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Baicalin, a flavonoid from Scutellaria baicalensis Georgi, activates large-conductance [Ca.sup.2+] -activated [K.sup.+] channels via cyclic nucleotide-dependent protein kinases in mesenteric artery.

ABSTRACT

Baicalin isolated from Scutellaria baicalensis is a traditional Chinese herbal medicine used for cardiovascular dysfunction. The ionic mechanism of the vasorelaxant effects of baicalin remains unclear. We investigated whether baicalin relaxes mesenteric arteries (MAs) via large-conductance [Ca.sup.2+]-activated [K.sup.+] ([BK.sub.Ca]) channel activation and voltage-dependent [Ca.sup.2+] channel (VDCC) inhibition. The contractility of MA was determined by dual wire myograph. [BK.sub.Ca] channels and VDCCs were measured using whole-cell recordings in single myocytes, enzymatically dispersed from rat MAs. Baicalin (10-100 [micro]M) attenuated 80 mM KCl-contracted MA in a concentration-related manner. L-NAME (30 [micro]M) and indomethacin (10 [micro]M) little affected baicalin (100 [micro]M)-induced vasorelaxations. Contractions induced by iberiotoxin (IbTX, 0.1 [micro]M), Bay K8644 (0.1 [micro]M) or PMA (10 [micro]M) were abolished by baicalin 100 [micro]M. In MA myocytes, baicalin (0.3-30 [micro]M) enhanced [BK.sub.Ca] channel activity in a concentration-dependent manner. Increased [BK.sub.Ca] currents were abolished by IbTX (0.1 [micro]M). Baicalin-mediated (30 [micro]M) [BK.sub.Ca] current activation was significantly attenuated by an adenylate cyclase inhibitor (SQ 22536, 10 [micro]M), a soluble guanylate cyclase inhibitor (ODQ, 10 [micro]M), competitive antagonists of cAMP and cGMP (Rp-cAMP, 100 [micro]M and Rp-cGMP, 100 [micro]M), and cAMP- and cGMP-dependent protein kinase inhibitors (KT5720, 0.3 [micro]M and KT5823, 0.3 [micro]M). Perfusate with PMA (0.1 [micro]M) abolished baicalin-enhanced [BK.sub.Ca] currents. Additionally, baicalin (0.3-30 [micro]M) reduced the amplitude of VDCC currents in a concentration-dependent manner and abolished VDCC activator Bay K8644-enhanced (0.1 [micro]M) currents. Baicalin produced MA relaxation by activating [BK.sub.Ca] and inhibiting VDCC channels by endothelium-independent mechanisms and by stimulating the cGMP/PKG and cAMP/PKA pathways.

[C]2010 Elsevier GmbH. All rights reserved.

ARTICLE INFO

Keywords:

Baicalin

Mesenteric artery

[BK.sub.Ca] channels

Patch clamp electrophysiology

Protein kinases

Introduction

The root of Scutellaria baicalensis Georgi (common name, Huang-qin) has been extensively used as a herbal therapy to treat cardiovascular dysfunction (Huang et al. 2005). However the exact mechanisms by which Huang-qin alters vascular reactivity remain unclear. Given the recent increases in the use of herbal medicines by all societies, understanding the possible mechanisms of action of Hung-qin is an urgent demand and may prove future clinic impact. Huang-qin has a multitude of pharmacological activities including antihypertensive (Tang and Zhou 1958), anti-inflammatory (Huang et al. 2006), antithrombotic (Kubo et al. 1985), antioxidant (Su et al. 2000), antihyperlipidemic (Huang et al. 2005), and anticarcinogenic effects (Chan et al. 2000). Huang-qin is also known to contain numerous flavone derivatives, including baicalin, baicalein and wogonin (Sekiya and Okuda 1982). Among them, baicalin (5,6-Dihydroxy-flavone-7-O-glucuronide, Fig. 1) is known to have excellent antiinflammatory effects (Lin and Shieh 1996; Lo et al. 2005) and provides potent free radical scavenging and xanthine oxidase inhibition (Shieh et al. 2000), thus improving endothelial function and conferring cardiovascular protective effects (Woo et al. 2005; Chang et al. 2007). One of previous reports showed that baicalin (1-100 [micro]M) and its aglycone baicalein (1-50 [micro]M) potentiated the MA contractile response to phenylephrine through inhibition of nitric oxide formation and/or release from the endothelium (Tsang et al. 2000). By contrast, high concentrations of baicalein (100-300 [micro]M) reduced the phenylephrine-induced tone in endothelium-intact vessels (Tsang et al. 2000). Baicalein (30-300 [micro]M) also reduced the protein kinase C (PKC)-mediated MA contractions in endothelium-denuded vessels (Chen et al. 1999). In this study, we found for the first time that baicalin (10-100 [micro]M) produced direct relaxation in KCl-contracted MA and we further investigated the ionic mechanism by which it produces relaxation.

[FIGURE 1 OMITTED]

Large-conductance [Ca.sup.2+]-activated [K.sup.+] ([BK.sub.Ca]) channels play a key role in regulating smooth muscle contractility and controlling the diameter of resistance arteries (Nelson and Quayle 1995). Previous reports showed that the functional role of [BK.sub.Ca] channels is enhanced in arterial smooth muscle during chronic hypertension. A similar phenomenon occurs throughout the vasculature, including the mesenteric artery (Asano et al. 1993), cerebral vascular beds (Paterno et al. 1997), and the aorta (Rusch et al. 1992). Therefore, increased [BK.sub.Ca] channel function in arterial smooth muscle cells may provide a protective mechanism against progressive increases in blood pressure.

The cAMP and the cGMP pathways are major regulators of smooth muscle contractility. It is widely accepted that activation of [BK.sub.Ca] channels via cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) contributes to relaxation (Zhou et al. 2001). Likewise, agents that elevate cAMP and cGMP have also been shown to modulate [BK.sub.Ca] channels and initiate vascular relaxation (Bayguinov et al. 2001; Zhou et al. 2001). Increases in cAMP and cGMP simultaneously activate PKA and PKG pathways resulting in the opening of [BK.sub.Ca] channels. In contrast, since PKC is known to inhibit [BK.sub.Ca] channel activity (Jaggar et al. 2000; Bayguinov et al. 2001), any activation of PKA or PKG would be likely to reduce PKC activation and increase [BK.sub.Ca] activity (Jaggar et al. 2000). These protein kinases interact with each other in modifying channel activity. Physiologically, increasing the activity of the [BK.sub.Ca] channel would result in the closure of voltage-dependent [Ca.sup.2+] channels (VDCCs) by membrane hyper-polarization (Nelson and Quayle 1995). Thus, [BK.sub.Ca] channels appear not only to integrate the output from several signaling cascades but also arrest VDCC activation to relax SMCs.

The main objective of this study was to investigate whether, and by what signaling and ionic mechanisms, baicalin relaxes resistance MA. To the best of our knowledge, this study provides the first evidence that the underlying mechanisms of baicalin-induced MA relaxation could be due to [BK.sub.Ca] channel activation and VDCC inhibition.

Materials and Methods

Chemicals

Baicalin (95% purity), Bay K8644, collagenase type la, dithioery-thritol, hyaluronidase, iberiotoxin (IbTX), KT5720, KT5823, [N.sup.[omega]]-nitro-L-arginine methyl ester (L-NAME), l,3-dihydro-l-[2-hydro-xy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one (NS1619), lH-[l,2,4]oxadiazolo[4,3-a]-quinoxalin-l-one (ODQ), papain, phorbol 12-myristate 13-acetate (PMA), Rp-cAMP, Rp-cGMP, 9-(terahydro-2-furanyl)-9H-purin-6-amine (SQ 22536) and tetraethylammonium chloride (TEA) were obtained from the Sigma-Aldrich Chemical Co. (St Louis, MO). All drugs and reagents were dissolved in distilled water unless otherwise noted. Baicalin, KT 5823, NS1619, ODQ and PMA (10 mM) were dissolved in dimethylsulf-oxide; indomethacin (10 mM) was dissolved in ethanol. Serial dilutions were made in phosphate-buffered solution.

Animal procedures and tissue preparations

All procedures and protocols were approved by the Animal Care and Use Committee at the Kaohsiung Medical University. Female Sprague-Dawley rats (200-250 g) were sacrificed with an overdose of urethane (2 g [kg.sup.-1]) by the intraperitoneal route. MAs were carefully removed and placed in cold oxygenated Krebs solution (in mM) 137 NaCl, 5.6 KCl, 1.8 [CaCl.sub.2], 1 [MgCl.sub.2], 4.17 [NaHCO.sub.3], 0.44 [NaH.sub.2][PO.sub.4], 0.42 [Na.sub.2][HPO.sub.4], 10 HEPES and 5 Glucose (pH 7.4). A segment of MA was dissected free of fat and connective tissue, and then cut into 2-3 mm long rings for isometric tension measurement or preparation of isolated arterial smooth muscle cells.

Contractile tension recordings

Resistance MA rings (~250 [micro]m internal diameter) were fitted with two stainless steel wires (40 [micro]m internal diameter) and mounted in a dual-channel Mulvany-Halpern myograph (DMT A/S, Model 410A, Aarhus, Denmark) for measurement of isometric tension. The rings were equilibrated with a resting tension of 5 mN for 90 min. After equilibrium, MA rings were contracted with a depolarizing concentration of KGl (80 mM), which was considered 100% of contraction. All experiments were performed in endothelium-intact arteries. Endothelium function was verified by the presence of relaxation response (> 70%) to acetylcholine (1 [micro]M) contracted by phenylephrine (10 [micro]M) as described previously (Wu et al. 2001). To determine whether baicalin-induced vasorelaxation involved nitric oxide synthase (NOS) or prostanoid related mechanisms, some rings were co-treated with baicalin (100 [micro]M) and L-NAME (30 [micro]M) or baicalin and indomethacin (10 [micro]M). Next, to evaluate whether baicalin (100 [micro]M)-induced vasorelaxation modulated the channel activity, it was added after [BK.sub.Ca] channel inhibitor iberiotoxin (IbTX, 0.1 [micro]M), VDCC activator Bay K8644 (0.1 [micro]M) or PKC activator PMA (10 [micro]M) induced MA contractions.

Preparation of mesenteric artery smooth muscle cells

SMCs from rat MAs were enzymatkally isolated. In brief, arterial segments were placed in a warm (37 [degrees]C) cell isolation medium containing (in mM) 137 NaCl, 5.6 KCl, 0.1 [CaCl.sub.2], 1 [MgCl.sub.2], 4.17 [NaHCO.sub.3], 0.44 [NaH.sub.2][PO.sub.4], 0.42 [Na.sub.2][HPO.sub.4], 10 HEPES and 5 glucose with 5 mg/ml albumin (pH 7.4) for 10 min. After this equilibration step, arterial segments were initially incubated (37 [degrees]C) in [Ca.sup.2+]-Free isolation medium, papain (0.3 mg [ml.sup.-1]) and dithioerythritol (1 mg [ml.sup.-1]) for 30 min. This was followed by a second incubation (37 [degrees]C) in isolation medium containing [Ca.sup.2+] (0.1 mM), collagenase type Ia (0.3 mg [ml.sup.-1]) and hyaluronidase (1 mg [ml.sup.-1]) for 15 min. After enzyme treatment, the tissue was washed at least three times in ice-cold isolation medium and triturated with a fire-polished pipette to release the myocytes. Cells were stored in ice-cold isolation medium for use on the same day.

Patch clamp electrophysiology

Ionic currents were recorded using conventional patch-clamp techniques at room temperature as described previously (Wu et al. 2005, 2007a, 2007b). In brief, mesenteric artery smooth muscle cells (MASMCs) were placed in a recording dish and perfused with a bath solution. Electrodes were made from borosilicate-thin wall filament capillary tubes (G150TF-4, Warner Instruments) using a Micropipette Puller (PP-S3, Narishige Instruments, Japan) and had a resistance of 3-7 M[OMEGA] when filled with internal solutions. Cells were subsequently voltage clamped (0 mV). Membrane currents were recorded on an Axopatch 700A amplifier (Axon Instruments, Union City, CA), filtered at 1 kHz using a low-pass Bessel filter, digitized at 5 kHz and stored on a computer for subsequent analysis with Clampfit 9.0. A 1 M NaCl-agar salt bridge between the bath and the Ag-AgCl reference electrode was used to minimize offset potentials.

Under [BK.sub.Ca] current recording, the bath solution contained (in mM) 60 NaCl, 80 Na-Gluconate, 5 KCl, 1.8 [CaCl.sub.2], 1 [MgCl.sub.2], 10 HEPES and 10 glucose (pH 7.4, NaOH). The pipette solution contained (in mM) 110 K-gluconate, 30 KCl, 0.5 [MgCl.sub.2], 1 EGTA, 5 HEPES, 5 [Na.sub.2]ATP and 1 GTP (pH 7.2, KOH). To study the [BK.sub.Ca] current, we inactivated [K.sub.v] channels by step depolarization to 0 mV. Under these conditions, [BK.sub.Ca] becomes the dominant outward current. The net current-voltage (I-V) relationship was determined at 5 min intervals by measuring the peak current at the end of the 300 ms pulse to voltages between 0 and +70 mV for [BK.sub.Ca] currents. To investigate whether the sGC/cGMP/PKG or AC/cAMP/PKA signal was involved in the baicalin-induced increases in [BK.sub.Ca] current, the whole cell configuration patch clamp was used.

Before each experiment, voltage-clamped cells were equilibrated for 10 min. Following equilibration, whole-cell [BK.sub.Ca] currents were monitored in the presence and absence of baicalin (30 [micro]M), or IbTX (0.1 [micro]M). MASMCs were preincubated for 15 min with SQ 22536 (10 [micro]M), ODQ (10 [micro]M), KT5720 (0.3 [micro]M), KT5823 (0.3 [micro]M), Rp-cAMP (100 [micro]M), Rp-cGMP (100 [micro]M) or PMA (0.1 [micro]M) before baicalin (30 [micro]M) was added. SQ 22536, ODQ, KT5720, KT5823 and PMA were continuously superfused in the bath, while Rp-cAMP and Rp-cGMP were added to the pipette solution. The effects of baicalin and various drug treatments were measured in the same cell. To measure voltage-dependent [Ca.sup.2+] currents (VDCCs), [K.sup.+] (140 [micro]M) inside the pipette solution was replaced with an equimolar CsCl and pH was adjusted to 7.2 with CsOH, whereas the bathing solution contained tetrodotoxin (1 [micro]M) and tetra-ethylammonium chloride (10 mM) (Wu et al. 2009). To evoke whole-cell [Ca.sup.2+] currents, cells were clamped at -40 mV with single pulse 10 mV and the currents were recorded in the presence and absence of baicalin (30 [micro]M) or baicalin (30 [micro]M) with Bay K8644 (0.1 [micro]M).

Measurement of cAMP and cGMP

Isolated MAs were prepared to determine the levels of cAMP and cGMP as previously described (Wu et al. 2001). Briefly, MAs were incubated with baicalin (10, 30, 100 [micro]M) in 95% [O.sub.2] and 5% [CO.sub.2] bubbled Krebs solution for 1 hr. After the incubation, the MAs were extracted with 1 ml of 0.1 N HC1. The concentrations of cAMP and cGMP in supernatant were measured using cAMP and cGMP EIA kits, following acetylation of the samples as described by the manufacturer (Assay Designs, Inc. Ann Arbor, MI).

Data analysis and statistics

Data are expressed as means [+ or -] SE, n indicating the number of cells. Repeated measures analysis of variance (ANOVA) compared values at a given voltage. When appropriate, Dunnett's test or Tukey Kramer pairwise comparison was used for post hoc analysis. P < 0.05 was considered statistically significant.

Results

Endothelium-independent vasodilation effects of baicalin

In isolated MA, the maximal tension obtained from 80 mM KCl was considered 100% of contraction (22.2 [+ or -] 3.2 mN, n = 9) in comparison with the basal tension (5 mN). The endothelium was verified by acetylcholine as described in Methods (Wu et al. 2001). Baicalin at concentrations [less than or equal to] 10 [micro]M had no significant effects on 80 mM KG-contracted MA. Baicalin at 30 and 100 [micro]M attenuated KCl-contracted MA in a concentration-dependent manner. The [BK.sub.Ca] channel activator NS1619 (100 [micro]M) also reduced KCl-induced MA contractions as a reference compound (Fig. 2A). To clarify the role of endothelium in baicalin-induced relaxation, the nitric oxide synthase (NOS) inhibitor L-NAME (30 [micro]M) and the cyclooxygenase (COX) inhibitor indomethacin (10 [micro]M) were used. Indomethacin or L-NAME per se did not affect KCl-induced MA contractions. Baicalin(100 [micro]M)-induced MA relaxation had little effect by L-NAME or indomethacin (Fig. 2B), and showed no significant changes from endothelium removal (data not shown), suggesting that it is endothelium-independent.

[FIGURE 2 OMITTED]

Role of [BK.sub.Ca] and [Ca.sup.2+] channels and PKC in baicalin-induced MA relaxation

To establish that baicalin-induced MA relaxation involved the [BK.sub.Ca] and [Ca.sup.2+] channels, the [BK.sub.Ca] channel inhibitor iberiotoxin (IbTX, 0.1 [micro]M) and the [Ca.sup.2+]-channel activator Bay K8644 (0.1 [micro]M) (Wu et al. 2009) were used. Baicalin (100 [micro]M) reversed IbTX-induced contractions (from 64.5 [+ or -] 12.5% to -11.4 [+ or -] 8% n = 6, P < 0.001 compared with IbTX) and nearly abolished Bay K8644-induced MA contractions (from 81 [+ or -] 8.2% to 8.4 [+ or -] 10% n = 6, P < 0.001 compared with Bay K8644) (Figs. 2C and D). The MA contraction induced by the PKC activator PMA (10 [micro]M) was also abolished by baicalin (from 73.1 [+ or -] 14.1% to 0.9 [+ or -] 4.8% n = 6, P < 0.001 compared with PMA) (Figs. 2C and D). Taken together, the results suggest that baicalin-induced MA relaxation could be mediated by modulating the [BK.sub.Ca] and [Ca.sup.2+] channels and PKC pathway.

Activation of [BK.sub.Ca] currents by baicalin

The regulation of outward [BK.sub.Ca] conductance by baicalin was measured using the conventional whole-cell patch clamp. Openings of [BK.sub.Ca] channels were identified based on characteristic single-channel conductance and blocked by IbTX as previously described (Jaggar et al. 2000). Rat MASMCs were voltage clamped at 0 mV (Fig. 3A) to inactivate voltage-dependent [K.sup.+] currents, and continuously superfused with an isotonic physiological solution containing 1.8 mM [Ca.sup.2+] [+ or -] baicalin (0.3, 3, 30 [micro]M). Concentration-dependent increases in outward currents by baicalin (+70 mV; 0.3 [micro]M: 5.6 [+ or -] 0.37 pA [pF.sup.-1]; 3 [micro]M: 6.8 [+ or -] 0.4 pA [pF.sup.-1]; 30 [micro]M: 8.1 [+ or -] 0.5 pA [pF.sup.-1], n = 5, P < 0.05 compared with control) (Figs. 3B and C) were inhibited by IbTX (+ 70 mV; 0.3 [micro]M: 1.4 [+ or -] 0.8 pA [pF.sup.-1]; 3 [micro]M: 1.6 [+ or -] 0.7 pA [pF.sup.-1]; 30 [micro]M: 2 [+ or -] 0.7 pA [pF.sup.-1], n = 5) (Figs. 3B and D). The IbTX-sensitive current was obtained from the difference in baicalin-enhanced outward current in the absence and presence of IbTX (Fig. 3E). These results ascertained that [BK.sub.Ca] channel activation may involve baicalin-induced vasorelaxation.

[FIGURE 3 MITTED]

Baicalin activates [BK.sub.Ca] currents via AC/cAMP- and sGC/cGMP-dependent mechanisms

To further investigate the signaling mechanisms of [BK.sub.Ca] channel activation, baicalin (30 [micro]M) was applied to voltage-clamped cells in the presence of ODQ a soluble guanylate cyclase (sGC) inhibitor. ODQ (10 [micro]M) attenuated baicalin-induced increases in [BK.sub.Ca] activity, indicating that the modulatory effect of this compound involved the sGC/cGMP pathway. Baicalin-enhanced [BK.sub.Ca] activity was also attenuated by the adenylate cyclase (AC) inhibitor SQ 22536 (10 [micro]M) (Fig. 4). Combination of ODQ and SQ 22536 abolished the enhancement of the [BK.sub.Ca] current by baicalin (Fig. 4). This result suggests that baicalin appears to modulate [BK.sub.Ca] channels through both the sGC/cGMP and AC/cAMP pathways.

[FIGURE 4 OMITTED]

Baicalin activates [BK.sub.Ca] currents via PKG and PKA pathways

Baicalin (30 [micro]M) enhanced [BK.sub.Ca] channel activity, which was attenuated in the presence of cAMP- and cGMP-dependent protein kinase inhibitors KT5720 (0.3 [micro]M) and KT5823 (0.3 [micro]M) (Fig. 5). Combination of KT5823 and KT5720 abolished the baicalin-enhanced [BK.sub.Ca] current (Fig. 5). Further experiments revealed that Rp-cAMP (100 [micro]M in the pipette), a competitive antagonist of cAMP, or Rp-cGMP (100 [micro]M in the pipette), a competitive antagonist of cGMP, fully prevented the baicalinenhanced [BK.sub.Ca] activity (Fig. 6). Moreover, the accumulation of cAMP and cGMP in MAs by baicalin at 10, 30, and 100 [micro]M were examined. Baicalin did not significantly affect cAMP and cGMP levels at 10 [micro]M, but it increased cGMP contents at 30 [micro]M. Both cAMP and cGMP were significantly increased by baicalin at 100 [micro]M (Table 1). Altogether, these results indicate that the PKA- and PKG-dependent signaling pathways are involved in the activation of [BK.sub.Ca] channels by baicalin.

[FIGURE 5 OMITTED]

[FIGURE 6 OMITTED]
Table 1

Baicalin enhanced cGMP and cAMP levels in MAs.

           Baicalin               Control          10 [micro]M

cGMP
(pmole [mg.sup.-1] protein)  22.7 [+ or -] 1.3  25.5 [+ or -] 1.6

cAMP
(nmole [mg.sup.-1] protein)  4.1 [+ or -] 0.2   4.1 [+ or -] 0.3

          Baicalin              30 [micro]M         100 [micro]M

cGMP
(pmole [mg.sup.-1] protein)  34.2 [+ or -] 3.1 *  42.4 [+ or -] 4.5 *

cAMP
(nmole [mg.sup.-1] protein)  4.3 [+ or -] 0.4     5.4 [+ or -] 0.3 *

Data shown are the means of three independent duplicate determinations.
* Significant difference from control (p < 0.05).


Baicalin activates [BK.sub.Ca] currents involved the PKC pathway

The PKC activator PMA (0.1 [micro]M) significantly attenuated [BK.sub.Ca] currents and this can be reversed by baicalin (30 [micro]M) (Fig. 7). These results suggest that [BK.sub.Ca] channels are modulated by baicalin in addition to cyclic nucleotide-dependent protein kinases, also mediated by a transduction process sensitive to PKC.

[FIGURE 7 OMITTED]

Inhibition of voltage-dependent [Ca.sup.2+] channels by baicalin

The experiment was conducted with a [Cs.sup.+]-containing solution. Perfusion with baicalin (0.3, 3, 30 [micro]M) was found to suppress the voltage-dependent [Ca.sup.2+] currents in a concentration-related manner ([IC.sub.50]: 0.11 [+ or -] 0.02 [micro]M, n = 7, P < 0.05 compared with control) (Figs. 8A, B and D). Baicalin (30 [micro]M) also abolished the current enhanced by Bay K8644 (0.1 [micro]M), a [Ca.sup.2+]-channel activator (Figs. 8A, C and E).

[FIGURE 8 OMITTED]

Discussion

The dried roots of Huang-qin are traditionally used for the treatment of hypertension and cardiovascular disease. Huang-qin has been shown to lower blood pressure in rats and cats (Lin et al. 1958; Kaye et al. 1997). Baicalin, one of the major bioactive constituents of Huang-qin, is a well-known anti-inflammatory and cardiovascular protective agent with diverse pharmacological activities (Sekiya and Okuda 1982; Chan et al. 2000; Shieh et al. 2000). However, the exact ionic mechanism of baicalin-induced vasorelaxation remains unclear. In this study, we provided the first evidence that the vasorelaxant effects of baicalin could be mainly attributed to VDCC inhibition and [BK.sub.Ca] channel activation through the PKA and PKG pathways.

KCl-induced depolarization elicits vasoconstriction exclusively by the activation of VDCCs (Kubo et al. 1985; Loutzenhiser et al. 1989; Huang et al. 2006), and we tested relaxant effects of baicalin on the isolated resistance MAs. In this experiment, baicalin attenuated 80 mM KCl-induced contractions in a concentration-related manner. Neither the COX inhibitor indomethacin nor the NOS inhibitor L-NAME affected baicalin-induced vasorelaxation. Since baicalin-induced relaxation was also not affected by endothelium denudation, it appears to be an endothelium-independent vasodilator. In this study, although baicalin ([less than or equal to] 10 [micro]M) had no significant effects on KCl-contracted MAs, it did abolish selective [BK.sub.Ca]-channel inhibitor IbTX, VDCC activator Bay K8644, and PKC activator PMA-induced contractions at concentrations [greater than or equal to] 30 [micro]M. These findings suggested that baicalin relaxes MA via modulation of the [BK.sub.Ca] and [Ca.sup.2+] channels and also mediates by the PKC associated pathway. Previous reports showed that the aglycone of baicalin, baicalein, exerts diverse actions on vessels not only dependent on the integrity of the endothelium but also on the drug concentrations involved (Chen et al. 1999; Tsang et al. 2000; Huang et al. 2002, 2004). However, we found that baicalin-induced MA relaxation is endothelium-independent. Additionally, baicalein-induced vasorelaxations appeared to be unrelated to [K.sub.ATP] and [BK.sub.Ca] channels, in contrast to our findings in baicalin, but it relaxed MA, in part due to inhibition of PKC-mediated contractile mechanism (Chen et al. 1999). It is generally accepted that VDCC inhibition and [BK.sub.Ca] channels activation offer a vasoprotective mechanism during pathological progression (Cox and Rusch 2002; Lu et al. 2006; Eichhorn and Dobrev 2007). Here, we demonstrated that baicalin could modulate the [BK.sub.Ca] and [Ca.sup.2+] channels resulting in vasorelaxation, and thus it could be useful in vascular dysfunction.

A great number of studies have suggested that high blood pressure and vascular dysfunction involve cellular signaling cascades that alter the expression of arterial [BK.sub.Ca] channels and voltage-dependent [Ca.sup.2+] channels to further modify vascular tone (Cox and Rusch 2002; Lu et al. 2006; Wu et al. 2007a; Burg et al. 2008; Dong et al. 2008). For example, in hypertension [BK.sub.Ca] signaling is completely diminished or reduced (Cox and Rusch 2002); in pulmonary hypertension decreased [BK.sub.Ca] channel activity leads to elevated cytosolic [[Ca.sup.2+]], cell proliferation, vascular contraction, and vascular remodeling (Burg et al. 2008); and in diabetes and atherosclerosis associated with alteredcurrent amplitude [BK.sub.Ca] channels are probably involved in the vascular dysfunction (Lu et al. 2006; Dong et al. 2008). Compensatory expression of [BK.sub.Ca] channels is thought to provide a counter-regulatory mechanism to help avert local vasospasms and ischemic episodes during cardiovascular disease (Eichhorn et al 2007).

Alterations in [BK.sub.Ca] channel activity play a central role in mediating vasoconstriction and vasodilation. As measured by whole-cell patch clamp electrophysiology, baicalin enhanced [BK.sub.Ca] currents in a concentration-dependent manner. The enhanced [BK.sub.Ca] currents were abolished by combining inhibitors of AC (SQ 22536) and sGC (ODQ), by combining inhibitors of PKA (KT5720) and PKG (KT5823), and PMA in MASMCs. In cyclic nucleotide assays, baicalin enhanced both cAMP and cGMP levels in MAs. Smooth muscle relaxants that increase cAMP and cGMP have been shown to activate [BK.sub.Ca] channels through direct phosphorylation effects on the channel protein (Robertson et al. 1993) and through elevation of [Ca.sup.2+] spark frequency. PKA and PKG may in part increase [Ca.sup.2+] sensitivity to [Ca.sup.2+] sparks by increasing the sarcoplasmic reticulum [Ca.sup.2+] load, which may enhance [BK.sub.Ca] activity (Jaggar et al. 2000). In addition, the increase in cellular cyclic nucleotides induced by baicalin could also contribute to its relaxation of vessels, which is important because dilation by cyclic nucleotides does not require [BK.sub.Ca] activation. In other words, some part of the relaxation may result from the activation of cAMP/cGMP independent of changes in membrane potential. Previous studies showed that any activation of PKA/PKG would be likely to cause a reduction in PKC activation, and an increase in [BK.sub.Ca] activity through cross-talk. Thus, cyclic nucleotides are required to initiate the cross-talk with PKC, thereby enhancing the [BK.sub.Ca] current (Jaggar et al. 2000). These results indicate that baicalin-induced [BK.sub.Ca] channel activation occurs not only as a result of PKA and PKG, but also as a result of cross-interaction with PKC (Fig. 9).

[FIGURE 9 OMITTED]

Physiologically, activation of [BK.sub.Ca] channels would hyperpolarize MASMCs, thereby closing VDCCs, reducing intracellular [Ca.sup.2+] and relaxing SMCs (Nelson and Quayle 1995). Its negative-feedback mechanism can modulate vascular tone, limit pressure-induced vasoconstriction and preserve local blood flow (Nelson and Quayle 1995). Similarly, baicalin could hyperpolarize MASMCs by activating [BK.sub.Ca] channels and inhibiting VDCCs. In this study, we observed that baicalin attenuated the VDCC current in a concentration-dependent manner, and it also abolished the [Ca.sup.2+] channel activator Bay K8644-enhanced currents. Since baicalin blocks VDCCs, it is not surprising that it relaxes KCl-induced contractions in MAs. Therefore, we suggest that baicalin-induced MA relaxations could be due to the reduction the activity of the [Ca.sup.2+] channel not only indirectly through [BK.sub.Ca]-channel producing hyperpolarization, but also directly by its [Ca.sup.2+] channel blocking action.

In this study, we provide the first evidence that baicalin activates [BK.sub.Ca] currents in rat MASMCs, which mainly mediates the PKA and PKG pathways, and therefore phosphorylates the channels or their associated regulatory proteins. This in turn enhances the [K.sup.+] effluxes and leads to membrane hyperpolarization and closure of VDCCs. Based on our findings, we suggest that baicalin could be a potential agent for the management of cardiovascular disorders.

Acknowledgements

The authors would like to thank Dr. Susan Olmstead-Wang at Johns Hopkins University for her editorial assistance with the manuscript. This work was supported by the National Science Council, Taiwan (grant NSC-95-2320-B-037-038-MY2); and the Cardiac Children's Foundation of the Republic of China (grant CCFT0804).

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* Corresponding author at: Department of Pharmacology and Department of Pediatrics, College of Medicine, Kaohsiung Medical University, 100 Shih-Chuan 1st Road, Kaohsiung 807, Taiwan. Fax: +886 7 3234686

E-mail addresses: binnan@kmu.edu.tw (B.-N. Wu), jirewu@kmu.edu.tw (J.-R. Wu).

Yi-Ling Lin (a), (b), Zen-Kong Dai (c), Rong-Jyh Lin (d), Koung-Shing Chu (e), Ing-Jun Chen (a), Jiunn-Ren Wu (c), *, Bin-Nan Wu (a), (b), *

(a) Department of Pharmacology, College of Medicine, Kaohsiung Medical University, 100 Shih-Chuan 1st Road, Kaohsiung 807, Taiwan

(b) Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan

(c) Department of Pediatrics, Division of Pediatric Pulmonology and Cardiology, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan

(d) Department of Parasitology, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan

(e) Department of Anesthesiology, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan

doi:10.1016/j.phymed.2010.01.003
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Author:Lin, Yi-Ling; Dai, Zen-Kong; Lin, Rong-Jyh; Chu, Koung-Shing; Chen, Ing-Jun; Wu, Jiunn-Ren; Wu, Bin-
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Aug 1, 2010
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