chosen to minimize
contamination of the slowly developing current with the rapidly
activating and inactivating current. Figure 1B shows a plot
of the current-voltage relationship for 21 cells. The fast current
activated at potentials positive to
40 mV and peaked at
10 mV,
characteristics typical of the L-type Ca2+
[ICa(L)] current previously described in these
cells (20). There was considerable variation in the amount
of current expressed between different cells. Thus the mean peak
Cl
current measured at
20 mV was
47 ± 9 pA
(n = 21) with a range of values from
5 to
178 pA.
Similarly, the peak Ca2+ current at
10 mV
averaged
65 pA ± 18 pA (n = 21) and ranged from
19 to
346 pA.
|
Effect of nifedipine and BAY K 8644.
If the slowly activating inward currents were dependent on the influx
of Ca2+ through L-type Ca2+ channels then it
should be possible to block these currents with nifedipine and enhance
them with the L-type Ca2+ channel agonist BAY K 8644. The
following experiments suggested that this was indeed the case. Figure
2A shows a current evoked by a
step from 60 to
20 mV before and during exposure to the L-type
Ca2+ channel antagonist nifedipine (1 µM). In the absence
of nifedipine, the fast and slow components of inward current could be
clearly resolved. Upon repolarization to
60 mV a slow inward
Itail was also apparent. After blockade of the
ICa(L), all three components of inward current
were abolished. Figure 2B shows a summary current voltage
relationship for the slowly activating current (measured at the end of
the 500-ms pulse) in six similar experiments in the absence and
presence of nifedipine. The slowly developing current was significantly
reduced by nifedipine (P < 0.05). To further
investigate the Ca2+ dependence of the sustained current,
the effect of BAY K 8644 (1 µM), an agent known to enhance
ICa(L), was examined. Figure 2C shows
the currents evoked in a cell in response to a voltage step from
60
to
20 mV. The rapidly and slowly activating components and the
Itail were clearly resolved in the absence of
the L-type channel agonist. Application of BAY K 8644 enhanced all
three components of inward current. Figure 2D shows a
summary from seven cells in which the effect of BAY K 8644 was examined
across a wide voltage range. The slowly developing current was
significantly enhanced by BAY K 8644, with peak current at
20 mV
increasing from
48 ± 16 to
100 ± 8 pA
(P < 0.05). These results suggest that the slowly
activating inward current and the Itail evoked by a depolarizing step are dependent on the influx of Ca2+
through L-type Ca2+ channels.
|
Effect of ion substitution on the slowly activating current.
Under the symmetrical Cl conditions used in the
experiments described above, the slowly developing inward current
reversed close to the ECl (0 mV). To determine
if the slowly activating current was carried by Cl
, the
effect of substituting Cl
with different anions was
examined. We first examined the effect of reducing external
Cl
from 135 to 49 mM by isosmotic substitution with
glutamate. The protocol used is shown in Fig.
3, top. A cell was stepped
from a holding potential of
60 to 0 mV for 500 ms. This was followed by a voltage ramp from
50 to +50 mV for 200 ms. Under symmetrical Cl
conditions (135 mM extracellular Cl
concentration), the step to 0 mV evoked only the
ICa(L), and the Itail
reversed at +2 mV. This was close to the ECl of
0 mV predicted by the Nernst equation. The external Cl
concentration was then reduced to 49 mM by substitution with glutamate
(see Solutions), giving a new calculated value for
ECl of +27 mV. Under these conditions, stepping
to 0 mV not only evoked ICa(L) but also a slowly
developing inward current indicative of a positive shift in the
Erev of the slowly developing current. The
Itail Erev moved to +22
mV, close to the new ECl. The effect of reducing
external Cl
concentration on Itail
Erev was examined in four other cells and
resulted in a mean shift in Erev from 1 ± 1.5 mV in symmetrical Cl
solutions to 23 ± 1.2 mV
(data corrected for junction potentials of
3 and +2 mV in normal and
reduced external Cl
solutions, respectively). Conversely,
when the external Cl
were substituted with
I
, the Erev of the slowly
activating inward current shifted negatively, as demonstrated in a
number of other studies (11, 14, 23). In four cells,
replacement of external Cl
with I
shifted
the Erev from
4.5 ± 1.6 to
34.3 ± 3.5 mV (data corrected for junctions potentials). These results
suggest that the slowly activating current and
Itail were predominantly carried by
Cl
.
|
Effect of 9-AC on currents.
The data presented so far support the idea that the slowly activating
current is a Ca2+-activated Cl current
[ICl(Ca)]. If this is the case, then the
current should be reduced by Cl
channel blockers such as
9-AC. The experiment shown in Fig.
4A suggested this was the
case. In this experiment, the cell was depolarized from
60 to
20
and 0 mV for 500 ms. Under control conditions, stepping to 0 mV evoked
ICa(L) and Itail, while
stepping to
20 mV evoked ICa(L), the slowly
developing current, and Itail. During
application of 1 mM 9-AC, stepping to 0 mV still activated ICa(L), but the Itail was
clearly reduced. Furthermore, when the cell was depolarized to
20 mV,
both the sustained current and the Itail were
abolished. Figure 4B shows a summary for nine cells in which
the effect of 9-AC was examined across the voltage range
80 to +50
mV. 9-AC significantly reduced the sustained current (P < 0.05). Although 9-AC significantly inhibited the Cl
current it did not significantly reduce the
ICa(L) (peak current at 0 mV was
63 ± 16 pA before compared with
48 ± 11 pA in 9-AC, n = 11, P = 0.28), suggesting that the inhibitory effect of
9-AC on Cl
current was not due to a decrease in
Ca2+ influx.
|
Effect of Ca2+ release from intracellular stores.
To establish if the release of Ca2+ from intracellular
stores could activate the Cl current in lymphatic smooth
muscle cells, the effect of caffeine was examined at a variety of
different voltages. Cells were held at a range of values from
80 to
+60 mV, and caffeine (10 mM) was administered for 10 s at each
potential. Approximately 90 s were allowed between consecutive
applications of caffeine to ensure repeatable responses. Application of
caffeine at negative potentials evoked inward currents that reversed
when the cell was held at potentials positive to
ECl (0 mV; Fig.
5A). The caffeine-evoked current decayed within 4-8 s of onset despite the continued
presence of caffeine. Figure 5B shows a plot of the peak
caffeine-evoked current against a holding potential in seven cells. The
caffeine-evoked current was inward at potentials negative to 0 mV and
reversed close to 0 mV, a finding consistent with the activation of a
Cl
conductance under the symmetrical Cl
conditions of the experiments. There was also evidence of outward rectification at the more positive potential, suggesting that this
current may show some voltage sensitivity. The Cl
current
showed a similar pharmacology to the slowly activating Cl
current evoked by a depolarizing step and thus was reversibly blocked
by 9-AC (Fig. 5C). The effect of 1 mM 9-AC on
caffeine-evoked current at
80 mV in four cells is illustrated in Fig.
5D. 9-AC significantly reduced the inward current from
117 ± 15 to
21 ± 11 pA (P < 0.05).
|
Effect of 9-AC on spontaneous activity in single cells.
In an attempt to examine the possible physiological role of such a
current in lymphatic smooth muscle, we examined electrical activity
under current-clamp mode using Cs+ pipettes. In these
experiments, it was necessary to inject a steady background current to
bring the membrane potential to 60 mV. In more than 1,000 cells
examined in the course of this and another related project
(24), spontaneous activity was observed in <1% of cells.
Spontaneously active cells exhibited transient depolarizations of
variable amplitude (9.8 ± 0.8 mV, n = 7 cells), a
rise time of 292 ± 28 ms (n = 7 cells), and were
occasionally large enough to raise the membrane potential to the
threshold value (approximately
40 mV) from which an action potential
was fired. Figure 6 shows typical
examples of STDs and action potentials recorded from a lymphatic smooth
muscle cell using a Cs+-filled pipette. Figure
6A, top, shows recordings from a cell in which
both small transient depolarizations and action potentials were
present. Figure 6A, bottom, shows an action
potential and STD displayed on an expanded time scale. The action
potential was biphasic, consisting of a small depolarization that
clearly preceded and initiated the more rapid depolarization. When the STD marked with the asterisk was scaled in amplitude, it was apparent that the time course of activation for the STDs and the initial part of
the action potential were very similar.
|
Effect of 9-AC on evoked action potentials in single cells.
Having demonstrated that spontaneous activity was abolished when
Cl channels were blocked, we next examined the effect of
9-AC on evoked action potentials using K+ pipettes. Figure
7 shows a typical experiment in which
action potentials were evoked every 20 s by the injection of
depolarizing current (100 pA, 40-ms duration). The action potentials
were triphasic in nature, consisting of an upstroke, repolarization,
and slowing after depolarization or plateau. Application of 9-AC (1 mM)
had no effect on either the upstroke or repolarization but reduced the
amplitude of the plateau, suggesting that this phase was dependent on
ICl(Ca). Similar effects were observed in three
cells.
|
Effect of 9-AC on spontaneous contractile activity of whole tissue.
The results from the above experiments on single cells suggest that the
Cl current contributes to both the generation of
spontaneous activity and the plateau phase of the action potential. To
establish a role for ICl(Ca) in the regular
spontaneous contractile activity of lymphatic smooth muscle, the effect
of blocking Cl
current was examined on a spontaneously
pumping segment of lymphatic smooth muscle. Figure
8 shows a recording of fluid output and outflow pressure before, during, and after application of 9-AC (1 mM).
Before application of any drugs, the lymphatic contracted at a
frequency of 6/min, and ~300 µl of fluid were expelled per minute.
Application of 9-AC (1 mM) at first slowed and then reversibly abolished spontaneous contractions. The decrease in spontaneous contractions was associated with a cessation of fluid output. This
inhibitory effect of 9-AC was observed in a total of five preparations
where contraction frequency was reduced from 6.2 ± 0.8/min before
compared with 2.7 ± 1.7/min in the presence of 9-AC. Similarly,
flow was reduced from 225 ± 40 to 58 ± 25 µl/min in the
presence of 9-AC (P < 0.02).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although it is now well established that lymphatics produce
regular spontaneous contractions in a variety of species (4, 6,
7, 12, 25, 31), little is known about the mechanisms underlying
this rhythmic activity. The demonstration of STDs in guinea pig
lymphatics suggests that a Cl conductance may act as the
pacemaker current in that tissue (31). The
ECl in smooth muscle is thought to be between
30 and
20 mV because of the relatively high intracellular
Cl
concentration in these cells (1).
Consequently, activation of a Cl
current will tend to
depolarize cells and may elicit action potentials. However, to date, no
study has demonstrated the presence of such a current in lymphatic
smooth muscle under voltage-clamp conditions.
Several pieces of evidence suggest that the slowly activating current
in sheep lymphatic myocytes is similar to the
ICl(Ca) found in a variety of smooth muscles
(reviewed in Ref. 23). The Erev of the current
in sheep lymphatics was close to the calculated Erev for Cl, changed in a
predictable manner when the Cl
were substituted with
either glutamate or I
, was blocked by the
Cl
channel blocker 9-AC, and was activated by either
Ca2+ influx through L-type Ca2+ channels or the
release of Ca2+ from intracellular stores.
To assess the relative permeability of the slowly activating inward
current to different anions, the shift in Erev
of the current was measured in response to substitution of the
Cl with either glutamate or I
. Replacement
of external Cl
with glutamate, an anion thought to be
relatively impermeant, shifted Erev positively
to a similar extent to that found in urethral and gastrointestinal
smooth muscle (8, 29). In contrast, replacement of
external Cl
with I
shifted
Erev negatively, as observed in other smooth
muscle preparations (3, 14, 28). When the permeability
ratios of these three anions were calculated using the
Goldman-Hodgkin-Katz equation (13, 18), values for the
permeability of I
vs. Cl
were equal to 3.2 and those for the permeability of glutamate vs. Cl
were
equal to 0.11; these values were obtained to give a permeability sequence of I
> Cl
> glutamate.
These values are in agreement with those found in rat lacrimal gland
cells (11), rabbit portal vein myocytes (14), and in other smooth muscle preparations (23). These data
are consistent with the view that the slowly activating current is carried mainly by Cl
through channels with similar
permeability properties to ICl(Ca) in other
types of tissue.
A number of agents that block Cl channels, such as 9-AC,
in smooth muscle have been identified (2, 3, 5, 19, 23). These agents vary in their potency and selectivity in different smooth
muscle types. 9-AC has been used to block agonist- and voltage
step-induced ICl(Ca) in rabbit portal vein and
esophagus and in rat portal vein, with IC50 values of
120-650 µM (2, 5, 19, 23). In the present study,
9-AC significantly blocked the depolarization-induced current,
Itail, and caffeine-evoked currents at a
concentration within the range used in other studies. These data
provide further evidence to suggest that the slowly activating current
and caffeine-evoked currents were ICl(Ca). When
the effect of 9-AC was investigated on ICa(L),
it did reduce the Ca2+ current in some cells, but this
effect was not significant and therefore was taken not to account for
blockade of ICl(Ca).
Under the conditions of our experiments we found that the majority of
lymphatic smooth muscle cells (~70%) possessed Cl
currents of variable amplitude although <1% showed regular
spontaneous activity. Although this was a surprising finding, it is not
without precedent. A recent study on ICl(Ca) in
sheep urethral smooth muscle cells demonstrated that regular
spontaneous activity was only observed in ~10% of their cells, even
though the majority of cells possessed Cl
current
(8). The reason why so few isolated lymphatic cells were
spontaneously active is puzzling given that recording conditions were
optimized to allow spontaneous activity to occur
(ECl set at 0 mV, K+ currents
blocked with Cs+). One possibility is that the isolated
smooth muscle cells are damaged in some way during the dispersal
procedure, although all of the cells studied were healthy looking
relaxed smooth muscle cells. A more likely explanation for the lack of
spontaneous activity in our experiments is that lymphatic smooth muscle
consists of a heterogeneous population of cells. A recent study on
isolated sheep mesenteric lymphatic cells has demonstrated the presence of a hyperpolarization-activated inward current in only 5% of cells,
suggesting that such heterogeneity does exist between cells in this
tissue (24). It is therefore possible that a minority of
cells differ in some way to the bulk of the smooth muscle cells and are
capable of firing spontaneous action potentials. However, it was
interesting to note that there were no obvious gross morphological differences between electrically quiescent cells and spontaneously active cells using light microscopy in this study.
When spontaneous electrical activity was observed in sheep lymphatic
myocytes, it was remarkably similar to that recorded with intracellular
microelectrodes in guinea pig mesenteric lymphatics. Thus the sheep
lymphatics produced STDs that had a mean amplitude of ~10 mV and a
rise time of ~300 ms, values that are similar to the time course and
amplitude of STDs in guinea pig lymphatics (31). The
action potentials in both guinea pig and sheep lymphatics also appeared
to have a large degree of overlap since they were biphasic, and the
rise time of the initial "bump" at the beginning of the action
potential was very similar to that of the STDs. The above results
support the idea that a Ca2+-activated Cl
conductance underlies the STDs and initiates the action potentials in
our single cell experiments. Similar STDs and action potentials have
also been recorded in the urethra of sheep and rabbits (8, 16,
17). In both of these tissues, it has been suggested that Ca2+ release from the sarcoplasmic reticulum is responsible
for the activation of the Cl
current that in turn
depolarizes the membrane and elicits Ca2+ action
potentials. Whether a similar mechanism acts as the pacemaking current
in sheep lymphatics is not clear from the present study, although there
is little doubt that release of Ca2+ from intracellular
stores can activate the Cl
current and blockade of this
current significantly decreases lymphatic contractility. It is
interesting that the Cl
current can also be activated by
Ca2+ influx through L-type Ca2+ channels. If
this were to happen during an action potential, then the
Cl
current could maintain Ca2+ influx by
clamping the membrane potential in the window current for
ICa(L), as has been proposed in the sheep
urethra (8). The results shown in Fig. 7 suggest that
elicited action potentials in sheep lymphatic smooth muscle have a
plateau phase that is sensitive to the Cl
channel blocker
9-AC. Consequently, the observed inhibitory effects of 9-AC on
lymphatic pumping could be explained by either the inhibition of STDs
or the inhibition of Ca2+ influx during the plateau phase
of the action potential.
To suggest that one specific current acts as the pacemaking current in lymphatic smooth muscle is, undoubtedly, an oversimplification. To date, a variety of potential depolarizing currents have been demonstrated in sheep lymphatic smooth muscle (20, 21, 24). We have previously shown that blockade of either the T-type Ca2+ current (7) or hyperpolarization-activated inward current (24) slows spontaneous contractions, suggesting that these currents contribute to pacemaking in sheep lymphatics. Thus it is likely that pacemaking in lymphatic vessels is generated not by a single current but relies on complex interactions between a number of currents as is the case in the heart (10). The exact details of how these currents interact with each other in lymphatic vessels are, as yet, far from clear. Future studies may focus on examining how the ICa(Ca) and other currents act together to produce the regular rhythmic contractions typical of lymphatic smooth muscle.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank the British Heart Foundation and the European Union for financial assistance and ABP Lurgan and John Robinson & Sons for supplying the tissue used in this study. Helen Toland was in receipt of a graduate award from the European Social Fund.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: M. Hollywood, Smooth Muscle Group, Dept. of Physiology, The Queen's Univ. of Belfast, 97 Lisburn Rd., Belfast BT9 7BL, UK (E-mail: m.hollywood{at}qub.ac.uk).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 22 March 2000; accepted in final form 24 May 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Aickin, CC.
Chloride transport across the sarcolemma of vertebrate smooth and skeletal muscle.
In: Chloride Channels and Carriers in Nerve, Muscle and Glial Cells. New York: Plenum, 1990, p. 209-249.
2.
Akbarali, HI,
and
Giles WR.
Ca2+ and Ca2+-activated Cl currents in rabbit oesophageal smooth muscle.
J Physiol (Lond)
460:
117-133,
1993[Abstract].
3.
Amedee, T,
Large WA,
and
Wang Q.
Characteristics of chloride currents activated by noradrenaline in rabbit ear artery cells.
J Physiol (Lond)
428:
501-516,
1990[Abstract].
4.
Armenio, S,
Cetta F,
Tanzini F,
and
Guercia C.
Spontaneous contractility in the human lymph vessels.
Lymphology
14:
173-178,
1981[ISI][Medline].
5.
Baron, A,
Pacaud P,
Loirand G,
Minonneau C,
and
Minonneau J.
Pharmacological block of Ca2+-activated Cl current in rat vascular smooth muscle cells in short term primary culture.
Pflügers Arch
419:
553-558,
1991[ISI][Medline].
6.
Campbell, T,
and
Heath T.
Intrinsic contractility of lymphatics in sheep and in dogs.
Q J Exp Physiol
58:
207-217,
1973[ISI].
7.
Convery, M,
Hollywood MA,
Cotton KD,
Thornbury KD,
and
McHale NG.
Role of inward currents in pumping activity of isolated sheep (Abstract).
J Physiol
501P:
110-111,
1997.
8.
Cotton, KD,
Hollywood MA,
McHale NG,
and
Thornbury KD.
Ca2+ current and Ca2+-activated chloride current in isolated smooth muscle cells of the sheep urethra.
J Physiol (Lond)
505:
121-131,
1997[Abstract].
9.
Cotton, KD,
Hollywood MA,
McHale NG,
and
Thornbury KD.
Outward currents in smooth muscle cells isolated from sheep mesenteric lymphatics.
J Physiol (Lond)
503:
1-11,
1997[Abstract].
10.
DiFancesco, D.
The contribution of the pacemaker current (If) to generation of spontaneous activity in rabbit sinoatrial node myocytes.
J Physiol (Lond)
434:
23-40,
1991[Abstract].
11.
Evans, MG,
and
Marty A.
Calcium-dependent chloride currents in isolated cells from lacrimal glands.
J Physiol (Lond)
378:
437-460,
1986[Abstract].
12.
Ferguson, MK,
and
DeFilippi VJ.
Nitric oxide and endothelium dependent relaxation in tracheobronchial lymph vessels.
Microvasc Res
47:
308-317,
1994[ISI][Medline].
13.
Goldman, DE.
Potential, impedence and rectification in membranes.
J Gen Physiol
27:
37-60,
1943
14.
Greenwood, IA,
and
Large WA.
Modulation of the decay of Ca2+-activated Cl currents in rabbit portal vein smooth muscle cells by external anions.
J Physiol (Lond)
516:
365-376,
1999
15.
Hamill, OP,
Marty A,
Neher E,
Sakmann B,
and
Sigworth FJ.
Improved patch clamp techniques for high resolution current recording from cells and cell-free membrane patches.
Pflügers Arch
391:
85-100,
1981[ISI][Medline].
16.
Hashitani, H,
and
Edwards FR.
Spontaneous and neurally activated depolarisations in smooth muscle cells of the guinea-pig urethra.
J Physiol (Lond)
514:
459-470,
1999
17.
Hashitani, H,
Van Helden DF,
and
Suzuki H.
Properties of depolarisations in circular smooth muscle cells of rabbit urethra.
Br J Pharmacol
118:
1627-1632,
1996[Abstract].
18.
Hodgkin, AL,
and
Katz B.
The effect of sodium ions on the electrical activity of the giant axon of the squid.
J Physiol (Lond)
108:
37-77,
1949[ISI].
19.
Hogg, RC,
Wang Q,
and
Large WA.
Effects of Cl channel blockers on calcium-activated chloride and potassium currents in smooth muscle cells from rabbit portal vein.
Br J Pharmacol
111:
1333-1341,
1994[Abstract].
20.
Hollywood, MA,
Cotton KD,
Thornbury KD,
and
McHale NG.
Isolated sheep mesenteric lymphatic smooth muscle cells possess both T- and L-type calcium currents (Abstract).
J Physiol (Lond)
501:
109P,
1997.
21.
Hollywood, MA,
Cotton KD,
Thornbury KD,
and
McHale NG.
Tetrodotoxin-sensitive sodium current in sheep lymphatic smooth muscle.
J Physiol (Lond)
503:
13-21,
1997[Abstract].
22.
Horn, R,
and
Marty A.
Muscarinic activation of ionic currents measured by a new whole-cell recording method.
J Gen Physiol
92:
145-159,
1988[Abstract].
23.
Large, WA,
and
Wang Q.
Characteristics and physiological role of the Ca2+-activated Cl conductance in smooth muscle.
Am J Physiol Cell Physiol
271:
C435-C454,
1996
24.
McCloskey, KD,
Toland HM,
Hollywood MA,
Thornbury KD,
and
McHale NG.
Hyperpolarisation-activated inward current in isolated sheep mesenteric lymphatic smooth muscle.
J Physiol (Lond)
521:
201-211,
1999
25.
McHale, NG,
and
Roddie IC.
The effect of transmural pressure on pumping activity in isolated bovine lymphatic vessels.
J Physiol (Lond)
261:
255-269,
1976[Abstract].
26.
Mironneau, J,
Arnaudeau S,
Macrez-Lepretre N,
and
Boittin FX.
Ca2+ sparks and Ca2+ waves activate different Ca2+-dependent ion channels in single myoctyes from rat portal vein.
Cell Calcium
20:
153-160,
1996[ISI][Medline].
27.
Neher, E.
Correction for liquid junction potentials in patch clamp experiments.
Methods Enzymol
207:
123-131,
1992[ISI][Medline].
28.
Ohta, T,
Ito S,
and
Nakazato Y.
Chloride currents activated by caffeine in rat intestinal smooth muscle cells.
J Physiol (Lond)
465:
149-162,
1993[Abstract].
29.
Pacaud, P,
Loirand G,
Lavie JL,
Mironneau C,
and
Mironneau J.
Calcium-activated chloride current in rat vascular smooth muscle cells in short term primary culture.
Pflügers Arch
413:
629-636,
1989[ISI][Medline].
30.
Rae, J,
Cooper K,
Gates P,
and
Watsky M.
Low access resistance perforated patch recordings using amphotericin B.
J Neurosci Methods
37:
15-26,
1991[ISI][Medline].
31.
Van Helden, DF.
Pacemaker potentials in lymphatic smooth muscle of the guinea-pig mesentery.
J Physiol (Lond)
471:
465-479,
1993[Abstract].