Institut für Pharmakologie, Freie Universität Berlin, 14195 Berlin, Germany
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ABSTRACT |
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To investigate the
possible role of members of the mammalian transient receptor potential
(TRP) channel family (TRPC1-7) in vasoconstrictor-induced
Ca2+ entry in vascular smooth muscle cells, we studied
[Arg8]-vasopressin (AVP)-activated channels in A7r5
aortic smooth muscle cells. AVP induced an increase in free cytosolic
Ca2+ concentration ([Ca2+]i)
consisting of Ca2+ release and Ca2+ influx.
Whole cell recordings revealed the activation of a nonselective cation
current with a doubly rectifying current-voltage relation strikingly
similar to those described for some heterologously expressed TRPC
isoforms. The current was also stimulated by direct activation of G
proteins as well as by activation of the phospholipase C-coupled
platelet-derived growth factor receptor. Currents were not activated by
store depletion or increased [Ca2+]i.
Application of 1-oleoyl-2-acetyl-sn-glycerol stimulated the current independently of protein kinase C, a characteristic property of
the TRPC3/6/7 subfamily. Like TRPC6-mediated currents, cation currents
in A7r5 cells were increased by flufenamate. Northern hybridization
revealed mRNA coding for TRPC1 and TRPC6. We therefore suggest that
TRPC6 is a molecular component of receptor-stimulated Ca2+-permeable cation channels in A7r5 smooth muscle cells.
transient receptor potential channel; calcium ion influx; receptor-operated channel
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INTRODUCTION |
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IN MAMMALIAN CELLS, stimulation of receptors linked to phospholipase C (PLC) leads to an increase in free cytosolic Ca2+ concentration ([Ca2+]i) mediated by release of Ca2+ from intracellular stores such as the endoplasmic reticulum (ER) and/or Ca2+ influx across the plasma membrane. The signal transduction cascade that links receptor activation to Ca2+ release has been thoroughly investigated (for review, see Ref. 3). In short, receptor stimulation results in PLC-dependent hydrolysis of membrane-bound phosphatidylinositol 4,5-bisphosphate (PIP2) into the soluble messenger inositol 1,4,5-trisphosphate (InsP3) and membrane-bound diacylglycerol (DAG). InsP3 mediates Ca2+ release by opening InsP3 receptor channels in the membrane of the ER Ca2+ stores, whereas DAG couples receptor stimulation to the activation of protein kinase C (PKC). On the other hand, the mechanisms underlying the regulation of Ca2+ entry from the extracellular space are less well understood. Multiple pathways appear to be involved and may operate in parallel (for review, see Refs. 2 and 6). In many cell types, depletion of intracellular Ca2+ stores stimulates Ca2+ influx via a pathway that is commonly referred to as the "capacitative" or "store-operated" Ca2+ entry pathway (for review, see Ref. 38). Important for the concept of store-operated Ca2+ entry is the fact that receptor activation is not an indispensable requirement. Any means of depleting the intracellular Ca2+ pool provides a full and sufficient signal for activation of Ca2+ entry, even in the absence of receptor stimulation or generation of InsP3. The best-characterized current through store-operated channels is the highly Ca2+-selective calcium release-activated current (ICRAC; Ref. 35). However, other less Ca2+-selective channels have also been described. Although current models of PLC-dependent Ca2+ entry mechanisms focus on the capacitative concept, there is increasing evidence that many Ca2+ entry pathways may be activated independently of store depletion by various messengers of the signal transduction cascade such as InsP3, inositol 1,3,4,5-tetrakisphosphate (InsP4), Ca2+, G proteins, DAG, PKC, or arachidonic acid (AA).
Despite intensive research, reliable evidence for the molecular identity of the Ca2+-permeable channels involved in receptor-stimulated Ca2+ entry is still lacking. Mammalian homologues of the Drosophila transient receptor potential (TRP) channel family (TRPC1-7) have been implicated as molecular candidates for channels mediating receptor-stimulated Ca2+ influx (for review, see Ref. 13). The properties and activation mechanisms of all family members have been extensively studied in heterologous expression systems. However, the gating mechanisms of the recombinant mammalian TRPCs remain highly controversial. Whereas some studies have proposed store-operated activation mechanisms for many family members (23, 36, 42, 48, 49), others report the involvement of store-independent pathways in the activation of TRPC2-7 (17, 27, 30, 33, 39), e.g., through direct activation by DAG in the case of the TRPC3/6/7 subfamily of TRP channels (17, 30, 33). The reasons for these contradictory findings remain elusive. The discrepancies could be due to differences in the expression systems used and/or different expression levels of the proteins (22). In contrast to the abundance of reports on TRP channels in heterologous expression systems, relatively little information is available on their role in native tissues. In most studies on the role of TRPCs in native tissues, these channels have been implicated as a component of store-operated channels (1, 9, 10, 29, 31, 37, 45-47). However, a role for TRPCs in receptor-regulated, store-independent channels has also been proposed (26).
Although smooth muscle cells are known to express voltage-activated Ca2+ channels, receptor-activated Ca2+-permeable channels stimulated downstream of PLC activation form an additional, important Ca2+ entry pathway. In A7r5 cells, a clonal cell line derived from embryonic rat thoracic aorta, [Arg8]-vasopressin (AVP) activates Ca2+ influx through store-dependent and -independent pathways (4, 5, 8, 44). The electrophysiological properties of different nonselective cation conductances have been described in these cells (19, 20, 25, 32, 43). In the present study, we investigated receptor-stimulated Ca2+ entry in A7r5 smooth muscle cells and tried to evaluate the role of TRP channels using combined Ca2+ imaging, electrophysiological, and molecular biological techniques. From the results, we propose a major role for TRPC6 in mediating vasopressin-induced Ca2+ entry in A7r5 cells.
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MATERIALS AND METHODS |
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Cell Culture
Vascular smooth muscle cells derived from embryonic rat thoracic aorta (A7r5) were purchased from American Type Culture Collection (Manassas, VA) and grown in 75-cm2 plastic tissue culture flasks (Greiner, Frickenhausen, Germany) at 37°C in a humidified atmosphere (7% CO2) in DMEM (4.5 g/l glucose) supplemented with 10% (vol/vol) fetal bovine serum (Life Technologies, Karlsruhe, Germany), 4 mM L-glutamine (Fluka, Taufkirchen, Germany), 100 U/ml penicillin, and 100 U/ml streptomycin (Biochrom, Berlin, Germany). For recordings, cells were plated onto glass coverslips. All experiments were performed between 1 and 3 days after plating.Molecular Biology
For PCR cloning and Northern blotting, total RNA was purified from A7r5 cells and rat brain and vomeronasal organ (VNO) with TRIzol LS (Life Technologies) according to the standard protocol. For cDNA synthesis, 1-5 µg of total RNA was reverse transcribed with 200 U of Superscript II reverse transcriptase (Life Technologies) and 5 pM primer 5'-CCAGTGAGCAGCAGAGTGACGAGGACTCGAGCTCAAGCTTTTTTTTTTTTTTTTT. The cDNAs were used as templates for PCR amplification of partial cDNA fragments of rat TRPC1-7. The following primer sets were used for amplification of reverse transcripts: rtrpc1, GGGAATTCGCCCCGGCTACACAGAGAAACC (sense)/GGGAATTCGGGAACACTTAAACCTGGGACATT (antisense); rtrpc2, GGGAATTCAGGGCCAGCTGGGTCTCGTG (sense)/GGGAATTCATGCCCCGGTAGGTGTTGATTC (antisense); rtrpc3, GGGAATTCGAACTGGGCATGGGTAACTCAAAG (sense)/GGGAATTCCGTCATGGCTGGTCGTAAAACA (antisense); rtrpc4, GCTGAGCAAAACGCAAACCA (sense)/AATTATTACGTCAAGATGGATGGAAGT (antisense); rtrpc5, GGCATCGCACAGCAGCACTCTAT (sense)/CAGCATGGGCAGCGTGTAAGC (antisense); rtrpc6, CGGTGGTCATCAACTACAATCA (sense)/TCCAAATGATCCAAGTTACCAGTT (antisense); and rtrpc7, TCCCTTTAACCTGGTGCCGAGTC (sense)/ATGTGCGTATGTTGGGGAGGAA (antisense).Cycling conditions were 2 min at 95°C, followed by 10 cycles of
30 s at 95°C, 15 s at 55-65°C, and 60-70 s at
72°C, followed by 20 cycles of 30 s at 95°C, 15 s at
55-65°C, and 60-70 s + 5 s/cycle at 72°C, and a
final extension at 72°C for 7 min. The PCR products were subcloned
into pCR2.1/TOPO or pcDNA3.1/TOPO vectors (Invitrogen, Karlsruhe,
Germany), and the sequences were confirmed by DNA sequencing.
EcoRI, BstX1, or NotI fragments (New England Biolabs, Frankfurt/Main, Germany), depending on the vector used
and on the presence of internal restriction sites, were radiolabeled by
random priming (Prime-It Rmt random primer labeling kit; Stratagene, Amsterdam, The Netherlands) with [-32P]dCTP (NEN Life
Science Products, Zaventem, Belgium) and purified by gel filtration
(ProbeQuant; Amersham Pharmacia Biotech Europe, Freiburg, Germany). For
Northern blotting, 40 µg of total RNA (or purified mRNA in the case
of TRPC1; Oligotex, Invitrogen) from the desired cells or tissues (48 µg of VNO total RNA) was separated by gel electrophoresis on a
formaldehyde-agarose gel and transferred to a nylon membrane
(Hybond-XL, Amersham Pharmacia Biotech Europe, or Tropilon-Plus,
Applied Biosystems, Weiterstadt, Germany). Four to five hours of
prehybridization in UltraHyb hybridization solution (Ambion,
Huntingdon, UK) was followed by overnight hybridization at
42-50°C with the same solution supplemented with 1 × 106 cpm/ml of labeled probe. Blots were washed with
high-stringency washing protocols [twice with 2× standard saline
citrate (SSC)-0.1% SDS at 50°C for 10 min, 60 min in 0.2× SSC-0.1%
SDS at 50°C, twice with 0.1× SSC-0.1% SDS at 55-60°C for 60 min]. Autoradiographs were obtained using a phosphoimaging system
(BAS-reader, Fuji) and exposure times of at least 24 h. As a
control for the integrity and the amount of the transferred RNA, blot
membranes were stained with methylene blue before hybridization.
Furthermore, after hybridization, blots were stripped and rehybridized
with a 888-bp cDNA fragment of rat glyceraldehyde-3-phosphate dehydrogenase.
Fluorometric Measurements of Intracellular Ca2+ Concentration
[Ca2+]i was monitored using the fluorescent Ca2+ indicator fura 2-AM. Cells were loaded in standard extracellular solution (in mM: 140 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 glucose, and 10 HEPES, pH 7.4 with NaOH) supplemented with 4 µM fura 2-AM and 0.005% Pluronic F-127 for 60-120 min at room temperature. Cells were then rinsed with standard extracellular solution and allowed to deesterify for at least 30 min at room temperature. Measurements of [Ca2+]i were performed with an inverted microscope (Axiovert 10; Zeiss, Göttingen, Germany) equipped with an imaging system (T.I.L.L. Photonics, Gräfeling, Germany). The illumination was generated by a xenon arc lamp. The excitation wavelength was alternated between 340 and 380 nm, and the fluorescence emission of selected areas within the A7r5 cell layer was long-pass filtered at 520 nm and recorded with a charge-coupled device camera. After correction for background fluorescence, the fluorescence ratio F340/F380 was calculated. Monochromator settings and data acquisition were controlled by Fucal 5.12 C software (T.I.L.L. Photonics). All experiments were performed at room temperature. Coverslips were mounted in a recording chamber on the microscope stage (chamber volume ~0.5 ml) and continuously superfused at a rate of ~5 ml/min by gravity feed. For Ca2+-free solutions, Ca2+ was replaced by 1 mM EGTA.Patch-Clamp Recordings
For electrophysiological recordings, cells were placed in a recording chamber (chamber volume ~0.5 ml) and continuously superfused with solution by gravity feed at a rate of 3 ml/min. Single cells were voltage-clamped in the whole cell mode (12) with an EPC-7 amplifier and Pulse software (HEKA, Lambrecht, Germany). Patch pipettes were made from borosilicate glass and had resistances of 3-6 MThe standard extracellular solution contained (in mM) 140 NaCl, 5 CsCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES (pH 7.4 with NaOH). Solutions with reduced Ca2+ concentrations were made by adjusting the Ca2+ content to the desired value. For Ca2+-free solutions, Ca2+ was omitted or replaced by 1 mM EGTA. For Na+-free solutions, Na+ was replaced by N-methyl-D-glucamine (NMDG+). In 0 Ca2+-0 Na2+ solutions, Na+ was replaced by NMDG+ and Cs+ and Ca2+ were omitted. Unless otherwise stated, nimodipine (10 µM) was present in the external solutions to block voltage-dependent Ca2+ currents activated by depolarizing ramps. The standard intracellular solution contained (in mM) 110 cesium methanesulfonate, 25 CsCl, 2 MgCl2, 3.62 CaCl2, 10 EGTA, and 30 HEPES (pH 7.2 with CsOH) with a calculated [Ca2+] of 100 nM. In some experiments, Ca2+-free pipette solutions (only 10 mM EGTA, no CaCl2) were used. For experiments with 10 µM Ca2+, the pipette solution contained 0.098 mM CaCl2 and 1 mM N-(2-hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid (HEDTA). All experiments were performed at room temperature (20-25°C).
Chemicals
AVP, flufenamate (FFA), guanosine 5'-O-(3-thiotriphosphate) (GTPStatistics
All data are given as means ± SE. The statistical significance of differences between mean values was assessed with Student's t-test. Differences were regarded as statistically significant for P < 0.05. ![]() |
RESULTS |
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Vasopressin-Induced Ca2+ Signals in A7r5 Cells
To compare the effects of AVP application on [Ca2+]i of single A7r5 cells with previous studies on this cell type (4, 5, 32), we first performed ratiometric imaging experiments with the Ca2+-sensitive dye fura 2. In the presence of extracellular Ca2+, addition of AVP (100 nM) to the extracellular solution elicited a substantial increase in F340/F380 consisting of a fast rise and a slower decline to baseline (Fig. 1A). The slow component of response decay was very heterogeneous in different cells. Some cells exhibited a clear plateau phase (n = 112 of 178; see also Fig. 1A, inset), whereas in others [Ca2+]i returned to baseline monophasically (n = 66 of 178; Fig. 1A, inset). Removal of the agonist did not notably affect the rate of decline. To separate Ca2+ release from Ca2+ entry across the plasma membrane, the effects of Ca2+ removal from the bath solution on response amplitude and kinetics were studied (Fig. 1B). In the absence of extracellular Ca2+, AVP was still able to increase [Ca2+]i, suggesting that Ca2+ release contributed to the observed Ca2+ response (n = 162). However, the response kinetics were changed. The increase in [Ca2+]i was now followed by a rapid return to prestimulation levels. When, in the continued presence of AVP, Ca2+ was added to the bath solution, an increase in F340/F380 was observed (n = 113). Hence, in addition to Ca2+ release, Ca2+ entry was involved in AVP-induced Ca2+ signaling in A7r5 cells. The above findings suggest that AVP increases the plasma membrane permeability for divalent cations in A7r5 smooth muscle cells. AVP-induced divalent cation entry is not mediated by voltage-gated Ca2+ channels because all experiments were performed in the presence of nimodipine (10 µM). Addition or removal of nimodipine did not change the slow component of the response (data not shown).
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Electrophysiological Recordings
Cation currents activated by vasopressin.
Whole cell voltage-clamp recordings were performed to study the nature
of the AVP-stimulated cationic currents underlying the observed
Ca2+ signals. To block currents mediated by voltage-gated
K+ channels and voltage-gated Ca2+ channels,
pipette and bath solutions contained Cs+ rather than
K+ and nimodipine was added to all bath solutions. In
~90% of the cells tested (n = 288), application of AVP
resulted in the rapid, transient activation of a noisy inward current
at a holding potential of 60 mV (Fig.
2A). The I-V
relations of the AVP-induced current had a characteristic doubly
rectifying form (Fig. 2B) similar to those described for
various members of the TRPC family in heterologous expression systems
(17, 18, 24, 33, 39, 41). The currents displayed a
reversal potential of ~0 mV. Replacement of external Na+,
Cs+, and Ca2+ by NMDG+ almost
completely abolished the inward current, whereas outward currents at
positive potentials were either unaffected or slightly increased (Fig.
2, C and D). The Cl
channel
blockers NPPB (50 µM; n = 8) and niflumic acid (100 µM; n = 7) did not block the AVP-induced current (data not
shown). Together, these results suggest that the AVP-induced current
observed in A7r5 cells is a cation current. Because the I-V
relationship of the AVP-induced current is markedly different from
those described for agonist-evoked cation currents in previous studies
on A7r5 cells (19, 20, 25, 43), we characterized the
electrophysiological properties of the AVP-induced current in more
detail. To relate the results to the possible involvement of TRPC
isoforms, we focused on the activation mechanism and current
inhibition.
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Dependence of cation currents on external
Ca2+ concentration
The experiments described above were performed in reduced
external Ca2+ concentration
([Ca2+]o; 50 or 200 µM) because
larger response amplitudes were obtained in these solutions. As
reported for norepinephrine-evoked currents in rabbit portal vein
smooth muscle cells (14), the amplitudes of the
AVP-induced currents in A7r5 cells were highly variable and displayed a
complex dependence on [Ca2+]o. Figure
3A shows a typical experiment
in which the AVP-induced current was first evoked in an external
solution containing 2 mM Ca2+. After the inward current at
60 mV had reached its peak, [Ca2+]o was
decreased to 50 µM. This decrease in
[Ca2+]o resulted in a substantial increase in
current amplitude. On readdition of 2 mM Ca2+ to the
extracellular solution, the current amplitude decreased again. These
data suggest that external Ca2+ inhibits the AVP-induced
cation current. The inhibitory effect of external Ca2+ was
observed at both negative and positive potentials (Fig. 3B). Adding to the complexity of the external Ca2+ dependence of
the AVP-induced current is the fact that when
[Ca2+]o was further decreased (nominally
Ca2+-free solutions or substituting EGTA for external
Ca2+) inward current amplitudes did not further increase
but were slightly decreased [n = 9; Fig. 3C;
current activation by infusion of AlF
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Effects of di- and trivalent cations and SKF-96365 on AVP-induced
currents.
The effects of other di- and trivalent cations commonly used to block
nonselective cation currents were also studied on AVP-induced currents
in A7r5 cells. As can be seen in Fig. 4,
A and B, Gd3+ (100 µM; n
= 4) completely blocked the AVP-induced current at all potentials
whereas a lower concentration of 1 µM was only partially inhibitory
(~40% inhibition at 60 mV; n = 4; data not shown).
Likewise, La3+ (100 µM) completely inhibited AVP-induced
inward currents by 100% (n = 6; data not shown). The
effects of both La3+ and Gd3+ at a
concentration of 100 µM were not readily reversible. Moreover, current noise was completely abolished in the presence of these ions.
In contrast, the divalent cations Ni2+ (1 mM; 80 ± 7% inhibition at
60 mV; n = 5), Co2+ [100
µM (n = 1), 500 µM (n = 2), and 2 mM (n
= 2): ~30%, ~50%, and ~90% inhibition, respectively, at
60 mV], and Mn2+ (2 mM; 92 ± 5% inhibition at
60 mV; n = 5) only partially blocked AVP-induced inward
currents in A7r5 cells (Fig. 4C). Inhibition of AVP-induced
currents was readily reversible on removal of these cations from the
bath solution. AVP-induced outward currents were inhibited to a lesser
extent than the inward currents, suggesting a voltage-dependent block
by these cations. On the other hand, Ba2+ (2 mM; n
= 5; data not shown) and Sr2+ (500 µM or 2 mM;
n = 4; Fig. 4C) did not change current amplitudes when added to the extracellular solution. Furthermore, Sr2+
(2 mM; n = 8) was able to prevent the inhibitory action of
complete external Ca2+ removal on AVP-induced inward
currents (Fig. 3C). Removal of Mg2+, which was
normally present at 1 mM in all extracellular solutions, slightly
augmented AVP-induced currents (Fig. 4D; n = 4),
whereas the I-V relation remained largely unchanged.
SKF-96365 (100 µM), a commonly used organic cation channel blocker,
inhibited AVP-evoked cation currents in A7r5 smooth muscle cells at
60 mV by 98 ± 2% (n = 5; data not shown).
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Single-Channel Conductance
For most members of the TRP channel family, single-channel conductances have been estimated from single-channel recordings or fluctuation analysis (17, 18, 23, 39, 41, 49). We wanted to compare these data with the single-channel properties of the AVP-evoked currents in A7r5 cells. Because of low current densities, we performed fluctuation analysis rather than single-channel recordings. For this purpose, whole cell currents at
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Mechanism of Current Activation
We investigated the mechanism of cation current activation in A7r5 smooth muscle cells in more detail. PLC
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To test for a role of intracellular store depletion in the activation
of AVP-induced cation currents in A7r5 cells, protocols were used that
had previously been shown to activate store-operated channels in other
preparations (7). Infusion of InsP3 (100 µM)
in a Ca2+-free 10 mM EGTA pipette solution did not change
the holding current (n = 9). Similarly, no current
activation could be obtained in solutions containing 100 nM free
Ca2+ buffered with 10 mM EGTA (n = 12; Fig.
7A). However, after 5 min of
InsP3 infusion, 10 of the 19 cells tested
responded to AVP with activation of cation currents. Likewise,
passive depletion of internal Ca2+ stores by addition of
the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)
inhibitor thapsigargin (1 µM; n = 3) did not result in
activation of a cation current in A7r5 cells (data not shown). Current
activation was not impaired by infusion of the InsP3
receptor antagonist heparin (5 mg/ml) for 5 min before AVP application (n = 4; Fig. 7B). Infusion of cells with high
Ca2+ (10 µM) revealed that the cation conductance in A7r5
cells was not Ca2+ activated (n = 7; Fig.
7C). In four cells, AVP was able to induce current
activation after 5 min of 10 µM Ca2+ infusion.
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The TRPC3/6/7 subfamily of TRP channels was shown to be directly
activated by DAG (17, 30, 33), whereas TRPC4 and -5 are
unresponsive to the lipid (39). To assess the role of DAG in cation current activation in A7r5 cells, the membrane-permeant DAG
analog OAG was added to the extracellular solution during whole cell
recordings. As shown in Fig. 8,
A and B, OAG evoked cation currents (n
= 45 of 53) with I-V relations identical to those of
AVP- and AlF
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As for AVP-induced currents, the amplitudes of the currents activated
by 100 µM OAG were highly variable, ranging from 0.1 to 8.9 pA/pF at
60 mV and 200 µM [Ca2+]o. The mean peak
current density in response to 100 µM OAG was 1.4 ± 0.3 pA/pF
(n = 36), a value smaller than, but not statistically significant from, that for AVP-induced currents under identical conditions.
To investigate whether the OAG-stimulated current accounts for all or only part of the AVP-induced current increase, we measured OAG-induced currents followed by the addition of AVP and vice versa. In both cases, the second substance was applied after the response to the first substance had reached its peak. When AVP was applied after OAG, three of seven cells showed no further increase in current on AVP application. The remaining four cells showed a very small increase in current amplitude that did not exceed the peak response to the preceding OAG application (data not shown). When OAG was applied after AVP, some cells showed no additional effect upon OAG application (n = 5 of 11), others displayed a very small increase in current (n = 4 of 11), and two cells exhibited a substantial increase in current amplitude that well exceeded the peak response to the preceding AVP application (data not shown).
We then tested for an involvement of PKC in AVP-induced Ca2+ entry. In fura 2 experiments, preincubation with PMA (500 nM) for several minutes reduced the response amplitudes to AVP applied subsequently (data not shown). PMA alone did not increase [Ca2+]i in A7r5 cells. Hence, activation of PKC, rather than activating Ca2+ influx, inhibits AVP-induced Ca2+ entry. Together, these data support a contribution of the TRPC3/6/7 subfamily of TRP channels to cation currents in A7r5 smooth muscle cells.
AA [100 (n = 8), 50 (n = 5), 20 (n = 2), and 10 (n = 8) µM] was tested for its ability to induce currents in A7r5 cells (data not shown). Experiments were complicated by the fact that AA rendered recordings unstable, leading to loss of the cell between 10 s and 4 min after application. AA did not activate a cation current with a doubly rectifying I-V relation in any of the cells tested.
Effect of FFA on Cation Currents
The nonspecific cation channel blocker FFA has been shown to reversibly enhance currents mediated by murine (m)TRPC6, whereas currents mediated by mTRPC3 and mTRPC7 were inhibited by the drug (18). To discriminate between the members of the TRPC3/6/7 subfamily that may be involved in agonist-induced cation currents in A7r5 cells, the effect of FFA on these currents was studied. FFA (100 µM) increased cation currents induced by infusion of AlFNorthern Hybridization Analysis
To identify the TRP channels expressed in A7r5 cells, we studied the expression of mRNA for TRPC1-7. By Northern hybridization analysis, we found that TRPC6 mRNA was abundantly expressed in A7r5 smooth muscle cells. Transcripts for TRPC2, -3, -4, -5, and -7 could not be detected in these cells, although prominent signals were present in the controls (Fig. 9). In contrast, expression of TRPC1 mRNA was detectable in A7r5 cells (Fig. 9). Detection of TRPC1 was only possible after purification of mRNA from the total RNA used in all other experiments. When total RNA was used, only blurred signals were obtained in A7r5 cells and in controls (data not shown). With purified mRNA, distinct bands were observed in brain and A7r5 cells (Fig. 9).
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For all autoradiographs except that for TRPC2 expression, rat brain RNA served as a positive control. In the case of TRPC2 expression analysis, no signal was detected in rat brain, and thus RNA derived from the rat VNO, in which abundant expression was described previously (27), was used as a positive control.
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DISCUSSION |
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In the present study, we provide evidence for the involvement of TRP channels in AVP-induced nonselective cation currents in A7r5 smooth muscle cells. In light of the combined Ca2+ imaging, electrophysiological, and molecular biological data, we suggest that TRPC6 is a candidate molecular component of the channels mediating these currents.
A number of observations support this conclusion. AVP-induced nonselective cation currents in A7r5 cells were found to display a characteristic doubly rectifying I-V relationship that resembles the I-V relationship described for recombinantly expressed human (h)TRPC3/6 (17, 24), mTRPC7 (33), and mTRPC4/5 (39, 41). In contrast, currents mediated by TRPC1 show a linear I-V relation (28, 40, 49). Activation of the cation current was shown to be dependent on PLC activation. However, neither InsP3 nor elevated [Ca2+]i directly activated the current. Likewise, store depletion protocols failed to activate the current. Current activation was also unaffected by infusion of heparin, ruling out an involvement of InsP3 receptors. Comparable PLC-dependent but store depletion-independent activation mechanisms have been described for mTRPC4/5 (34, 39, 41), hTRPC3/6 (17), and mTRPC7 (33). An important feature of the cation current in A7r5 cells that supports the involvement of TRPCs and provides a pointer to the TRPC isoforms that may be involved is its activation by the membrane-permeant DAG analog OAG. The effects of OAG on A7r5 cells seem to be direct and not mediated by subsequent activation of PKC. PKC-independent activation of TRP channels by application of DAG analogs is a characteristic feature of the TRPC3/6/7 subfamily of TRP channels (17, 33) not shared with the TRPC4/5 subfamily (39).
The fact that in the majority of cells tested AVP- and OAG-induced currents were not additive supports the notion that they are mediated by the same channels. Considering that both substances exert their effect at different sites of the signal transduction cascade leading to current activation (OAG presumably much more distally than AVP), it is not surprising that small additive current increases were seen in some experiments. Furthermore, the fact that only OAG applied after AVP but not vice versa could in rare cases (n = 2 of 11) substantially increase current levels supports the idea that current activation by OAG is more direct than activation by AVP. Hence, we conclude that the AVP-induced current does not consist of components other than those that can be elicited by the application of OAG.
The susceptibility of the cation currents in A7r5 cells to block by lanthanides also supports an involvement of the TRPC3/6/7 subfamily (11, 33) rather than the TRPC4/5 subfamily, for which La3+ has been shown to potentiate currents in two studies (39, 41) but not in another (34). Hence, similarities in the electrophysiological properties and the activation mechanism of recombinantly expressed TRPC3/6/7 and the AVP-induced currents in A7r5 smooth muscle cells strongly suggest that one or more of these TRP channel isoforms is likely to be a molecular component of the endogenous cation channel expressed in A7r5 smooth muscle cells.
Interestingly, the cation currents observed in A7r5 cells were reversibly enhanced by FFA, an inhibitor of most nonspecific cation channels that was recently shown to selectively increase currents mediated by mTRPC6 but to inhibit currents mediated by the other two members of the TRPC3/6/7 subfamily (18). This finding provides a pointer to the possible involvement of TRPC6 in agonist-induced cation currents in A7r5 cells.
The involvement of TRPC6 is also strongly supported by the molecular biological data provided in the present study. Northern hybridization analysis demonstrated that, of the structurally related isoforms TRPC3, -6, and -7 that respond to DAG, only mRNA coding for TRPC6 is expressed in A7r5 cells. In addition to TRPC6, TRPC1 mRNA was present, whereas mRNA for isoforms TRPC2, -4, and -5 was not detectable. By analogy with the transmembrane topology of voltage-gated Na+ and Ca2+ channels, which have four linked domains of six transmembrane segments, it is likely that TRPCs form homo- or heterotetramers. From the data on TRPC isoform expression, together with the activation of currents by OAG and the potentiating effect of FFA, it is likely that the AVP-activated, TRP-like cation channels in A7r5 cells are formed by homomers of TRPC6 or possibly heteromers of TRPC1 and TRPC6. To date, however, there is no evidence that heteromultimerization of TRPC1 and -6 can occur. Furthermore, the electrophysiological properties of the AVP-induced currents in A7r5 smooth muscle cells presented here are strikingly similar to those of currents observed in cells heterologously overexpressing TRPC6 alone, in which homomultimers are presumably formed. For other TRPC isoforms, TRPC1 and TRPC4/5 (41) or TRPC1 and TRPC3 (28), heteromultimers show markedly different current properties compared with homomultimers. On the other hand, a role for TRPC1 alone in cation channel formation in A7r5 cells is unlikely because the electrophysiological properties of TRPC1, i.e., its linear I-V relationship (28, 40, 49) and low single-channel conductance (49), as well as its store depletion-dependent activation mechanism (48, 49), are inconsistent with the properties of the nonselective cation currents in A7r5 cells described in this study.
Interestingly, the current observed in A7r5 smooth muscle cells exhibits a dual dependence on [Ca2+]o. The current is partially inhibited by [Ca2+]o in the physiological range, and the amplitude increases when [Ca2+]o is decreased. On the other hand, complete removal of external Ca2+ does not further potentiate the currents but rather leads to a decrease of the amplitude of inward currents. A similar complex dependence of agonist-evoked cation currents on [Ca2+]o has been reported for norepinephrine-evoked currents in rabbit portal vein smooth muscle cells (14, 18). For recombinant TRP channels, a potentiating effect of decreasing [Ca2+]o has been described for hTRPC1 (28, 40, 49) hTRPC3 (28), and mTRPC7 (33). In contrast, current responses in mTRPC4/5-expressing cells were shown to be increased when extracellular [Ca2+] was raised above physiological levels (34, 39). Likewise, a potentiation of currents mediated by recombinant mTRPC6 in Ca2+-containing versus Ca2+-free solutions was described recently (18).
Although we cannot provide a reliable direct demonstration that the cation channels that mediate the AVP response are permeable to Ca2+, because of the inhibition of cation currents by Ca2+ and other divalent cations, similarities in the properties of the AVP-induced Ca2+ signal and the cation currents suggest that they are mediated by the same channel. Both responses are transient, and they have similar time courses. Previous studies showed that AVP-induced Ca2+ entry in A7r5 cells has two components, a capacitative component and a noncapacitative component (4, 5). The current observed in the present study most closely resembles the latter component, both in its insensitivity to store depletion and in its sensitivity to Gd3+. Like noncapacitative Ca2+ entry, the cation current is inhibited by 100 µM Gd3+ but not by 1 µM Gd3+, which completely and irreversibly inhibits the capacitative component (4). Like Iwasawa et al. (20), we could not detect any additional current component that would correspond to capacitative Ca2+ entry in our A7r5 cells under any of the experimental conditions used. Because the identification of a store-operated current component was not the main focus of our study, we did not try experimental protocols commonly used to reveal store-operated currents like ICRAC. We cannot, therefore, rule out the existence of an additional store-operated current. In our hands, the nonselective cation current was activated by OAG, but we could not demonstrate current activation by AA. This contrasts with the results of Broad et al. (4), who showed that the noncapacitative Ca2+ entry pathway was activated by AA.
The activation of nonselective cation currents by AVP in A7r5 smooth muscle cells has been described in a number of previous studies (19, 20, 25, 32, 43). In all but one early study by Nakajima et al. (32), the currents presented show linear I-V relationships. In this early study by Nakajima et al. (32), however, the reported I-V relation of the AVP-induced current was very similar to that described in the present study. Under the conditions used in our study, we never observed AVP-induced currents with linear I-V relations. Furthermore, one study (19) suggests that several types of nonselective cation channels are activated in A7r5 cells in response to endothelin-1. From our experiments, we have no evidence for multiple components of the cation currents in A7r5 cells. The reasons for the described differences are unclear. Interestingly, other properties of the current or Ca2+ entry pathway, including PLC-dependent but store depletion-independent activation (25, 32), single-channel conductance (32), inhibition by di- and trivalent cations and SKF-96365 (19, 25, 32), and negative feedback of PKC activation (4, 21), are essentially consistent with our findings.
The AVP-induced cation currents in A7r5 cells described in this study show a striking similarity to receptor-activated cation currents in smooth muscle cells from the rabbit portal vein (4, 14-16, 21). These similarities include, for example, I-V relation shape, single-channel conductance, dual dependence on [Ca2+]o and [Sr2+]o, and PKC-independent activation by OAG. During the preparation of this manuscript, Inoue et al. (18) provided convincing evidence that TRPC6 shows similar properties to, and is an important component of, norepinephrine-stimulated cation channels in smooth muscle cells. In contrast to A7r5 cells, portal vein cells, however, also show significant levels of mRNA for TRPC3 and TRPC4. Given these findings and our own results, it can be concluded that TRPC6 plays an important role in agonist-stimulated nonselective cation currents both in vascular smooth muscle cells from portal vein and in muscle cells derived from the embryonic aorta. It will be interesting to determine whether an involvement of TRPC6 in agonist-stimulated Ca2+ influx is a general feature of vascular smooth muscle cells or is restricted to certain vessel types.
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ACKNOWLEDGEMENTS |
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We thank Inge Reinsch and Sabina Naranjo Kuchta for excellent technical assistance and Dr. Torsten Schöneberg and Dr. Angela Schulz for helpful comments on Northern hybridization. We are grateful to Dr. Stephan Klug for providing the rat tissues.
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FOOTNOTES |
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S. Jung is a scholar of the Studienstiftung des deutschen Volkes e.V. This work was supported by grants from the Deutsche Forschungsgemeinschaft (FG341) and Fonds der Chemischen Industrie.
Address for reprint requests and other correspondence: T. Plant, Institut für Pharmakologie, Freie Universität Berlin, Thielallee 69-73, 14195 Berlin, Germany (E-mail: tplant{at}zedat.fu-berlin.de).
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.
First published November 14, 2001; 10.1152/ajpcell.00283.2001
Received 22 June 2001; accepted in final form 12 October 2001.
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