Introduction

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Carvedilol, an antihypertensive agent, possesses the pharmacological properties of -adrenoceptor blocking action and direct vasodilating action (Ruffolo et al., 1990). A series of investigations revealed that this agent possesses multiple actions, including those characteristic of a potent antioxidant, such as inhibition of superoxide release from human neutrophils and suppression of lipid peroxidation in rat brain homogenates (Yue et al., 1992), cardioprotective action (Feuerstein and Ruffolo, 1996), and inhibition of vascular smooth muscle cell proliferation (Ohlstein et al., 1993). Carvedilol is also reported to have neuroprotective activity via inhibition of glutamate release and the N-methyl-D-aspartate receptor (Lysko et al., 1992, 1994). However, it has not been reported whether/how carvedilol affects ion channels in cardiac, vascular, or neuronal cells.

Voltage-dependent Na⁺ channels are responsible for the rising phase of the action potential in the membranes of neurons and most electrically excitable cells (for review, see Catterall, 1992). Na⁺ channels consist of the principal -subunit, which may be associated with a noncovalently attached ₁-subunit, and a disulfide-linked ₂-subunit (Klugbauer et al., 1995; Catterall, 2000). The -subunits issued from a large multigene family contain the ion-pore and the toxin binding sites, i.e., site 1 for tetrodotoxin (TTX) and saxitoxin (STX), site 2 for veratridine, site 3 for -scorpion toxin, site 4 for -scorpion toxin, and site 5 for Ptychodiscus brevis toxin-3 (PbTx-3) (Wada et al., 1992; Catterall, 2000). Structures of ₁-subunit are similar among various tissues, but ₂-subunit is cloned only in the brain (Isom et al., 1995).

In adrenal medullary cells (Yamamoto et al., 1996, 1997), the Na⁺ channel -subunits are homologous to the human neuroendocrine-type Na⁺ channel -subunit (hNE-Na) (Klugbauer et al., 1995), and the Na⁺ channel ₁-subunits are structurally similar to that of rat brain (Oh and Waxman, 1994). Bovine adrenal medullary Na⁺ channels share many physiological and pharmacological properties with those of the human cells (Wada et al., 1985, 1992). In the cells, veratridine caused catecholamine secretion, which was dependent on veratridine-induced ⁴⁵Ca²⁺ influx via voltage-dependent Ca²⁺ channels as well as ²²Na⁺ influx via voltage-dependent Na⁺ channels (Wada et al., 1985). In the regulation of Na⁺ channel expression, cyclic AMP-dependent protein kinase (protein kinase A) (Yuhi et al., 1996) or activation of insulin receptors (Yamamoto et al., 1996) increased the cell surface density of Na⁺ channels without elevating Na⁺ channel mRNA levels. In contrast, chronic treatment with antiepileptic valproic acid increased both the density and mRNA of Na⁺ channels (Yamamoto et al., 1997).

In a previous study (Morita et al., 1989), carvedilol inhibited the secretion of catecholamines induced by various secretagogues, by its stabilizing action on the plasma membranes rather than by its blocking action on Ca²⁺ influx in bovine adrenal medullary cells. The aim of the present study was to investigate whether carvedilol directly interferes with Na⁺ influx mediated through voltage-dependent Na⁺ channels in cultured bovine adrenal medullary cells. In addition, the effects of prolonged exposure to carvedilol on the density of cell surface Na⁺ channel proteins and levels of Na⁺ channel mRNA were also evaluated in adrenal medullary cells. As a result, we found that carvedilol acutely reduces the functional activity of Na⁺ channels, followed by an increase in cell surface Na⁺ channels, without any change in levels of mRNA.

	Experimental Procedures

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Materials. Chemicals used were obtained from the following sources: Eagle's minimum essential medium (MEM), Nissui Pharmaceutical, Tokyo, Japan; calf serum, Nacalai Tesque, Kyoto, Japan; brefeldin A (BFA), cycloheximide, TTX, carbachol, veratridine, -scorpion venom (Leiurus quinquestriatus quinquestriatus), and -scorpion venom (Centruroides sculpturatus), Sigma-Aldrich, St. Louis, MO; PbTx-3, Latoxan, Westbury, NY; and collagenase, Nitta Zerachin Inc., Osaka, Japan. Carvedilol was kindly donated from Daiichi Pharmaceutical Co. Ltd., Tokyo, Japan. Plasmids containing hNE-Na (Klugbauer et al., 1995) and rat brain Na⁺ channel ₁-subunit cDNA (Oh and Waxman, 1994) were generously donated by Dr. F. Hofmann (Technischen Universität München, Munich, Germany) and Dr. Y. Oh (University of Alabama, Birmingham, AL), respectively.

Isolation and Primary Culture of Adrenal Medullary Cells and Drug Treatment. Bovine adrenal medullary cells were isolated by collagenase digestion and maintained in Eagle's MEM, containing 10% calf serum and antibiotics (4 × 10⁶ cells/dish; Falcon 35-mm dish), as reported previously (Yanagihara et al., 1994). To remove nonchromaffin cells, such as fibroblasts or epithelial cells, the differential plating procedure was used, and the final cell preparation contained at least 80 to 90% chromaffin cells. The cells were cultured under 5% CO₂/95% air in a CO₂ incubator and used for experiments within 3 to 5 days of culture. For prolonged exposure to carvedilol, cells were incubated with or without 10 µM carvedilol in Eagle's MEM containing 10% calf serum for the various periods indicated. After treatment, the cells were washed three times with 1 ml of Krebs-Ringer phosphate buffer (see below) and used for the experiments. There was not a significant change in cell viability between control cells and cells treated with carvedilol. Carvedilol was dissolved in dimethyl sulfoxide (DMSO). To avoid the possible influence of DMSO on cells, all reaction media, including the control media, were standardized at a final concentration of 0.25% DMSO.

²²Na⁺ and ⁴⁵Ca²⁺ Influx. Oxygenated Krebs-Ringer phosphate (KRP) buffer was used throughout. It had the following composition: 154 mM NaCl, 5.6 mM KCl, 1.1 mM MgSO₄, 2.2 mM CaCl₂, 0.85 mM NaH₂PO₄, 2.15 mM Na₂HPO₄, and 10 mM glucose, adjusted to pH 7.4. Cells were incubated with 1.5 µCi of ²²NaCl (6-17 Ci/mmol; PerkinElmer Life Sciences, Boston, MA) or 1.5 µCi of ⁴⁵CaCl₂ (0.5-2.0 Ci/mmol; Amersham Biosciences UK, Ltd., Little Chalfont, Buckinghamshire, UK) at 37°C for 5 min with or without various secretagogues, - and -scorpion venoms, and/or PbTx-3 in KRP buffer. After reaction, the cells were quickly washed four times with 1 ml of ice-cold KRP buffer. Then, the cells were solubilized with 1 ml of 10% Triton X-100. Radioactivity of ²²Na⁺ and ⁴⁵Ca²⁺ in the cells was counted by an Aloka ARC-2005 gamma counter (Aloka Co., Ltd., Tokyo, Japan) and a Beckman LS-7000 liquid scintillation counter (Beckman Coulter Inc., Fullerton, CA), respectively. The influx of ²²Na⁺ and ⁴⁵Ca²⁺ was calculated from the initial specific radioactivity of these ions in the incubation medium.

Catecholamine Secretion. Cells were incubated at 37°C for 5 min with or without carvedilol in the presence or absence of 100 µM veratridine (final volume 1.0 ml). After the reaction, the incubation medium was immediately transferred to a test tube containing 4 ml of ice-cold 0.5 M perchloric acid. Catecholamines (norepinephrine and epinephrine) secreted into the incubation medium were adsorbed to aluminum hydroxide and estimated by the ethylenediamine condensation method with a fluorescence spectrophotometer (Hitachi 650-10S; Hitachi Ltd., Tokyo, Japan) with an excitation wavelength of 420 nm and an emission of 540 nm.

[³H]STX Binding. Cells were washed with ice-cold KRP buffer and incubated with 1 to 25 nM [³H]STX (20-40 Ci/mmol; Amersham Biosciences) in 1 ml of KRP buffer at 4°C for 15 min in the absence (total binding) or presence (nonspecific binding) of 1 µM TTX. The cells were immediately washed, solubilized in 10% Triton X-100, and counted for radioactivity to determine specific binding, which was calculated as the total binding minus nonspecific binding. A mere addition of carvedilol to the binding assay medium per se did not alter [³H]STX binding.

mRNA Isolation and Electrophoresis. Total cellular RNA was isolated from cells treated with or without carvedilol by acid guanidine thiocyanate-phenol-chloroform extraction, using TRIzol reagent (Invitrogen, Carlsbad, CA). Poly(A)⁺ RNA was purified using Oligotex-dT30<Super> (Nippon Roche Co., Ltd., Tokyo, Japan), electrophoresed on a 1% agarose gel containing 6.3% formaldehyde in the running buffer [40 mM 3-(N-morpholino)propanesulfonic acid, pH 7.2, 0.5 mM EDTA, and 5 mM sodium citrate], then transferred to a nylon membrane (Hybond-N; Amersham Biosciences) in 20× saline-sodium citrate (SSC; 1× SSC = 0.15 M NaCl and 0.015 M sodium citrate) overnight, and cross-linked using a UV cross-linker (Funakoshi, Tokyo, Japan).

Northern Blot. cDNA fragments of hNE-Na (435-2666) and ₁-subunit (457-790), as well as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1.1 kb pair) were labeled with [-³²P]deoxycytidine 5'-triphosphate (>4000 Ci/mmol; Amersham Biosciences) using the BcaBEST labeling kit (Takara, Kyoto, Japan). The membrane was prehybridized and hybridized with hNE-Na probe at 42°C for 18 h in 6× SSC, 10× Denhardt's solution (2% bovine serum albumin fraction V, 2% polyvinylpyrrolidone, and 2% Ficoll 400), 50% formamide, 0.5% SDS, and 50 µg/ml salmon sperm DNA; it was washed at 65°C in 2×, 1×, and 0.2× SSC containing 0.1% SDS, each for 30 min twice, and subjected to autoradiography. The same membrane was sequentially hybridized to probes for ₁-subunit and GAPDH (BD Biosciences Clontech, Palo Alto, CA) after it was thoroughly washed in 0.1% SDS at 100°C to remove the former probe. Autoradiogram was quantified by a BAS 2000 bioimage analyzer (Fujifilm, Tokyo, Japan).

Statistical Analysis. Data are expressed as mean ± S.D. The statistical evaluation of the data was performed by analysis of variance, followed by post hoc test. Values were considered statistically different when P was less than 0.05. Student^'s t test was used when two means of group were compared.

	Results

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Effect of Carvedilol on Veratridine-Induced ²²Na⁺ Influx in Cultured Bovine Adrenal Medullary Cells. Veratridine (100 µM), an activator of voltage-dependent Na⁺ channels, caused the influx of ²²Na⁺ (42.5 ± 3.3 nmol/10⁶ cells) (Fig. 1A). Carvedilol reduced veratridine-evoked ²²Na⁺ influx in a concentration-dependent manner (IC₅₀ = 1.8 µM). A significant reduction by carvedilol was observed at 1 µM, and a maximal reduction was observed at 100 µM. The basal ²²Na⁺ influx was not changed by carvedilol at any concentration used.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Effects of carvedilol on veratridine-induced 22Na+ influx in cultured bovine adrenal medullary cells. Cultured cells were incubated for 5 min in KRP buffer containing 22Na+ (1.5 µCi) and 100 µM veratridine in the presence of increasing concentrations of carvedilol (A), or with increasing concentrations of veratridine in the presence () or absence () of 10 µM carvedilol (B). Cells were then washed and solubilized, and influx of 22Na+ to the cells was measured. Basal values obtained at 37°C were subtracted. Data are mean ± S.D. of three to five separate experiments carried out in triplicate. , P < 0.001 versus veratridine alone.

Effect of Carvedilol on Veratridine-Induced ⁴⁵Ca²⁺ Influx and Catecholamine Secretion. As shown in Fig. 2, veratridine produced ⁴⁵Ca²⁺ influx (1.25 ± 0.05 nmol/10⁶ cells) and catecholamine secretion (1.05 ± 0.04 µg/10⁶ cells), respectively. Carvedilol (1-100 µM) also attenuated veratridine-induced ⁴⁵Ca²⁺ influx (IC₅₀ = 1.9 µM) and catecholamine secretion (IC₅₀ = 2.3 µM) in a concentration-dependent manner similar to that of ²²Na⁺ influx. Carvedilol did not affect any basal responses.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Effects of carvedilol on 45Ca2+ influx (A) and catecholamine secretion (B) induced by veratridine. Cultured cells were incubated for 5 min with 45Ca2+ (1.5 µCi) and increasing concentrations of carvedilol in the presence of 100 µM veratridine. Then, influx of 45Ca2+ to the cells and catecholamines secreted in the incubation medium were assayed. Basal values were subtracted. Data are mean ± S.D. of three to five separate experiments carried out in triplicate. , P < 0.05; , P < 0.01; , P < 0.001 versus veratridine alone.

Effect of Carvedilol on Carbachol-Evoked ²²Na⁺ Influx and High K⁺-Evoked ⁴⁵Ca²⁺ Influx. Carbachol (0.1 mM), an activator of nicotinic acetylcholine receptor-ion channels, caused ²²Na⁺ influx (43.3 ± 1.3 nmol/10⁶ cells). Carvedilol at 3 to 100 µM significantly reduced carbachol-evoked ²²Na⁺ influx in a concentration-dependent manner (IC₅₀ = 4.3 µM) (Fig. 3A). On the other hand, 56 mM K⁺, an activator of voltage-dependent Ca²⁺ channels, stimulated the influx of ⁴⁵Ca²⁺ (2.12 ± 0.13 nmol/10⁶ cells) (Fig. 3B). Carvedilol at concentrations of 0.1 to 10 µM had no effect on 56 mM K⁺-induced ⁴⁵Ca²⁺ influx. Only at 100 µM did carvedilol slightly suppress ⁴⁵Ca²⁺ influx caused by 56 mM K⁺. The basal ²²Na⁺ influx and ⁴⁵Ca²⁺ influx were not changed by carvedilol at any concentrations used.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Effects of carvedilol on carbachol-evoked 22Na+ influx (A) and 56 mM K+-evoked 45Ca2+ influx (B). Cultured cells were incubated for 5 min with increasing concentrations of carvedilol and 0.1 mM carbachol (A) or 56 mM K+ (B) in the presence of 22Na+ and 45Ca2+, respectively. Influx of 22Na+ or 45Ca2+ to the cells was measured. Basal values were subtracted. Data are mean ± S.D. of four to five separate experiments carried out in triplicate. , P < 0.05; , P < 0.001 versus carbachol or 56 mM K+ alone.

Effect of Prolonged Exposure to Carvedilol on Veratridine-Induced ²²Na⁺ Influx in Combination with Either - or -Scorpion Venom and/or PbTx-3. We examined whether/how prolonged exposure to carvedilol may alter ²²Na⁺ influx induced by veratridine, using several distinct classes of toxins of Na⁺ channels (Wada et al., 1992). Cells were treated for 12 h with or without 10 µM carvedilol, washed three times with 1 ml of KRP buffer, and used for ²²Na⁺ influx assay. -Scorpion venom, which binds to site 3 between segment (S) 3 to 4 of domain IV (Rogers et al., 1996), enhanced ²²Na⁺ influx evoked by veratridine, a toxin acting at site 2 of S6I (Trainer et al., 1996) (Fig. 4). Similarly, -scorpion venom, which interacts with site 4 (Catterall, 1992), and PbTx-3, which binds to site 5 (Trainer et al., 1994) between S5IV and S6I, augmented veratridine-induced ²²Na⁺ influx. Furthermore, PbTx-3 in the presence of either - or -scorpion venom enhanced ²²Na⁺ influx induced by veratridine to a greater extent than did either toxin/venom alone. Pretreatment with carvedilol, however, rather suppressed veratridine-induced ²²Na⁺ influx. Furthermore, veratridine-induced ²²Na⁺ influx was also decreased in the presence of - or -scorpion venom and/or PbTx-3 in cells pretreated with carvedilol compared with nontreated cells. Nevertheless, these four toxins still enhanced veratridine-induced ²²Na⁺ influx even in the cells treated with carvedilol.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Effects of prolonged exposure to carvedilol on 22Na+ influx caused by veratridine, - or -scorpion venom, and PbTx-3. Cells were treated with (closed column) or without (open column) 10 µM carvedilol for 12 h. After treatment, the cells were washed three times with 1 ml of KRP buffer and then incubated with 22Na+ (1.5 µCi) at 37°C for 5 min in the presence (+) or absence () of 30 µM veratridine, 100 µg/ml -scorpion venom, 100 µg/ml -scorpion venom, and 1 µM PbTx-3. Basal values of 22Na+ influx without any toxin or venom were subtracted from the data. Data are mean ± S.D. of three separate experiments carried out in triplicate.

Effect of Prolonged Exposure to Carvedilol on Cell Surface [³H]STX Binding. As shown in Fig. 4, prolonged exposure to carvedilol reduced the functional activity of Na⁺ channels that showed pharmacological properties similar to that of nontreated cells. Next, we examined the cell surface expression of Na⁺ channels by using [³H]STX binding. Surprisingly, when cells were treated with 10 µM carvedilol for 3 to 72 h, increases in [³H]STX binding were found between 3 and 72 h and reached a plateau at 12 h with the maximal increase of 61.2% (Fig. 5A). Carvedilol also increased [³H]STX binding in a concentration-dependent manner (Fig. 5B).

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Effect of prolonged exposure to carvedilol on [3H]STX-specific binding. After treatment of cells for the indicated periods with () or without () 10 µM carvedilol (A), or for 12 h with increasing concentrations of carvedilol (B), cells were washed three times with 1 ml of KRP buffer and incubated at 4°C for 15 min with 25 nM [3H]STX in the presence or absence of 1 µM unlabeled TTX. Data are mean ± S.D. of three separate experiments. , P < 0.05; , P < 0.01; , P < 0.001 versus nontreated cells.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Scatchard plot of [3H]STX-specific binding. Binding was assayed with 1 to 25 nM [3H]STX in the presence or absence of 1 µM unlabeled TTX after treatment of the cells with () or without () 10 µM carvedilol for 12 h. Scatchard plot of [3H]STX binding to the cells. Data are typical of three independent experiments, with similar results. B/F, bound/free.

Effect of Carvedilol Treatment on Na⁺ Channel - and ₁-Subunit mRNA Levels. From the results of Scatchard analysis (Fig. 6), carvedilol-induced increase of [³H]STX binding may implicate an up-regulation of Na⁺ channel proteins through its transcriptional events. Therefore, we estimated the steady-state levels of Na⁺ channel - and ₁-subunit mRNAs in cells that had or had not been treated with 10 µM carvedilol for 0, 1, 3, 6, 12, and 24 h. cDNA probes for hNE-Na and ₁-subunit hybridized, respectively, to one major -subunit mRNA (9.4 kb) and to a single ₁-subunit mRNA (1.5 kb), as reported previously (Oh and Waxman, 1994; Klugbauer et al., 1995; Yamamoto et al., 1997). When levels of - and ₁-subunit mRNAs were normalized against those of GAPDH mRNA, densitometric analysis revealed that treatment by carvedilol did not significantly change the levels of -subunit mRNA (102 ± 4, 100 ± 4, 99 ± 4, 100 ± 5, and 106 ± 7% of control) (n = 3) and ₁-subunit mRNA (98 ± 5, 100 ± 4, 102 ± 7, 102 ± 3, and 105 ± 3% of control) (n = 3) at 1, 3, 6, 12, and 24 h, respectively (data not presented).

Effects of Cycloheximide, BFA, H-7, and H-89 Treatment on Carvedilol-Induced Increase in [³H]STX Binding. We used various inhibitors of translational or post-translational events of membrane proteins to know which step(s) on carvedilol-induced increase in [³H]STX binding is affected by carvedilol. Treatment with 10 µg/ml cycloheximide, an inhibitor of protein synthesis, abolished per se [³H]STX binding stimulated by carvedilol (Fig. 7). BFA is an inhibitor of guanine nucleotide-exchange protein for ADP-ribosylation factor 1, a monomeric GTPase of the Ras superfamily, and blocks vesicle-mediated externalization of newly synthesized proteins from the trans-Golgi network (Moss and Vaughan, 1995; Morinaga et al., 1997). Exposure to 10 µg/ml BFA lowered basal [³H]STX binding by 50.4% but nullified the rise of [³H]STX binding caused by carvedilol. Furthermore, to test an involvement of protein kinases in the carvedilol effect, we used H-7 and H-89 as inhibitors of protein kinase C and cyclic AMP-dependent protein kinase, respectively. H-7 (100 µM) did not affect the stimulatory effect of carvedilol, whereas H-89 (30 µM) completely suppressed it.

View larger version (24K): [in this window] [in a new window]   Fig. 7.   Effects of cycloheximide, brefeldin A, H-7, and H-89 on carvedilol-induced increase of [3H]STX binding. Cultured cells were pretreated for the first 0.5 h with or without cycloheximide (CHX), BFA, H-7, or H-89. Then, they were incubated for an additional 12 h with (closed column) or without (open column) 10 µM carvedilol in the presence or absence of brefeldin A (10 µg/ml), cycloheximide (10 µg/ml), H-7 (100 µM), or H-89 (30 µM) and subjected to [3H]STX binding assay. Data are mean ± S.D. of three separate experiments. , P < 0.05; , P < 0.01; , P < 0.001 versus carvedilol-treated cells in control group.

	Discussion

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Suppression of Na⁺ Channel Activity by Carvedilol. In the present study, we demonstrated that carvedilol (1-100 µM) reduced ²²Na⁺ influx evoked by veratridine in cultured bovine adrenal medullary cells (Fig. 1A). Our study also provides evidence that the reduction of veratridine-induced ²²Na⁺ influx by carvedilol was not reversed by increasing concentrations of veratridine (Fig. 1B), suggesting that carvedilol does not share common binding sites (site 2) with veratridine. Carvedilol decreased the veratridine-induced ⁴⁵Ca²⁺ influx and catecholamine secretion similar to that for the ²²Na⁺ influx (Fig. 2). In our previous study, veratridine-induced Na⁺ influx mediated through Na⁺ channels increased Ca²⁺ influx via activation of voltage-dependent Ca²⁺ channels and produced the exocytotic secretion of catecholamines (Wada et al., 1985). Thus, it appears that carvedilol reduces veratridine-induced Na⁺ influx which, in turn, suppresses Ca²⁺ influx through voltage-dependent Ca²⁺ channels and finally produces the reduction of catecholamine secretion.

Up-Regulation of Na⁺ Channels by Carvedilol Treatment. Prolonged exposure of adrenal medullary cells to carvedilol enhanced [³H]STX binding, which reached a plateau at 12 h with the increase of 61% (Fig. 5). Scatchard analysis revealed that carvedilol raised the number of [³H]STX binding sites without altering the K_d value (Fig. 6).