Articles in PresS. Am J Physiol Cell Physiol (March 31, 2004). 10.1152/ajpcell.00537.2003
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Hypotonic swelling stimulates L-type Ca2+ channel activity in vascular smooth muscle cells
through PKC
Running Title: Cell swelling and vascular Ca2+ channel
Yanfeng, Ding 1, Dean Schwartz 1, Philip Posner 2, Juming Zhong 1*
Department of Anatomy, Physiology, & Pharmacology, Auburn University College of
Veterinary Medicine, Auburn, AL 36849 1
Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee,
FL 32306 2
*Correspondences:
Dr. Juming Zhong
Department of Anatomy, Physiology, & Pharmacology
Auburn University, College of Veterinary Medicine
Auburn, AL 36849
Tel:
334-844-6739
Fax:
334-844-5388
[email protected]
1
Copyright © 2004 by the American Physiological Society.
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ABSTRACT
It has been suggested that L-type Ca2+ channels play an important role in cell swellinginduced vasoconstriction. However, there is no direct evidence that Ca2+ channels in vascular
smooth muscle are modulated by cell swelling. We tested the hypothesis that L-type Ca2+
channels in rabbit portal vein myocytes are modulated by hypotonic cell swelling via protein
kinase activation. Ba2+ currents (IBa) through L-type Ca2+ channels were recorded in smooth
muscle cells freshly isolated from rabbit portal vein using the conventional whole-cell patch
clamp technique. Superfusion of cells with hypotonic solution reversibly enhanced Ca2+ channel
activity, but did not alter the voltage-dependent manner of Ca2+ channel. Bath application of
selective inhibitors of protein kinase C (PKC), Ro 31-8425 or Go 6983, prevented IBa
enhancement by hypotonic swelling, while the specific protein kinase A inhibitor KT 5720 had
no effect. Bath application of phorbol 12,13-dibutyrate (PDBu) significantly increased IBa under
isotonic conditions and prevented current stimulation by hypotonic swelling. However, PDBu
did not have any effect on IBa when cells were first exposed to hypotonic solution. Furthermore,
down-regulation of endogenous PKC by overnight treatment of cells with PDBu prevented
current enhancement by hypotonic swelling. These data suggest that hypotonic cell swelling can
enhance Ca2+ channel activity in rabbit portal vein smooth muscle cells through activation of
PKC.
Keywords:
Ca2+ channel, cell swelling, vascular smooth muscle, protein kinases
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INTRODUCTION
Mechanical stretch as well as cell volume change has been shown to activate ion channels
in a variety of cells. Cell swelling induced by a decrease of extracellular osmolarity results in
activation of Cl- channels in rabbit cardiac cells (9), canine pulmonary smooth muscle cells (39),
and rabbit portal vein smooth muscle cells (13). Delayed rectifier K+ channels in guinea pig
ventricular myocytes (33), Ca2+ activated K+ channels in rabbit pulmonary smooth muscle cells
(18), and non-selective cation channels in guinea-pig gastric smooth muscle cells (38) and rat
osteoblasts (35) have also been shown to be modulated by cell swelling. In rabbit cardiac cells,
current through L-type Ca2+ channels was reversibly increased by osmotic cell swelling and by
cell inflation via the patch pipette (22). In vascular smooth muscle cells, it has been suggested
that the L-type Ca2+ channel might be involved in osmotic swelling induced vasoconstriction (2).
Exposure of guinea pig aortic strips to hypotonic extracellular fluid leads to a rapid
depolarization followed by marked vasoconstriction. These responses are inhibited by the Ca2+
channel blocker, D-600 (19). In addition, exposure of isolated portal vein to hypotonic solution
has been reported to elicit vasoconstriction, which was blunted by a reduction of extracellular
Ca2+ concentration (2). However, until now there has been no direct measurement of Ca2+
channel activity in these cells while exposed to a hypotonic condition.
The cellular and molecular mechanisms that link changes in cell volume to the activation
of different ion channels remains of great interest. In guinea cardiac myocytes and canine
pulmonary arterial smooth muscle cells, cell swelling-induced activation of Cl- channels is
thought to result from inhibition of protein kinase C (PKC) or enhancement of phosphatase (8;
42). In rabbit pulmonary arterial myocytes, activation of Ca2+- activated K+ channels appears to
occur by a direct effect of stretch on the channels (18). Modulation of L-type Ca2+ channels in
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rabbit cardiac cells by osmotic cell swelling and by cell inflation via the patch pipette was neither
dependent on PKA nor on intracellular Ca2+ (22). L-type Ca2+ current in smooth muscle cells of
the human stomach is also enhanced by hypotonic cell swelling, but the mechanism is not clear
(17).
The purpose of the present study was to delineate the mechanism underlying the effect of
hypotonic cell swelling on L-type Ca2+ channels in rabbit portal vein smooth muscle cells. Using
the conventional whole-cell technique and various protein kinase activators and inhibitors, we
have demonstrated that exposure of cells to a hypotonic bath solution significantly increases Ltype Ca2+ channel activity. Furthermore, protein kinase C appears to play an important role in the
hypotonic swelling induced activation of these channels.
MATERIALS AND METHODS
Isolation of rabbit portal vein smooth muscle cells
Myocyte isolation was performed as previously reported (40; 41). Male albino rabbits
(2.0 - 3.0 kg) were killed with an intravenous overdose of sodium pentobarbital (50 mg/kg), and
the portal vein was rapidly removed and cleaned of connective tissue in ice-cold Krebs solution
(in mmol/L): 125 NaCl, 4.2 KCl, 1.2 MgCl2, 1.8 CaCl2, 11 glucose, 1.2 K2HPO4, 23.8
NaHCO3, and 11 HEPES; pH 7.4 with NaOH. The portal vein was then cut into small segments
and pre-incubated for 60 minutes in a shaking water bath at 35 0C in a dispersion solution
(enzyme-free, in mmol/L): 90 NaCl, 1.2 MgCl2, 1.2 K2HPO4, 20 glucose, 50 taurine, and 5
HEPES; pH 7.2 with NaOH. The segments were then incubated in the dispersion solution
containing 2 mg/ml collagenase type I (Sigma), 0.5 mg/ml protease type XXVII (Sigma), and 2
mg/ml bovine serum albumin for 10-14 minutes at 35 0C. Following the digestion period, the
segments were rinsed with enzyme-free dispersion solution, and cells were separated by gentle
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trituration using a wide tipped fire-polished Pasteur pipette. Following dispersion, cells were
stored in enzyme-free dispersion solution containing 100 µM CaCl2 at 4 0C. Cells were used
within 10 hrs except in the experiments with overnight PDBu treatment. The animal use protocol
was approved by the Institutional Animal Use and Care Committee at Auburn University.
Electrophysiology
Inward Ba2+ current in vascular myocytes was measured using the whole-cell patch
clamp technique at room temperature (40). A drop of cell suspension was added to a small
recording chamber mounted on the stage of an inverted microscope (Nikon TS-100). Cells in the
chamber were superfused by gravity at a constant rate (~ 1-2 ml/min). Patch electrodes were
made from borosilicate glass pulled with a micropipette puller (PP-830, Narishige, Japan) and
fire-polished with a microforge (MF-830, Narishige, Japan). Pipette resistance was 3-5 MΩ
when filled with the appropriate solution. After establishing the whole-cell configuration,
membrane capacitance and series resistance were recorded using a 20 mV hyperpolarization
potential and were partially compensated. Inward current was elicited by stepping voltage from
the holding potential of –70 mV to 0 mV at 30 sec intervals using an Axopatch 200B patchclamp amplifier and pClamp 8 (Axon Instruments, USA). The leak currents at both isotonic and
hypotonic states were not subtracted. The standard isotonic bath solution (~290 mOsm kg-1 H2O)
used to record inward Ba2+ currents in portal vein cells was composed of (in mmol/L): 80 NaCl,
10 tetratethylammonium chloride (TEA-Cl), 5 BaCl2, 0.5 MgCl2, 5.5 glucose, 5 CsCl, 10
HEPES, and 70 D-mannitol, pH 7.40 with NaOH. Both TEA-Cl and CsCl were used to block K+
currents. The standard hypotonic solution was made from the above isotonic bath solution by
removing D-mannitol (~220 mOsm kg-1 H2O). The standard hypertonic bath solution was made
from the above isotonic solution by adding 70 mM D-mannitol (total D-mannitol 140 mM, ~360
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mOsm kg-1 H2O). The pipette solution contained (in mmol/L): 80 CsCl, 20 TEACl, 5 glucose, 2
MgCl2, 5 ATP, 1 GTP, 5 EGTA, and 80 D-mannitol, pH 7.2 with CsOH (40).
PKC-ε mRNA Expression.
RT-PCR was used to evaluate the expression of PKC-ε mRNA. Total RNA was isolated
by a monophasic solution of phenol and guanidine isothiocynate with TRIzol according to the
manufacturer’s instructions (Life Technologies, Grand Island, NY). Rabbit portal vein smooth
muscle rings were homogenized in TRIzol. Phase separation was carried out using chloroform
(0.2ml chloroform per 1ml of TRIzol) and centrifuged at 12,000 x g for 15 min at 4o C. The
aqueous phase was removed, and the RNA precipitated with isopropanol and washed 2 times
with 75% ethanol.
The RNA was resolved in diethyl pyrocarbonate-treated water.
RNA
concentration was measured spectrophotometrically at 260 nm.
One step RT-PCR was performed using the cMaster RTplus PCR System (Eppendorf,
Westbury, NY). Total RNA (0.5 µg) was incubated with RTplus PCR buffer containing Mg2+
(2.5 mM), dNTP (200 µM each), cMaster RT enzyme (0.15 U/µl), cMaster PCR enzyme
(0.05U/µl) and PKC-ε forward and reverse primers (400 nM) in a total volume of 20 µl. The
PCR primer sequences were: PKC-ε (forward) 5’-GCTCTGGCGCGGAAACACCCTTAT-3’;
PKC-ε (reverse) 5’-GATGGCTGGGCAGCCTCCCTTT-3’. Primers were derived from the
human PKC-ε gene (Accession # NM_005400) and produces an amplification product of 440 bp.
Control reactions were performed in the absence of RT. The RT-PCR was carried out in a BioRad iCycler. The RT step was performed at 50 0C for 45 min. The PCR reaction was: 94 oC 2
min followed by 35 cycles of 94 0C for 15 sec, 60 0C for 30 sec and 68 0C for 45 sec. Primer
extension was carried out at 68 0C for 3 min. RT-PCR amplification products were analyzed on
a 1.2% agarose gel and stained with ethidium bromide. The gels were visualized by ultraviolet
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light and photographed using a Bio-Rad Fluor S MultiImager. To confirm identity of the PCR
product, the band was cut out and eluted with the PerfectPrep Gel Cleanup Kit (Eppendorf) and
TA cloned into pCR2.1 using the Original TA Cloning Kit (Invitrogen, Calsbad, CA). One Shot
chemically competent cells (INVαF’, Invitrogen, Calsbad, CA) were transformed and plasmid
DNA isolated for sequencing. Sequencing was performed at the Auburn University Genomic
and Sequencing Laboratory.
Drugs and Reagents
Phorbol 12,13-dibutyrate (PDBu), Ro-31 8425, Go 6983, and KT 5720 were purchased from
Calbiochem (La Jolla, CA). Isoproterenol, nicardipine, niflumic acid, and DIDS were purchased
from Sigma (St. Louis, MO). Those drugs not soluble in water were first dissolved in dimethylsulphoxide (DMSO) and then further diluted in the appropriate solution with the final concentration
of DMSO less than 0.2%. DMSO alone at 0.2% had no effect on Ca2+ currents. The PKC
translocation inhibitory peptides βC2-4 (SLNPEWNET, corresponding to the residues 218-226 of
βPKC), εV1-2 (EAVSLKPT, corresponding to the residues 14-21 of εPKC), and scrambled εV1-2
(LSETKPAV) were purchased from CalBiochem (La Jolla, CA).
Statistical Analysis
Values were reported as mean + SE and n as the number of cells studied. Single-point
data between control and treated cells was compared using two-tailed unpaired Student’s t-test.
Comparisons between multiple groups were done using a two-way ANOVA with a StudentNewman-Kuels post-test. P value < 0.05 is considered significantly different.
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RESULTS
Hypotonic superfusion increased the inward currents through L-type Ca2+ channels.
In order to test the effect of osmolarity change on L-type Ca2+ channels in vascular
smooth muscle cells, rabbit portal vein myocytes were first superfused with isotonic solution
(~290 mOsm kg-1 H2O) while Ba2+ current was recorded continuously. When steady state current
amplitude was obtained under isotonic condition (~3 min), the superfusate was switched to the
standard hypotonic solution (~220 mOsm kg-1 H2O). Exposure of cells to the hypotonic solution
did not significantly change the access resistance of the pipettes (4.9 ± 0.3 mΩ, isotonic; 5.2 ±
0.3 mΩ, hypotonic; p = 0.56, n = 18). However, exposure to the hypotonic solution increased cell
size within 3 min as assessed roughly by measuring the two-dimensional nominal cell length and
width under the microscope. The mean length and the width of cells were 131 ± 7 and 7.9 ± 0.4
µm, respectively, under isotonic condition, and 146 ± 9 µm and 9.3 ± 0.7 µm after superfusion
with hypotonic solution (n = 6). Concomitant with the cell swelling, IBa was increased in all the
cells examined. As shown in Fig. 1A, switching the superfusate from an isotonic to the standard
hypotonic solution significantly increased IBa within 5 min (34 ± 7%, n = 20), which could be
reversed by switching the superfusate back to the isotonic solution. The increase of IBa was
closely proportional to the degree of reduction in osmolarity. Figure 1B demonstrates the gradual
increase of peak IBa in response to a proportional reduction of osmolarity. The degree of hypoosmolarity was adjusted by adding various amount of D-manmitol into the standard hypotonic
solution. On the other hand, exposure of cells to hypertonic solution did not have a significant
effect on inward current up to 10 min after the superfusate was switched (n= 6, data not shown).
To evaluate if the currents recorded under our experimental conditions were through Ltype Ca2+ channels in rabbit portal vein smooth muscle cells, we tested the effect of the L-type
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Ca2+ channel blocker nicardipine (NIC) on the inward currents recorded under isotonic or
hypotonic conditions. NIC (10 µM) completely abolished the inward current when applied into
the isotonic bath solution, and further prevented any detectable change of the current when cells
were exposed to the hypotonic solution (Fig. 2A, n = 7). Furthermore, when cells were first
exposed to hypotonic solution, NIC also completely abolished the increased inward current (Fig.
2B, n = 6).
In rabbit portal vein smooth muscle cells, a volume-regulated Cl- current (ICl-swell) has
been identified that is completely eliminated by either DIDS or niflumic acid (13). To prevent
possible contamination of IBa by ICl-swell in our experiments, we set the test potential at 0 mV,
which is the theoretical equilibrium potential for ICl-swell under our experimental conditions (13).
In addition, we tested the effects of DIDS and niflumic acid on the currents recorded under our
experimental conditions. Bath application of DIDS (100 µM) or niflumic acid (100 µM) had no
detectable effect on either basal currents under isotonic conditions or current enhanced by
hypotonic swelling. Peak inward currents were elevated 34 ± 5% by hypotonic swelling in the
presence of DIDS (Figure 2 C, n = 8), and 33 ± 4% in the presence of niflumic acid (Figure 2D,
n = 11). Thus, 100 µM DIDS was routinely added into the bath solutions in subsequent
experiments.
To examine the effect of hypotonic cell swelling on the voltage-dependent characteristics
of L-type Ca2+ channels, we compared the current-voltage (I-V) relationships and the steady state
inactivation under isotonic and hypotonic conditions. The I-V relationship was measured when
the test membrane potentials were stepped between –60 to +60 mV with increments of 10 mV
from the holding potential of –70 mV in the presence of either DIDS or niflumic acid. As shown
in Fig. 3 A, peak IBa was significantly higher at test potentials between –40 and +30 mV after
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hypotonic exposure in the presence of DIDS. The test potentials for threshold current and the
maximal peak current were also shifted towards the left after hypotonic exposure in the presence
of DIDS. Similarly, exposure of cells to hypotonic solution significantly increased the peak
currents at the test potentials between –30 to + 30 mV in the presence of niflumic acid. However,
hypotonic exposure did not change the voltage-dependent patterns of the current in this group of
experiments (Fig. 3 B). The left shift of the I-V relationship by hypotonic exposure in the first set
of experiments suggested possible contamination of IBa by ICl-swell at the negative test potentials in
the presence of DIDS, as DIDS is more potent to block ICl-swell at positive membrane potentials
(13). On the other hand, niflumic acid has been reported to be equally potent at blocking ICl-swell
at all membrane potentials (13). Our data suggest that hypotonic exposure did not change the
shape of the I-V relationship while it significantly enhanced IBa at most test potentials.
The effect of hypotonic cell swelling on steady state inactivation was tested using a twopulse protocol (40). The resting membrane potential was held at –70 mV. The conditioning prepulses ranged from –60 to +40 mV in 10 mV increments and were applied for 500 ms. The test
pulses were stepped to 0 mV for 200 ms. The two pulses were separated by an inter-pulse resting
interval of 5 ms. The relative availability of peak IBa was calculated by dividing the peak IBa with
the peak IBa.max (peak IBa /peak IBa.max), where the peak IBa is the peak IBa measured at the test
pulse after different prepulses, and the peak IBa.max is the peak IBa measured at the test pulse with
a prepulse of –60 mV. Increasing the potential of the conditioning prepulse reduced IBa elicited
by the following test pulse in cells. However, hypotonic swelling did not affect the relative
availability of peak IBa under different prepulse potential conditions (Fig. 3C). These data
suggest that although hypotonic cell swelling significantly increased the activity of Ca2+
channels, it did not change the voltage-dependent characteristics of these channels.
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PKC plays an important role in hypotonic swelling-induced activation of Ca2+ channels
In vascular smooth muscle cells, both PKA and PKC are able to stimulate L-type Ca2+
channels while PKG has an inhibitory effect (for review see (16)). To examine the mechanism
underlying the hypotonic-swelling induced stimulation of Ca2+ channel activity in vascular
smooth muscle cells, we first tested if protein kinase A plays a role in the signal transduction
pathway. Cells were treated with a specific PKA inhibitor, KT 5720, before and during exposure
to hypotonic solution. Previous report (41) indicated that KT 5720 had no detectable effect on
basal Ca2+ channel currents but eliminated the stimulatory effect of PKA in rabbit portal vein
smooth muscle cells. Figure 4A indicates that at the maximal concentration needed to inhibit
PKA activity, KT 5720 (200 nM) did not prevent the increase of IBa induced by hypotonic
exposure. The increase in IBa induced by hypotonic exposure were 31 ± 3% and 29 ± 4%
respectively, in the absence (n = 16) and presence (n = 11) of KT 5720. In another group of
experiments, effects of the β-adrenergic receptor agonist, isoproterenol (ISO), on IBa was
examined after cell exposure to hypotonic solution. As shown in Fig 4B, exposure to hypotonic
solution increased peak IBa about 30%. Application of ISO (1 µM) under hypotonic conditions
further increased peak IBa by 30%. Peak IBa in response to hypotonic exposure plus ISO was 158
± 7% of that under isotonic conditions without ISO treatment (n = 8). When these experiments
were repeated in the presence of KT 5720, exposure of cells to hypotonic solution significantly
increased peak IBa. However, the stimulatory effect of ISO on IBa under hypotonic condition was
completely prevented (Fig. 4C, n = 7). These data suggest that PKA is not involved in the cellswelling induced increase of IBa.
To evaluate the involvement of PKC in the stimulation of vascular Ca2+ channels by
hypotonic swelling, cells were treated with specific PKC inhibitors before and during exposure
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to hypotonic bath solution. Application of the PKC inhibitor Go 6983 (200 nM) did not have any
detectable effect on IBa under isotonic conditions. However, Go 6983 prevented the hypotonicswelling induced increase of IBa without affecting the cell size change. Cell length and width
were 126 ± 5 and 7.6 ± 0.4 µm, respectively, under isotonic condition, and 144 ± 8 µm and 9.4 ±
0.8 µm under hypotonic condition, in the presence of Go 6983 (n = 5). Similarly, the structurally
different PKC inhibitor Ro 31-8425 (200 nM) also blocked the hypotonic-swelling induced
increase of IBa. Figure 5A depicts the representative recordings of peak IBa in the presence or
absence of Ro 31-8425 before and after cell exposure to hypotonic solution. Figure 5B
demonstrates the effects of different PKC inhibitors on the hypotonic swelling induced increase
in IBa.
In rabbit portal vein smooth muscle cells, three isoforms of PKC, including PKC-α, ε,
and ξ, have been reported (3). To determine which isoform of PKC is involved in the activation
of Ca2+ channels following hypotonic swelling, we tested the effects of selective PKC isozyme
inhibitor peptides on the hypotonic swelling-induced increase of Ca2+ channel current. Cells
were dialyzed with either εV1-2, εV1-2S, or βC2-4, before exposure to hypotonic solution. εV12 is a short peptide derived from the V1 region of PKC-ε that inhibits translocation of PKC-ε.
εV1-2S is a scrambled peptide of εV1-2 and served as a negative control. βC2-4 is a short
peptide derived from the C2 domain of PKC-β and inhibits translocation of PKC-α and β (24;
42). Dialyzing cells with either εV1-2S or βC2-4 had no effect on hypotonic swelling induced
activation of Ca2+ channels. On the other hand, dialyzing cells with εV1-2 prevented the current
increase in response to exposure to hypotonic solution (Fig. 5 B). Furthermore, RT-PCR analysis
identified the expression of PKC-ε mRNA in portal vein smooth muscle cells (Fig. 5 C). The
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cloned DNA fragment showed 90% and 88% similarity, respectively, with human and mouse
PKC-ε.
Involvement of PKC in the hypotonic swelling induced stimulation of IBa was further
evaluated using the PKC activator, phorbol 12,13-dibutyrate (PDBu, 200 nM). Application of
PDBu under isotonic conditions significantly increased IBa which reached a steady state in about
5 min. Switching the superfusate from isotonic to hypotonic solution in the presence of PDBu
did not further change the amplitude of inward current. (Fig. 6 A & B). In another set of
experiments, cells were treated with PDBu after hypotonic exposure. Again, peak IBa was
significantly increased when cells were exposed to hypotonic solution. Application of PDBu
under hypotonic conditions did not further increase Ca2+ channel activity (Fig. 6 C & D).
Long-term exposure of cells to phorbol esters is a common method used to down-regulate
endogenous PKC activity, and an alternative method to test possible involvement of PKC in a
signaling pathway (31; 41). We also employed this method to further confirm the involvement of
PKC in hypotonic swelling induced stimulation of Ca2+ channels in vascular smooth muscle
cells. Cells were pretreated with phorbol ester (PDBu, 200 nM) or its vehicle (time-matched
control) and stored at 4 0C in the enzyme-free dispersion solution (100 µM CaCl2) for more than
18 hrs before IBa recording. Pretreatment of cells with PDBu did not have any significant effect
on the basal currents. Peak IBa under isotonic conditions in cells pretreated with PDBu (-201 ± 12
pA, n = 7) was comparable to that in time-matched control cells (-196 ± 13 pA, n = 6). Exposure
of time-matched control cells to hypotonic solution increased IBa (Fig. 7). On the other hand,
over-night pretreatment of cells with PDBu eliminated IBa response to cell exposure to hypotonic
solution (Fig. 7), as well as IBa response to an acute application of PMA (200 nM) under isotonic
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condition (n = 8, data not shown). These data further confirmed that PKC plays an important role
in the activation of Ca2+ channels by hypotonic cell swelling.
DISCUSSION
Activation of ion channels by hypotonic cell swelling has been reported in a wide range
of cell types. However, whether and how hypotonic cell swelling affects L-type Ca2+ channel
activity in vascular smooth muscle cells is not well understood. The present study demonstrates
that in rabbit portal vein smooth muscle cells, L-type Ca2+ channels are stimulated by hypotonic
cell swelling through a PKC-dependent pathway. In the present study, contamination of recorded
currents by K+ channels was eliminated using Cs+ and TEA-Cl in bath and pipette solutions, and
the possible activation of ICl-swell was prevented with DIDS. In addition, the specific L-type Ca2+
channel blocker nicardipine completely abolished the inward currents recorded in our
experiments and prevented the further increase of current induced by hypotonic superfusion.
These data indicate hypotonic cell swelling activates an inward current through L-type Ca2+
channels. Whether the increased channel activity induced by hypotonic swelling is associated
with an increase in the number of functional channels, channel opening probability, or channel
conductance was not tested in the present study and deserves further evaluation.
Hypotonicity of the extracellular environment occurs in several situations including:
reduced [Na+]e seen in normal pregnancy (11), over-hydration following intense exercise (1; 12),
and during treatment with citalopram (15). Other hypo-osmotic states are found in situations
involving deficits in plasma proteins secondary to hepatic dysfunction and nutritional deficits. Ltype Ca2+ channels play a central role in the excitation-contraction coupling in vascular smooth
muscle cells and has been suggested that activation of L-type Ca2+ channels is responsible for the
vasoconstriction induced by osmotic swelling (2). Ca2+ channels are also activated by membrane
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depolarization through volume-regulated chloride channels or stretch-activated non-selective
cation channels (4; 19; 23; 26; 37). Our data demonstrated a proportional increase in peak Ba2+
currents in response to gradual reductions in osmolalities. These data are consistent with the
reports that graded decreases of extracellular osmolarity lead to a proportional increase in the
tension of guinea pig aortic strips (19) and rat portal vein rings (2). Our data are also consistent
with other reports demonstrating that L-type Ca2+ channels in vascular smooth muscle cells are
stimulated by inflating cells with positive pressure through a pipette electrode (4; 20; 23). Thus,
activation of Ca2+ channels may play an important role in the myogenic response under
physiological and patho-physiological conditions. Although the 25% reduction of extracellular
osmolarity used in this study may not occur under normal physiological conditions, it has been
used as a common experimental procedure for the study of volume-regulated channels by many
research groups (5; 13; 21; 37; 42).
Previous studies on various volume-regulated anion channels in different cell types have
demonstrated that PKA activation plays an important role in the cell volume change-induced
modulation of these channels (14; 6; 10; 34). In the present study, we evaluated the possible
involvement of PKA in the modulation of L-type Ca2+ channels in vascular smooth muscle cells by
hypotonic cell swelling. Exposure of cells to hypotonic bath solution did not prevent, but rather
blunted, the stimulatory effects of isoproterenol on IBa. In addition, pretreatment of cells with the
PKA inhibitor KT 5720 did not prevent the increase in IBa induced by hypotonic swelling, but
prevented further increase of IBa by isoproterenol under hypotonic condition. These data are
consistent with the previous reports that isoproterenol induced more than a 50% increase in Ca2+
channel currents in both rabbit and rat portal vein smooth muscle cells through activation of both
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PKA and PKC (36; 41). Thus, it is unlikely that hypotonic swelling stimulates Ca2+ channel activity
through activation of PKA in vascular smooth muscle cells.
Modulation of volume-regulated ion channels by PKC has also been reported. For example,
PKC inhibitors enhanced the basal ICl-swell under isotonic condition and further abolished the
swelling-induced activation of ICl-swell in canine pulmonary artery smooth muscle cells (42) and
guinea pig cardiac myocytes (8). In contrast, phorbol esters dose-dependently increased the
amplitude of ICl-swell in canine atrial myocytes (7). More pertinent to our study is a recent report
demonstrating that in rabbit portal vein smooth muscle cells, phorbol esters increased, while PKC
inhibitors decreased, the amplitude of ICl-swell (10). Thus, hypotonic-swelling may activate ICl-swell
through activation (7; 10) or inhibition (8; 42) of PKC in different cells. The discrepancy between
the PKC-dependent modulation of ICl-swell might be related to the species variation. In the present
study, stimulation of endogenous PKC by PDBu strongly increased peak IBa under isotonic
conditions and prevented further current enhancement by hypotonic swelling while PKC inhibitors
completely abolished IBa stimulation by hypotonic cell swelling. Long-term treatment of cells with
PDBu to down regulate endogenous PKC activity also abolished Ba2+ current stimulation induced
by hypotonic cell swelling. These data strongly suggest that a hypotonic induced cell volume
change can stimulate L-type Ca2+ channels by activating PKC in rabbit portal vein cells.
Furthermore, our results demonstrated that the selective inhibitory peptide against PKC-ε, εV1-2,
was able to prevent the activation of Ca2+ channels by hypotonic swelling, where as neither the
scrambled εV1-2 nor the selective cPKC inhibitor αC2-4 was effective. Thus, PKC-ε may play an
important role in the hypotonic swelling induced activation of Ca2+ channels.
While our results do not directly answer the question as to how cell swelling stimulates PKC
activity in this cell type, results from other groups have demonstrated a PKC isozyme-specific
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interaction with F-actin (29; 30) and caveolae (27; 32). In addition, possible redistribution or
reorganization of F-actin and caveolar micro domains during cell swelling has been suggested (25;
28). If F-actin or caveolin serve as essential anchoring proteins for specific PKC isozymes,
alteration of F-actin or caveolin during cell swelling may change PKC translocation and activity to
its specific targets. Whether this same mechanism can account for cell swelling-induced stimulation
of L-type Ca2+ channels in rabbit portal vein smooth muscle cells has not yet been elucidated and
deserves further study. In addition, further studies should also answer the question of whether the
cell swelling induced increase of Ba2+ current through these channels is related to the increase of
single channel conductance, open channel probability, or the number of functional channels.
In summary, results from the present study demonstrate for the first time that hypotonic
cell swelling can enhance L-type Ca2+ channel activity in rabbit portal vein smooth muscle cells.
Furthermore, PKC, but not PKA, plays an important role in the cell swelling induced stimulation
of L-type Ca2+ channels. Thus, stimulation of Ca2+ channels as well as stimulation of volumeregulated Cl- channels by hypotonic cell swelling may subsequently enhance the contractility of
blood vessels and be a mechanism for modulating afterload in arteriolar vessels or preload
(capacitance) in venous vessels.
ACKNOWLEDGMENTS
The authors would like to express their gratitude to Cathy Galle and Ruijiao Zou for their
technical support, and to Dr. Vitaly Vodyanoy for scientific review of this manuscript. This work
was supported by research grants from National Health Foundation, USA (H2002016, J.Zhong),
American Heart Association Southeast Affiliation (0255030B, J.Zhong), and Auburn University
(ALAV356, J.Zhong).
17
C-00537-2003.R2
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C-00537-2003.R2
FIGURE LEGENDS
Fig. 1. Hypotonic swelling increased Ba2+ current. A, time course of peak current
measurements from a cell under isotonic and hypotonic conditions. Currents were
recorded every 30 sec when the membrane potential was stepped to 0 mV from a holding
potential of –70 mV. The insert shows representative current traces recorded at 5- (1), 10(2), and 20- min (3) under different conditions. B, percent increase of peak Ba2+ currents
under various hypotonic conditions. Cells were first superfused with isotonic bath
solution, followed by gradual reduction in osmolarity of bath solutions (n = 8).
Fig. 2. Nicardipine completely abolished, while ICl-swell inhibitors had no effect on the
currents under either isotonic or hypotonic conditions. Fig. 2. Nicardipine
completely abolished, while ICl-swell inhibitors had no effect on the currents under
either isotonic or hypotonic conditions. A, time course of peak current measurements
from a cell in the presence of nicardipine (NIC, 10 µM). NIC was applied before cell
exposure to hypotonic solution, as indicated by the horizontal line. B, time course of peak
current measurements from a cell in the presence of nicardipine (NIC, 10 µM). NIC was
applied after cell exposure to hypotonic solution, as indicated by the horizontal line. C,
time course of peak current measurements from a cell in the presence of DIDS (100 µM).
DIDS was added into the bath solutions as indicated by the horizontal lines. D, time
course of peak current measurements from a cell in the presence of niflumic acid (NFA,
100 µM). Niflumic acid was added into the bath solutions as indicated by the horizontal
lines.
Fig. 3. Hypotonic swelling did not change the voltage-dependent characteristics of Ca2+
channel. A, peak currents measured under isotonic and hypotonic conditions at different
23
C-00537-2003.R2
test potentials in the presence of DIDS (n = 8). Peak currents were significantly higher at
the test potentials between –40 and +30 mV (p < 0.05). B, peak currents measured under
isotonic and hypotonic conditions at different test potentials in the presence of niflumic
acid (NFA, n = 11). Peak currents were significantly higher at the test potentials between
–30 and +30 mV (p < 0.05). C, relative peak IBa-prepulse potential relationship for cells
before (n = 6) and after (n = 5) superfusion with hypotonic bath solution in the presence
of DIDS. The insert is a representative recording of steady state IBa inactivation in a cell
superfused with isotonic bath solution using a two-pulse protocol (see text). Values in A,
B, and C represent mean ± SE.
Fig. 4. KT 5720 did not block hypotonic swelling-induced IBa increase. A, time course of
peak IBa measurements from a cell in the presence of KT 5720 and a cell in the absence
of KT 5720. KT 5720 (0.2 µM) was added in both isotonic and hypotonic solutions in the
first case. B, time course of peak IBa measurements from a cell treated with isoproterenol
(ISO, 1 µM) after exposure to hypotonic solution. ISO was added into hypotonic solution
as indicated with the horizontal line. C, time course of peak IBa measurements from a cell
treated with ISO (1 µM) after exposure to hypotonic solution in the presence of KT 5720
(0.2 µM). In all the experiments presented in A, B, and C, cells were superfused with
isotonic bath solution followed by hypotonic bath solution, as indicated with the
horizontal lines.
Fig. 5. PKC inhibitors prevented hypotonic swelling induced IBa increase. A, time course of
peak IBa measurement from a control cell and a cell treated with Ro 31-8425. Cells were
superfused with isotonic bath solution followed with hypotonic solution, as indicated
with the horizontal lines. B, averaged percent increase of peak IBa by hypotonic swelling
24
C-00537-2003.R2
in cells treated without any PKC inhibitor (Ctl, n = 16), or with either Ro 31-8425 (Ro, n
= 11), Go 6983 (Go, n = 6), PKC inhibitory peptide αC-2-4 (n = 9), εV1-2 (n = 17), or
scrambled εV1-2 (εV1-2S, n = 11). Values represent mean ± SE and * represents
significantly different from the control value (p < 0.05). C, RT-PCR reaction using gene
specific primers for human PKC epsilon. One step RT-PCR was performed using the
cMaster RTplus PCR System. Lane 1 is the indicated bp ladder. Lane 2 is the product
of the RT-PCR. Lane 3 is the RT-PCR reaction minus the RT enzyme.
Fig. 6. Hypotonic swelling prevented PDBu stimulation of IBa. A, peak currents measured
from a cell treated with PDBu before and after exposure to hypotonic bath solution. B,
averaged percent increase of peak IBa by PDBu before and after hypotonic swelling (n =
9). Values represent mean ± SE. C, peak currents measured from a cell treated with
PDBu after exposure to hypotonic bath solution. Cell was superfused with isotonic
solution and then followed with hypotonic bath solution as indicated with the horizontal
lines. PDBu was added into the hypotonic bath solution when peak IBa reached a steady
state. D, averaged percent increase of peak IBa by hypotonic swelling with or without
PDBu (n = 15).
Fig. 7. Overnight pretreatment with PDBu prevented hypotonic swelling-induced
stimulation of Ca2+ channels. A, peak currents measured from a cell pre-treated with
200 nM PDBu for more 18 hrs and a time-matched control cell. Cell was exposed to
isotonic and hypotonic solutions as indicated by the horizontal lines. B, averaged percent
increase after hypotonic exposure. Values represent mean ± SE for PDBu-pretreated cells
(n = 7) and time-matched control cells (n = 6). * represents significantly different from
the values under isotonic conditions (p < 0.05).
25
A
500 pA
Peak Ba
2+
current (pA)
-200
1
3
-300
50 msec
2
-400
isotonic
-500
isotonic
hypotonic
-600
0
5
10
15
20
Time (min)
% change of peak current
B
60
40
20
0
0
5
10
15
20
25
30
% reduction of osmolarity
Fig. 1. Hypotonic swelling increased Ba2+ current. A, time course of peak current
measurements from a cell under isotonic and hypotonic conditions. Currents were recorded every
30 sec when the membrane potential was stepped to 0 mV from a holding potential of –70 mV.
The insert shows representative current traces recorded at 5- (1), 10- (2), and 20- min (3) under
different conditions. B, percent increase of peak Ba2+ currents under various hypotonic
conditions. Cells were first superfused with isotonic bath solution, followed by gradual reduction
in osmolarity of bath solutions (n = 8).
Peak Ba2+ current (pA)
A
B
0
0
-200
-200
NIC
-400
-400
isotonic
hypotonic
isotonic
-600
-600
2
4
6
8
10
12
14
16
C
0
2
4
6
8
10
12
14
16
14
16
D
-200
-200
-400
-400
Peak Ba
2+
current (pA)
NIC
hypotonic
-600
-600
DIDS
NFA
hypotonic
isotonic
hypotonic
isotonic
-800
-800
2
4
6
8
10
Time (min)
12
14
16
2
4
6
8
10
12
Time (min)
Fig. 2. Nicardipine completely abolished, while ICl-swell inhibitors had no effect on the
currents under either isotonic or hypotonic conditions. A, time course of peak current
measurements from a cell in the presence of nicardipine (NIC, 10 µM). NIC was applied before
cell exposure to hypotonic solution, as indicated by the horizontal line. B, time course of peak
current measurements from a cell in the presence of nicardipine (NIC, 10 µM). NIC was applied
after cell exposure to hypotonic solution, as indicated by the horizontal line. C, time course of
peak current measurements from a cell in the presence of DIDS (100 µM). DIDS was added into
the bath solutions as indicated by the horizontal lines. D, time course of peak current
measurements from a cell in the presence of niflumic acid (NFA, 100 µM). Niflumic acid was
added into the bath solutions as indicated by the horizontal lines.
Peak Ba2+ current (pA)
A
200
0
-200
-400
-600
-80
isotonic-DIDS
hypo-DIDS
-60
-40
-20
0
20
40
60
80
Test potential (mV)
200
Peak Ba
2+
current (pA)
B
0
-200
-400
-600
-80
isotonic-NFA
hypo-NFA
-60
-40
-20
0
20
40
60
80
Test potential (mV)
C
% Change Of Peak IBa
120
100
80
60
40
20
0
-80
isotonic-DIDS
hypo-DIDS
-60
-40
-20
0
20
40
60
Pre-conditioning Potential (mV)
Fig. 3. Hypotonic swelling did not change the voltage-dependent characteristics of Ca2+
channel. A, peak currents measured under isotonic and hypotonic conditions at different test
potentials in the presence of DIDS (n = 8). Peak currents were significantly higher at the test
potentials between –40 and +30 mV (p < 0.05). B, peak currents measured under isotonic and
hypotonic conditions at different test potentials in the presence of niflumic acid (NFA, n = 11).
Peak currents were significantly higher at the test potentials between –30 and +30 mV (p < 0.05).
C, relative peak IBa-prepulse potential relationship for cells before (n = 6) and after (n = 5)
superfusion with hypotonic bath solution in the presence of DIDS. The insert is a representative
recording of steady state IBa inactivation in a cell superfused with isotonic bath solution using a
two-pulse protocol (see text). Values in A, B, and C represent mean ± SE.
Peak Ba
2+
current (pA)
A
-200
control
KT 5720
-300
-400
-500
isotonic
hypotonic
-600
2
4
6
8
10
12
14
16
14
16
-200
-400
Peak Ba
2+
B
current (pA)
0
-600
hypotonic
-800
0
2
4
6
8
10
12
-200
KT 5720
-400
Peak Ba
2+
current (pA)
C
ISO
isotonic
Col 1 vs current
-600
ISO
isotonic
hypotonic
-800
0
2
4
6
8
10
12
14
16
Time (min)
Fig. 4. KT 5720 did not block hypotonic swelling-induced IBa increase. A, time course of
peak IBa measurements from a cell in the presence of KT 5720 and a cell in the absence of KT
5720. KT 5720 (0.2 µM) was added in both isotonic and hypotonic solutions in the first case. B,
time course of peak IBa measurements from a cell treated with isoproterenol (ISO, 1 µM) after
exposure to hypotonic solution. ISO was added into hypotonic solution as indicated with the
horizontal line. C, time course of peak IBa measurements from a cell treated with ISO (1 µM)
after exposure to hypotonic solution in the presence of KT 5720 (0.2 µM). In all the experiments
presented in A, B, and C, cells were superfused with isotonic bath solution followed by
hypotonic bath solution, as indicated with the horizontal lines.
A
Peak Ba
2+
current pA)
-200
control
Ro 31-8425
-300
-400
-500
isotonic
hypotonic
-600
0
2
4
6
8
10
12
14
16
Time (min)
B
% change of peak current
50
40
30
20
10
0
-10
Ctl
C
*
*
*
Ro
Go
1
2
αC2-4
εV1-2 εV1-2S
3
500 bp
400 bp
300 bp
Fig. 5. PKC inhibitors prevented hypotonic swelling induced IBa increase. A, time course of
peak IBa measurement from a control cell and a cell treated with Ro 31-8425. Cells were
superfused with isotonic bath solution followed with hypotonic solution, as indicated with the
horizontal lines. B, averaged percent increase of peak IBa by hypotonic swelling in cells treated
without any PKC inhibitor (Ctl, n = 16), or with either Ro 31-8425 (Ro, n = 11), Go 6983 (Go, n
= 6), PKC inhibitory peptide αC-2-4 (n = 9), εV1-2 (n = 17), or scrambled εV1-2 (εV1-2S, n =
11). Values represent mean ± SE and * represents significantly different from the control value
(p < 0.05). C, RT-PCR reaction using gene specific primers for human PKC epsilon. One step
RT-PCR was performed using the cMaster RTplus PCR System. Lane 1 is the indicated bp
ladder. Lane 2 is the product of the RT-PCR. Lane 3 is the RT-PCR reaction minus the RT
enzyme.
A
B
% increase of peak IBa
50
-300
-400
Peak Ba
2+
current (pA)
-200
PDBu (200 nM)
-500
isotonic
40
30
20
10
hypotonic
-600
0
0
2
4
6
8
10
12
14
isotonic
+ PDBu
16
Time (min)
D
-350
50
-400
40
% increase of peak IBa
2+
current (pA)
C
Peak Ba
hypotonic
+ PDBu
-450
-500
-550
PDBu (200 nM)
isotonic
30
20
10
hypotonic
-600
0
0
2
4
6
8
10
Time (min)
12
14
16
hypotonic
hypotonic
+ PDBu
Fig. 6. Hypotonic swelling prevented PDBu stimulation of IBa. A, peak currents measured
from a cell treated with PDBu before and after exposure to hypotonic bath solution. B, averaged
percent increase of peak IBa by PDBu before and after hypotonic swelling (n = 9). Values
represent mean ± SE. C, peak currents measured from a cell treated with PDBu after exposure to
hypotonic bath solution. Cell was superfused with isotonic solution and then followed with
hypotonic bath solution as indicated with the horizontal lines. PDBu was added into the
hypotonic bath solution when peak IBa reached a steady state. D, averaged percent increase of
peak IBa by hypotonic swelling with or without PDBu (n = 15).
A
Peak Ba
2+
current (pA)
0
PDBu
control
-50
-100
-150
-200
-250
isotonic
hypotonic
-300
0
2
4
6
8
10
12
Time (min)
B
40
% change of Peak IBa
*
30
20
10
0
-10
PDBu
control
Fig. 7. Overnight pretreatment with PDBu prevented hypotonic swelling-induced
stimulation of Ca2+ channels. A, peak currents measured from a cell pre-treated with 200 nM
PDBu for more 18 hrs and a time-matched control cell. Cell was exposed to isotonic and
hypotonic solutions as indicated by the horizontal lines. B, averaged percent increase after
hypotonic exposure. Values represent mean ± SE for PDBu-pretreated cells (n = 7) and timematched control cells (n = 6). * represents significantly different from the values under isotonic
conditions (p < 0.05).