Heart and Circulatory Physiology

TTX-sensitive voltage-gated Na+ channels are expressed in mesenteric artery smooth muscle cells

Roberto Berra-Romani, Mordecai P. Blaustein, Donald R. Matteson


The presence and properties of voltage-gated Na+ channels in mesenteric artery smooth muscle cells (SMCs) were studied using whole cell patch-clamp recording. SMCs from mouse and rat mesenteric arteries were enzymatically dissociated using two dissociation protocols with different enzyme combinations. Na+ and Ca2+ channel currents were present in myocytes isolated with collagenase and elastase. In contrast, Na+ currents were not detected, but Ca2+ currents were present in cells isolated with papain and collagenase. Ca2+ currents were blocked by nifedipine. The Na+ current was insensitive to nifedipine, sensitive to changes in the extracellular Na+ concentration, and blocked by tetrodotoxin with an IC50 at 4.3 nM. The Na+ conductance was half maximally activated at −16 mV, and steady-state inactivation was half-maximal at −53 mV. These values are similar to those reported in various SMC types. In the presence of 1 μM batrachotoxin, the Na+ conductance-voltage relationship was shifted by 27 mV in the hyperpolarizing direction, inactivation was almost completely eliminated, and the deactivation rate was decreased. The present study indicates that TTX-sensitive, voltage-gated Na+ channels are present in SMCs from the rat and mouse mesenteric artery. The presence of these channels in freshly isolated SMC depends critically on the enzymatic dissociation conditions. This could resolve controversy about the presence of Na+ channels in arterial smooth muscle.

  • vascular smooth muscle
  • freshly isolated cells
  • whole cell voltage clamp
  • batrachotoxin

stimulation of vascular smooth muscle cells (SMCs) results in a rise in the cytosolic Ca2+ concentration ([Ca2+]cyt), which triggers contraction. These cells express several distinct types of ion channels and transporters that play a role in regulating [Ca2+]cyt. Ca2+ enters the cell mainly through l-type voltage-gated Ca2+ channels (26). However, other Ca2+ entry pathways include receptor-operated Ca2+ channels (3), store-operated channels (1), and the Na+/Ca2+ exchanger (NCX) (15, 39). Ca2+ entry is a steep function of the membrane potential (VM), due mainly to the voltage dependence of l-type voltage-gated Ca2+ channel open probability (6, 11, 26). Therefore, a small change in VM should have a large effect on Ca2+ entry (26). The primary function of voltage-gated Na+ channels (VGNCs) in most cells is to depolarize the membrane. Thus the presence of these channels in vascular SMCs could have important consequences for cell function. In addition, Na+ influx through VGNCs could raise the cytosolic Na+ concentration ([Na+]cyt) and thereby activate Ca2+ influx via NCX.

The presence of VGNCs in vascular SMCs has been controversial. There are several reports of VGNCs in cultured vascular SMCs (8, 9, 16, 18, 30, 34). VGNCs have been found in freshly dissociated SMC from the rabbit main pulmonary artery (29) and from several veins (24, 25, 27, 28). Two groups of investigators reported that VGNCs were not present in mesenteric artery SMCs from the guinea pig, rabbit, or rat (9, 27). Here, we report for the first time that VGNCs are expressed in freshly dissociated SMCs from the rat and mouse mesenteric artery. A likely explanation for the difference between our findings and those of others (9, 27) is our observation that the presence of VGNCs in freshly isolated SMCs is critically dependent on the enzymatic dissociation conditions. This could explain why these channels have not been detected previously in mesenteric artery SMCs.


Myocyte Isolation

Male C57BL/6NCrlBR mice (3–4 mo old) and male Sprague-Dawley rats (200–250 g) were purchased from Charles River (Wilmington, MA). The mice and rats were euthanized by cervical dislocation. The animal protocol was approved by University of Maryland School of Medicine Institutional Animal Care and Use Committee. The main mesenteric artery was rapidly removed and transferred to a chamber containing ice-cold low-Ca2+ physiological salt solution (PSS) with the following composition (in mM): 140 NaCl, 5.36 KCl, 0.34 Na2HPO4, 0.44 K2HPO4, 10 HEPES, 1.2 MgCl2, 0.05 CaCl2, and 10 glucose, pH 7.2 (adjusted with Tris). The artery was cleaned of fat and connective tissue, and SMCs were isolated using one of the following two dissociation protocols.

Protocol 1.

Before enzymatic dissociation, the artery was placed in fresh low-Ca2+ PSS at 37°C for 30 min. The tissue was digested in low-Ca2+ PSS containing (in mg/ml) 2 collagenase type XI (Sigma-Aldrich; St. Louis, MO); 0.16 elastase type IV (Sigma-Aldrich), and 2 BSA (fat free; Sigma-Aldrich) for 35 min at 37°C. After digestion, the tissue was washed three times with low-Ca2+ PSS at 4°C. This protocol is similar to that used by Janiak et al. (17).

Protocol 2.

The dissociation solution contained (in mM) 55 NaCl, 80 Na glutamate, 6 KCl, 2 MgCl2, 10 HEPES, and 10 glucose, adjusted with NaOH to pH 7.3. Rat or mouse arteries were placed in dissociation solution containing 0.3 mg/ml papain (Worthington Biochemical) and 1 mg/ml DTT at 37°C for 40 or 30 min, respectively. Arteries were then transferred to dissociation solution containing 100 μM CaCl2, collagenase F (0.7 mg/ml, Sigma-Aldrich), and collagenase H (0.3 mg/ml, Sigma-Aldrich) for a period of 8 min for the rat and 6 min for the mouse at 37°C. The arteries were washed several times in dissociation solution at 4°C. The details of this cell isolation protocol have been described previously (31).

A suspension of single cells was obtained after either dissociation protocol by gently triturating the tissue with a fire-polished Pasteur pipette in low-Ca2+ PSS for protocol 1 and using a Ca2+-free dissociation solution for protocol 2. Dispersed cells obtained with both protocols were used immediately or stored at 4°C and used within 4 h. For an experiment, four to five drops of cell suspension were placed on a coverslip and allowed to sit for 10 min at 4°C to permit the cells to adhere. The coverslips were placed in a perfusion chamber mounted on an inverted phase-contrast microscope (Nikon Diaphot; Tokyo, Japan). The cells were superfused at 2 ml/min with external solution. Only cells with elongated morphology were studied.

Electrophysiological Recording

Membrane currents were recorded from freshly dissociated SMCs in the whole cell patch-clamp configuration using an Axopatch 200 patch-clamp amplifier (Axon Instruments; Union City, CA). Fire-polished micropipettes (1–3 MΩ resistance) were manufactured from borosilicate capillary tubing (Garner Glass; Claremont, CA) using a micropipette puller (model P-97, Sutter Instruments; Novato, CA). An Ag-AgCl reference electrode was connected to the bath using an agar salt bridge containing 1 M KCl. The offset voltage was zeroed immediately before seal formation. The pipette was placed in contact with the cell surface, and gentle suction was applied to form a high resistance (1–1.2 GΩ) seal. Additional suction was applied to break the membrane under the pipette tip, allowing low resistance access to the intracellular space. The holding potential was −70 mV in all experiments unless stated otherwise. Leak and capacity transients were subtracted using a P/4 protocol. Series resistance was compensated to give the fastest possible capacity transient without producing oscillations. Membrane currents were recorded with 12-bit analog-to-digital converters (Digidata 1322A, Axon Instruments). Data were sampled at 500 kHz (unless otherwise stated), filtered at 5 kHz with a 902LPF low-pass Bessel filter (Frequency Devices; Haverhill, MA), and stored using personal computers for subsequent analysis. All records were obtained at room temperature (25–26°C).


To isolate inward Na+ current (INa) and Ca2+ current, K+ currents were eliminated using pipettes filled with high-Cs+ solution of the following composition (in mM): 115 CsCl, 2.5 MgCl2, 10 HEPES, and 10 EGTA, with pH adjusted to 7.2 with CsOH. In experiments where the INa reversal potential was determined, 15 mM CsCl was replaced with equimolar NaCl. To record Ca2+ channel currents, the bath solution contained (in mM) 140 NaCl, 2.7 KCl, 10 BaCl2, and 10 HEPES, with pH adjusted to 7.4 with NaOH. In all other experiments, the bath solution contained (in mM) 150 NaCl, 10 HEPES, and 1.8 CaCl2, with pH adjusted to 7.4 with NaOH. In ion substitution experiments, NaCl was replaced with equimolar N-methyl-d-glucamine (NMDG) chloride, and the pH was adjusted with HCl. The osmolarity of all solutions was routinely maintained at 270–300 mosmol/l, measured with a model 5500 vapor pressure osmometer (Wescor; Logan, UT).


TTX was obtained from Calbiochem (San Diego, CA). Batrachotoxin (BTX) was a generous gift from Dr. John W. Daly (National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD). Nifedipine was purchased from Sigma-Aldrich. TTX was dissolved in deionized water, BTX in ethanol, and nifedipine in DMSO.

Data Analysis

Data analysis was performed using pCLAMP software (version 9.0, Axon Instruments). INa peak amplitudes were measured relative to the current level before the pulse. Data are expressed as means ± SE.

The TTX dose-response data were fit to an equation of the form Math(1) where Y is the response (relative INa amplitude), [TTX] is the TTX concentration, and IC50 is the [TTX] that reduces the response to 50% of control.

The data for the voltage dependence of Na+ channel activation or inactivation were fit with a Boltzmann equation of the form Math(2) For inactivation, Y is the fraction of channels not inactivated at any VM (in mV), and constant s equals +1. For activation, Y is the fraction of channels activated at VM and s equals −1. V1/2 is the VM at which the conductance is half-maximal, and k is a constant.


Cell Morphology

Two dissociation protocols, using different combinations of enzymes, were tested in order to find a method that produced viable cells for whole cell patch clamping. Typical rat or mouse SMCs obtained with each protocol are shown in Fig. 1. Mouse cells dissociated with protocol 2 (Fig. 1B) were long, slender, and fusiform. Those dissociated with protocol 1 (Fig. 1A) were somewhat shorter and fatter but still elongated. Similar results were obtained with the rat mesenteric artery (Fig. 1, C and D). With either protocol, SMCs from rat or mouse mesenteric arteries maintained the same morphology described above when they were superfused with external solution containing 1.8 mM Ca2+.

Fig. 1.

Freshly isolated mesenteric artery smooth muscle cells. A and B: mouse cells dissociated with protocol 1 (A) or protocol 2 (B). C and D: rat cells dissociated with protocol 1 (C) or protocol 2 (D). Cells in A and C are in external solution containing 0.05 mM Ca2+. Cells in B and D are in Ca2+-free external solution. The scale bar shown in A applies to A–D.

Inward Currents in Murine Mesenteric SMCs

Inward currents were initially recorded with 10 mM Ba2+ in the external solution to increase the amplitude of L-type Ca2+ channel currents. When single SMCs dissociated from the mouse or rat mesenteric artery with protocol 1 were voltage clamped at 0 mV, a current such as that shown in Fig. 2A was recorded. The inward current consists of two distinct components: an initial fast transient inward current superimposed upon a slowly activating, noninactivating component. Inward currents were recorded from about 95% of the rat or mouse cells isolated with protocol 1; they all had the pattern illustrated in Fig. 2A. Surprisingly, in either rat or mouse cells dissociated with protocol 2, the fast, transient inward current component was absent (Fig. 2B). The fast current was never observed in 26 cells (from 5 different arteries) isolated with protocol 2.

Fig. 2.

Inward currents in mouse mesenteric artery smooth muscle cells dissociated with two different protocols. The currents were generated by 5-ms steps to 0 mV from a holding potential of −70 mV. A: inward current from a cell isolated with protocol 1. B: inward current from cell isolated with protocol 2. C: same cell as in A after the addition of 10 μM nifedipine. D: same cell as in B after the addition of 10 μM nifedipine. The external solution contained 10 mM Ba2+. The dashed line indicates zero current.

Cells that express both voltage-gated Na+ and Ca2+ channels generate inward currents with a time course similar to that shown in Fig. 2A (19, 23). Because arterial SMCs are known to have L-type Ca2+ channels, the effect of the L-type Ca2+ channel blocker nifedipine (10 μM) was tested. As shown in Fig. 2C, nifedipine blocked the slowly activating component but not the fast component in cells dissociated with protocol 1. This implies that L-type Ca2+ channels are responsible for the slowly activating component. In cells dissociated with protocol 2, nifedipine almost completely blocked all the inward current in all cells studied, indicating that only L-type Ca2+ channels were present. The fast component remaining in the presence of nifedipine in cells dissociated with protocol 1 kinetically resembles VGNC current (13). It reached a peak in 0.44 ± 0.02 ms and decayed to zero during the next 2 ms at 0 mV. To identify further the nifedipine-insensitive fast component of the current, we studied its dependence on Na+ and its sensitivity to TTX.

Sodium Dependence of the Fast Current

If the fast component is carried by Na+, its reversal potential should change with the sodium equilibrium potential (ENa). To change ENa, the external Na+ concentration ([Na+]o) was decreased using equimolar substitution with the impermeant cation NMDG. Inward currents recorded during 5-ms steps to 0 mV in the presence of 155, 105, or 55 mM [Na+]o are superimposed in Fig. 3A. Lowering [Na+]o decreased the magnitude of the inward current, suggesting that Na+ is the charge carrier. Current-voltage relationships for the fast current component are shown in Fig. 3B. The current activates at voltages positive to −40 mV, reaches a maximum at about 0 mV, and reverses at 68 mV in 155 mM [Na+]o. The reversal potential of the fast component was shifted in the negative direction as [Na+]o was lowered, and the magnitude of the shift is consistent with the change in ENa. The INa reversal potential in cells superfused with 155, 105, or 55 mM NaCl was 68.0 ± 0.8 (n = 11), 57.8 ± 2.5 (n = 5), and 38.4 ± 2.1 (n = 10) mV, respectively. These reversal potentials are close to the theoretical Nernst equilibrium potentials for Na+ under these conditions: 60, 51, and 33.4 mV, respectively. Thus Na+ is the main charge carrier during the fast, transient current, and this current will be referred to as INa.

Fig. 3.

Fast inward current depends on extracellular Na+ concentration ([Na+]o). A: inward currents were generated by 5-ms steps to 0 mV from a holding potential of −70 mV. Superimposed are 3 currents recorded from the same cell in the presence of 155, 105, or 55 mM external Na+. To change [Na+]o, external Na+ was replaced with an equimolar concentration of N-methyl-d-glucamine. B: currents were generated by 5-ms steps to a range of potentials from −50 to +120 mV. The peak amplitude of the current is plotted as a function of membrance potential (VM) in the presence of 155, 105, or 55 mM Na+.

TTX Sensitivity of INa

Specific subtypes of VGNCs differ in their sensitivity to block by TTX (37). The superimposed current traces shown in Fig. 4A show the effect of increasing the concentration of TTX from 1 to 10 nM on INa in mesenteric artery SMCs. The addition of 1 nM TTX decreased the amplitude of the inward current by about 23%, and inward current was almost abolished at a TTX concentration of 10 nM. Removal of TTX from the bath solution restored INa completely within a few minutes (not shown). The relationship between TTX concentration and INa amplitude, averaged for three to five cells from six different arteries, is shown in Fig. 4B. The smooth curve is the fit of Eq. 1 to the data, with an IC50 at 4.3 nM. Thus the VGNCs in mesenteric artery SMCs have a high affinity for TTX.

Fig. 4.

Na+ current (INa) is blocked with high affinity by TTX. A: currents were generated by 5-ms steps to 0 mV. Superimposed are currents from the same cell in the absence of TTX (control) and after the addition of 1 or 10 nM TTX. The current amplitude decreases in a concentration-dependent manner. Dashed lines represent the zero-current level. B: dose-response relationship for TTX inhibition of INa. The peak magnitude of INa was measured during a 5-ms step to 0 mV. The amplitude of INa, expressed as a percentage of the control INa recorded in the absence of TTX, is plotted against TTX concentration. Data points are means ± SE; n = 3–6. The continuous curve was obtained by fitting the data to Eq. 1, with IC50 = 4.3 nM.

Steady-State Activation and Inactivation

VGNCs activate and then inactivate as VM becomes progressively more positive. A plot of the normalized Na+ conductance (gNa) as a function of VM shows the voltage dependence of activation (Fig. 5). gNa was calculated from the following equation: Math(3) where INa is the peak magnitude of the current generated by a 5-ms step to VM and Vrev is the INa reversal potential. The gNa vs. VM curve indicates that VGNCs begin to open when VM is positive to −40 mV.

Fig. 5.

Steady-state voltage dependence of voltage-gated Na+ channel (VGNC) activation and inactivation. For activation, conductance (gNa) was calculated as described in the text, normalized relative to its maximum value (gNa,max), and plotted as a function of VM (○). For inactivation, a 2-s prepulse to a VM in the range of −120 to −20 mV was used to inactivate the channels and was followed by a 5-ms step to 0 mV. INa during the test pulse is a measure of noninactivated channels, and its value (normalized to the maximum INa at negative prepulse potentials) is plotted as a function of the prepulse VM (▪). Data are plotted as means ± SE; n = 5. The smooth curves are fits of a Boltzmann distribution (Eq. 2) to the data. For activation, VM at which conductance is half-maximal (V1/2) = −15.7 mV and the constant (k) = 6.8 mV. For inactivation, V1/2 = −52.9 mV and k = 7.5 mV.

The voltage dependence of VGNC inactivation was determined using a double-pulse protocol. The VM was initially stepped to a level in the range of −120 to −20 mV for 2 s (“the prepulse”) to inactivate the channels. The prepulse was followed by a 5-ms test pulse to 0 mV, and the INa during the test pulse was used as a measure of the channels that were not inactivated. A plot of the normalized test pulse current vs. VM shows the steady-state voltage dependence of inactivation (Fig. 5). VGNCs begin to inactivate at about −80 mV and become fully inactivated at about −20 mV. The overlap between the activation and inactivation curves in Fig. 5 suggests the possibility of Na+ channel “window current,” i.e., there is a narrow range of membrane potentials, from about −40 to −20 mV, where some VGNCs could be open in the steady state.

Effects of BTX on INa in Mesenteric Artery SMCs

A number of lipid-soluble toxins, such as BTX and veratridine, bind to receptors on VGNCs and alter channel gating such that the channels open at a more negative VM and they do not inactivate (36). If these toxins open VGNCs in arterial SMCs in a similar way, the channels could be opened at the normal resting potential to investigate if they play any role in arterial SMC function. Because BTX binds preferentially to the open conformation of VGNCs (35), BTX binding was facilitated by applying a train of voltage-clamp steps (to +40 mV for 5 ms) at a frequency of 20 Hz for 5 min.

Na+ channel gating was modified by 1 μM BTX in three significant ways. First, BTX shifted the activation of the Na+ channels in the hyperpolarizing direction (Fig. 6). After BTX modification, inward current was clearly present at −50 mV (Fig. 6A, right), a potential at which no current was present in control cells (Fig. 6A, left). In BTX, INa reached its maximum value at about −30 mV, a potential at which current was barely detectable under control conditions in the absence of BTX. The peak amplitudes of both the control and BTX-modified currents are plotted as functions of VM in Fig. 6B. The control INa began to activate at −30 mV, and the BTX-modified INa became evident at −50 mV. To analyze further the voltage shift for the activation of modified channels, gNa was calculated for both normal and modified channels; gNa is plotted as a function of VM in Fig. 7. The gNa-VM curve was shifted about 27 mV in the hyperpolarizing direction by BTX. The midpoint of the gNa-VM relationship shifted from −10.7 mV in control to −37.9 mV in the presence of BTX.

Fig. 6.

Batrachotoxin (BTX) modifies the gating of VGNCs in mesenteric artery smooth muscle cells. A: typical whole cell INa recorded in control conditions (left traces) and in the presence of 1 μmol/l BTX (right traces). The traces show currents evoked by 5-ms steps to −50, −30, −10, and +10 mV. B: current-voltage relationships for the same cells as in A. Data points show peak amplitudes of INa elicited by 5-ms steps to potentials from −90 to +50 mV and are plotted as a function of VM. Holding potential was −100 mV.

Fig. 7.

BTX shifts the relationship between gNa and VM in the hyperpolarizing direction. The conductance was calculated from the current-voltage relationships shown in Fig. 6B for control and in the presence of BTX. The smooth curves are fits of a Boltzmann equation to the data. For control, V1/2 = −10.7 mV and k = 9.1 mV. For BTX, V1/2 = −37.9 mV and k = 9.0 mV.

A second effect of BTX was to decrease Na+ channel inactivation. In control SMCs, INa inactivates when VM is depolarized (Fig. 6A, left). BTX almost completely abolished this fast inactivation of INa at all potentials (Fig. 6A, right).

A third effect of BTX was to slow Na+ channel deactivation kinetics significantly. Under control conditions, Na+ channels deactivate rapidly: at −70 mV, the deactivation time constant (τd) was 0.18 ± 0.02 ms (n = 11). After BTX modification of the channels, τd at −70 mV was 1.56 ± 0.40 ms (n = 4; Fig. 8).

Fig. 8.

BTX slows Na+ channel deactivation kinetics. A: in control conditions, VGNCs were opened by a 0.5-ms step to 0 mV from a holding potential of −100 mV. A subsequent step to a VM in the range of −90 to −50 mV was used to close the channels. The tail current recorded during the second step was fit with an exponential, and the time constant of the exponential (τd) was used as a measure of deactivation kinetics. The pulse protocol is shown as the solid line at the top. B: after VGNCs are modified with 1 μM BTX, the tail currents at a VM in the range of −70 to −110 mV are much slower than those in controls. All of the current with BTX was blocked by 200 nM TTX (BTX + TTX). C: τd plotted as a function of VM in control medium and in medium containing 1 μM BTX.


INa Dependence on Isolation Protocol

This report reveals that mouse SMCs from the mesenteric artery dissociated with protocol 1 exhibit both INa and Ca2+ current. In contrast, cells dissociated with protocol 2 exhibit only Ca2+ currents, as shown by the ability of nifedipine to block almost completely the inward current in these cells. The precise cause(s) of this discrepancy is unknown but does not seem to be correlated with the different saline solutions used in protocols 1 and 2. When collagenase and elastase were used with the saline from protocol 2, INa as well as Ca2+ current also were observed (data not shown). This suggests that the results are more likely related to differences in the proteolytic actions of the enzymes employed for dissociation.

We observed differences in the morphology of the cells obtained with protocols 1 and 2. SMCs dissociated with protocol 2 (Fig. 1, B and D) were long, slender, and fusiform, and those dissociated with protocol 1 were shorter and fatter (Fig. 1, A and C). A possible explanation for these morphological differences is that in protocol 2 the cell dispersion was performed in Ca2+-free solution where the cells were fully relaxed. In contrast, in protocol 1, the Ca2+ concentration in the dispersion solution was 0.05 mM; at this concentration, during dispersion, a small amount of Ca2+ could enter the cells, thereby causing partial contractions. Despite these morphological differences, we obtained similar Ca2+ currents in cells isolated with both protocols, indicating that the cells were not drastically damaged by either enzyme cocktail. The fact that only INa was absent in cells dissociated with protocol 2 suggests that Na+ channels could be more sensitive to certain enzymatic conditions than other voltage-gated channels. This could explain the controversial data about the absence of INa in the mesenteric artery (9, 27) and maybe in other types of SMCs. Our results suggest that some dissociation protocols may have subtle detrimental effects on the cells that may not normally be recognized. This may clearly alter the cell physiology and influence data interpretation.

INa Characteristics

The present investigation is, to our knowledge, the first report of the presence of voltage-gated INa in mesenteric artery SMCs under voltage-clamp conditions. V1/2 is approximately −16 mV, and the half-inactivation voltage is approximately −53 mV. These values are more positive than those reported for cardiac (4, 20, 32), and nerve cells (13) but similar to or slightly more positive than those reported in various vascular SMC types (18, 25, 29). The reversal potential of INa was 38.4 mV (Fig. 3B) in cells dialyzed with 15 mM NaCl and superfused with 55 mM NaCl, close to the theoretical ENa calculated with the Nernst equation under these conditions (+33.4 mV). Increasing the [Na+]o to 105 and 155 mM increased the reversal potential to 57.8 and 68.0 mV (Fig. 3B), respectively. This was ∼8 mV more positive than the Nernst reversal potential calculated for these conditions (+51 and +60 mV, respectively). This small discrepancy between experimental and theoretical reversal potentials could be due to an unsubtracted component of the capacitative current. This component may interfere with the accurate measurement of INa especially at high command potentials where the channel kinetics are very fast.

TTX Sensitivity

The marine guanidinium toxin TTX has played a crucial role in the study of VGNCs. Na+ channels in excitable tissues have been categorized as TTX sensitive or insensitive. TTX-sensitive channels, found in nerve and adult skeletal muscle, are blocked by nanomolar concentrations of the toxin with an apparent Kd ∼ 1–10 nM (10). In contrast, TTX-insensitive Na+ channels in cardiac muscle require a much higher concentration of TTX for block (∼10−5 M) (22). In our experiments, the Kd for TTX block of Na+ channels in the mouse mesenteric artery was 4.3 nM. Similar Kd values have been reported in other types of vascular SMCs: 8.7 nM in the pulmonary artery (29), 8 (30) and 6.5 nM (18) in the coronary artery, and 3.15 (24) and 10 nM (28) in the portal vein. A lower affinity for TTX was reported in cultured neonatal azygous veins (Kd = 30 μM) (34). These data suggest that different subtypes of VGNCs may be present in different vascular SMCs, although it seems possible that preparation methods might alter the TTX binding site.

BTX Effects

BTX, a steroidal alkaloid toxin extracted from the skin of the Colombian frog Phyllobates terribilis (2), is one of the most potent and specific activators of Na+ channels. BTX shifts the voltage dependence of activation to more negative potentials, disables the inactivation process, and alters the channel conductance and selectivity. As a result, Na+ channels open persistently and irreversibly in the presence of BTX, even at the resting VM (7, 14).

Shinjoh et al. (33) report that BTX induces endothelium-independent contractions in the isolated rat aorta, suggesting that the influx of Na+ depolarized the plasma membrane and thereby activated voltage-gated Ca2+ channels to cause contraction. To our knowledge, however, there are no reports of the effects of BTX on INa under voltage-clamp conditions in vascular SMCs. In this study, we report that 1 μM BTX profoundly changes Na+ channel behavior in mesenteric artery SMCs. The conductance-voltage relationship was shifted by 27 mV in the hyperpolarizing direction, and inactivation was almost completely abolished. Together, the results obtained with TTX and BTX clearly reveal the presence of “classic” VGNCs in mesenteric artery SMCs.

Functional Role of INa

The primary function of VGNCs in most cells is to depolarize the membrane and generate the upstroke of the action potential. Intracellular microelectrodes have been used in the mesenteric artery to demonstrate the presence of action potentials (12, 21, 38). In all cases, the action potentials appear to be generated by Ca2+ current: 1) action potentials induced by K+ channel blockade with tetraethylammonium ion were insensitive to changes in [Na+]o (12); 2) action potential amplitude was increased by raising extracellular Ca2+ concentration, and the action potentials were blocked by the L-type Ca2+ channel blockers verapamil, Mn2+ (12), and nifedipine (21); and 3) action potentials were not affected by TTX (12, 21, 38).

A physiological role of INa in vascular SMCs has been suggested, however, by experiments on aortas and coronary arteries. Veratridine and BTX cause a gradual contraction in rat aorta rings (33). This suggests that activation of Na+ channels in SMCs induces a contraction that is probably mediated by depolarization and Ca2+ influx through voltage-gated Ca2+ channels. Recently, Boccara et al. (5) demonstrated the presence of a TTX-sensitive INa in primary cultured human coronary myocytes using the patch-clamp technique. In addition, they measured [Na+]cyt with sodium-binding benzofuran isophthalate and [Ca2+]cyt with fura-2. They reported that 1) veratridine increases [Na+]cyt and 2) veratridine, toxin V from Anemonia suculata, and N-bromoacetamide all increase [Ca2+]cyt. These effects were blocked by TTX or by the absence of extracellular Na+ and Ca2+. The Ca2+ channel blocker nicardipine partially blocked the effect of veratridine, and replacement of external Na+ with Li+ blocked the residual component. They concluded that INa regulates [Ca2+]cyt via voltage-gated Ca2+ channels and, to a lesser extent, via the NCX. These studies suggest an important role for INa in the excitation-contraction coupling processes in vascular SMCs. Na+ channels could play a similar role in mesenteric artery SMCs because the voltage dependence of INa activation and steady-state inactivation indicates a “window” current between −40 and −20 mV (Fig. 5), where some Na+ channels would be open in the steady state.

Molecular Species of VGNCs in Vascular Smooth Muscle

INa has been identified in several types of vascular SMCs. Nevertheless, the presence of specific Na+ channel genes in vascular tissue has only recently been determined. Jo et al. (18) showed abundant expression of SCN9A in cultured cells isolated from the human bronchus, main pulmonary artery, and coronary artery. The Na+ channel types expressed in the mesenteric artery remain unknown. Our results demonstrate that VGNCs in mesenteric artery SMCs bind TTX with high affinity. Therefore, the low TTX-affinity SCN5 channels normally expressed in cardiac myocytes do not appear to be involved in mesenteric artery SMCs.

In conclusion, the present data reveal the presence of VGNCs in rat and mouse mesenteric artery SMCs. The current is insensitive to nifedipine, sensitive to changes in [Na+]o, and blocked by TTX (100 nM); its gating is modified by BTX. INa characteristics were similar to those described in other vascular SMCs. An especially noteworthy observation is that the TTX-sensitive Na+ channels appear to be very sensitive to the conditions used to dissociate the SMCs.


This study was supported by National Heart, Lung, and Blood Institute Grant HL-45215.


We thank Drs. Mark T. Nelson, Adrian D. Bonev, and Sean M. Wilson for providing detailed information about cell dissociation protocols, Drs. Edson X. Albuquerque and John W. Daly for samples of BTX, and Dr. Daniel Weinreich and Stacey McCulle for technical advice and assistance.


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