- Email: [email protected]
Effects of berberine on potassium currents in acutely isolated CA1 pyramidal neurons of rat hippocampus
Brain Research 999 (2004) 91 – 97 www.elsevier.com/locate/brainres
Research report
Effects of berberine on potassium currents in acutely isolated CA1 pyramidal neurons of rat hippocampus Fang Wang a,*, Gang Zhao b, Lan Cheng a, Hong-Yi Zhou a, Li-Ying Fu a, Wei-Xing Yao a a
Department of Pharmacology, Tongji Medical College, Huazhong University of Science and Technology, Hong Kong Road 13, Wuhan, Hubei 430030, PR China b Pancreatic Surgery Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, PR China Accepted 24 November 2003
Abstract The effects of berberine, an isoquinoline alkaloid with antiarrhythmic action, on voltage-dependent potassium currents were studied in acutely isolated CA1 pyramidal neurons of rat hippocampus by using the whole-cell patch-clamp techniques. Berberine blocked transient outward potassium current (IA) and delayed rectifier potassium current (IK) in a concentration-dependent manner with EC50 of 22.94 F 4.96 AM and 10.86 F 1.06 AM, Emax of 67.47 F 4.00% and 67.14 F 1.79%, n of 0.77 F 0.08 and 0.96 F 0.07, respectively. Berberine 30 AM shifted the steady-state activation curve and inactivation curve of IA to more negative potentials, but mainly affected the inactivation kinetics. Berberine 30 AM positively shifted the steady-state activation curve of IK. These results suggested that blockades on K+ currents by berberine are preferential for IK, and contribute to its protective action against ischemic brain damage. D 2003 Elsevier B.V. All rights reserved. Theme: Excitable membranes and synaptic transmission Topic: Potassium channel physiology, pharmacology, and modulation Keywords: Berberine; Hippocampus; CA1 neuron; Patch-clamp technique; Potassium current
1. Introduction Berberine is an isoquinoline alkaloid with a long history of medicinal used in both Ayurvedic and Chinese medicine. It presents in Hydrastis canadensis (goldenseal), Coptis chinensis (Coptis or goldenthread), Berberis aquifolium (Oregon grape), Berberis vulgaris (barberry), and Berberis aristata (tree turmeric). The berberine alkaloid can be found in the roots, rhizomes, and stem bark of the plants [14]. These medicinal plants have been used as folk medicine in treatment of jaundice, dysentery, hypertension, inflammation and liver diseases [1,4]. Berberine possesses potent antiarrhythmic activities in animal models [13,34]. Electrophysiological studies have demonstrated that berberine prolonged the action potential duration (APD) and effective refractory period (ERP) in Purkinje fibers and ventricular muscle [5,25]. The previous studies also have shown that berberine could block the delayed rectifier potassium current (IK),
* Corresponding author. Tel.: +86-27-8369-2033. E-mail address: [email protected] (F. Wang). 0006-8993/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2003.11.036
inward rectifier potassium current (IK1) and L-type calcium current (ICa,L) in guinea pig ventricular myocytes [2,12,18]. Recently, Wu et al. [32] reported that berberine prevented ischemic brain damage induced by carotid ligation. Wei et al. [30] also reported berberine prolonged the survival time of mice under the condition of closed normobaric hypoxia. Elevations of [K+]0 have been observed in pathological states of the central nervous system, such as hypoxia, ischemia, and so on. It has been shown recently that the early increase in [K+]0 in the hippocampus of rats which accompanies short periods of hypoxia, results from the opening of voltagedependent potassium currents, rather than from the lack of energy provision for the ATP-dependent Na+ – K+-pump [17,36]. Yu et al. have recently demonstrated that the apoptosis of mouse neocortical neurons induced by serum deprivation or by staurosporine was associated with the early enhancement of the delayed rectifier potassium current and loss of total intracellular K+ [31]. However, to date, the neuroprotective mechanism of berberine still remains unknown. And there are no data available to the relationship between ion currents in CNS neurons and the neuroprotective effect of berberine. In the
92
F. Wang et al. / Brain Research 999 (2004) 91–97
present study, the effects of berberine on whole-cell potassium currents recorded from acutely isolated rat hippocampal CA1 neurons were investigated.
2. Materials and methods 2.1. Cell preparation All experiments were performed on hippocampal CA1 pyramidal cells which were acutely isolated from 14 –20 days old Sprague – Dawley (SD) rats according to the method
described by Kay and Wong [15]. Briefly, hippocampal CA1 region was cut into 300 – 500 Am thick slices and incubated for 40 –60 min at 30 jC in artificial cerebrospinal solution (ACS). Slices were transferred into ACS containing 1.5 mg/ml protease E at 30 jC for 20 min. Throughout the entire procedure the media were continuously saturated with a 95% O2 and 5% CO2 gas mixture to maintain a pH of 7.4. Single cells were isolated by successive trituration of the tissue pieces in this solution through several fire polished glass pipettes with opening diameters from 0.1 to 0.5 mm. The pyramidal neurons with a diameter of 15– 30 Am were identified by their characteristic bright pyramidal-shaped
Fig. 1. Effects of berberine on IA and IK in rat hippocampal neurons. (A) Represent tracings in rat CA1 hippocampal neurons illustrate a typical example of IA and IK at + 30 mV from a holding potential of 100 mV during the control condition and after berberine 10, 30 AM. (B) Dose – response curve for the effects of berberine on IA. (C) Dose – response curve for the effects of berberine on IK. Number in parentheses indicates the number of cells used. x¯ F s.
F. Wang et al. / Brain Research 999 (2004) 91–97
soma under a phase contrast microscope and two or three short branched dendrites and a long axon. Neurons with bright and smooth appearance and no visible organelles were selected for recording. 2.2. Patch-clamp recording A programmable vertical puller (pp-83, Narishige, Japan) was used to pull the electrodes. The resistance of the
93
capillary glass electrode (GC150TF-10, England) used was 2– 4 MV when filled with internal solution. A patchclamp amplifier (EPC-9, Germany) was used to record whole-cell currents with four-pole Bessel filter set at 1 kHz, digitized at 5 kHz. The protocols for patch-clamp and data analysis were established with routines using pClamp 6.0 software (Axon Instruments, USA), and data were stored on computer for subsequent analysis. Drug actions were measured only after steady-state conditions
Fig. 2. Effects of berberine on I – V relationship of IA and IK in rat CA1 hippocampal neurons. (A) Families of IA and IK recorded with changes in the absence or presence of berberine 30 A`M. The voltage steps used to elicit IA and IK were shown in the inset. (B) I – V relations of the control IA (closed circles) and IA in the presence of berberine 30 A`M (open circles) constructed from original currents presented in (A). (C) I – V relations of the control IK (closed circles) and IK in the presence of berberine 30 AM. n = 8. x¯ F s. *P < 0.05, **P < 0.01 vs. control.
94
F. Wang et al. / Brain Research 999 (2004) 91–97
reached, which were judged by the amplitudes and time courses of currents remaining constant with further perfusion of drug.
cal significance of differences between means. A value of P < 0.05 was considered to be statistically significant.
2.3. Drugs and solutions
3. Results
Berberine hydrochloride was obtained from Yichang Pharmaceutical Company of China as base powders and dissolved in distilled water. Berberine was added to bath solution for extracellular application. All drugs were from Sigma unless otherwise indicated. ACS contained (in mM): NaCl 124, KCl 5, NaHCO3 26, NaH2PO4 1.25, CaCl2 2.4, MgSO4 1, and Glucose 10 (pH 7.4). The bath solution contained (in mM): NaCl 144, KCl 4.0, CaCl2 1.8, MgCl2 0.53, Na2HPO4 0.33, HEPES 10 and Glucose 10 (pH 7.3). The patch pipette solution contained (in mM): KCl 130, K2ATP 5.0, creatine phosphate 5.0 and HEPES 5.0 (pH 7.4). To record potassium current, 1 AM TTX and 0.2 mM CdCl2 were added to extracellular solution.
3.1. Effects of berberine on IA and IK
2.4. Data analysis All values were presented as mean F S.E.M. and error bars were plotted as S.E.M. All data were analyzed by the use of Sigmaplot 6.0 (Jandel Scientific, San Rafael, CA) software. Student’s t-test was used to evaluate the statisti-
The traces shown in Fig. 1A were obtained when the cell was generated by depolarizing pulse to + 30 mV for 160 ms from a holding potential 100 mV. Step depolarization to above 50 mV from 100 mV activated two components of outward currents. First, a rapidly activating and inactivating current, sensitive to 4-AP, referred to IA, and a delayed sustained current inactivating minimally during the 160 ms depolarization, sensitive to TEA, named as IK. Therefore, IA was estimated as the peak current, and IK was determined as late current at 158 ms step depolarization. In control experiments, there was a little decrease in the amplitude of both IA and IK in time-dependent, and IA and IK recorded by the end of 20 min were decreased by 6.51 F 4.20% (n = 10), 6.12 F 4.43% (n = 10) at + 30 mV, respectively. The percentage block was defined as (ICon Iberberine)/ICon and plotted as a function of logarithm [berberine] in Fig. 1B,C. Concentration –response curves were fitted by the Hill equation: Inhibition of current (%) = Emax/[1+(EC50/C)nH], Where EC50 is the concentra-
Fig. 3. Effects of berberine on IK with a holding potential 50 mV in rat CA1 hippocampal neurons. (A) Current traces are shown before and after application of berberine 30 AM. The voltage steps used to elicit IK were shown in the inset. (B) The influence on I – V curves for IK by berberine 30 AM. n = 8. x¯ F s. *P < 0.05, **P < 0.01 vs. control.
F. Wang et al. / Brain Research 999 (2004) 91–97
tion of berberine for half-maximum block, C is the concentration of berberine, and nH, the Hill coefficient. Berberine 1– 300 AM exhibited a concentration-dependent blockade on IA and IK. The EC50 values for block of IA and IK were 22.94 F 4.96 and 10.8 F 61.06 AM, respectively. The Emax were 67.47 F 4.00% and 67.14 F 1.79%, with nH of 0.77 F 0.08 and 0.96 F 0.07, respectively. The effect of the drug was poorly reversible after washout. As shown in Fig. 1A, at + 30 mV, berberine 10, 30 AM decreased the amplitudes of IA from 2820.78 F 292.45 to 2181.27 F 212.65 and 1799.44 F 177.21 pA, respectively. And the inhibition rates were 22.65% F 4.30% and 36.19% F 5.40%, respectively. The inhibitory effects at the same concentrations on IK were from 1866.72 F 148.96 to 1297.43 F 121.48 and 930.57 F 96.71 pA, respectively. And the inhibition rates were 30.47 F 6.42% and 50.13 F 6.88%, respectively. Consistent observations represented by the examples in Fig. 1 revealed that the following features of the action of berberine: the initial phase of the total outward current (including its peak value) is less affected by the drug as compared to the steady-state correspondingly. These observations suggested that berberine could elicit different effects on the early and delayed potassium current components.
95
3.4. Effects of berberine on the activation kinetics of IA and IK The steady-state activation curves for IA and IK under control (closed circles) and after exposure to berberine 30 AM (open circles) were shown in Fig. 4. The membrane was held at 100 mV and depolarized from 80 to + 90 mV for 160 ms with a 10-mV increment. The normalized currents were fitted by the Boltzmann function as follows: I/Imax = 1/ (1 + exp((V1/2 V)/k)), where V is the conditioning voltage, V1/2 is the voltage at which half-maximal effect is obtained, and k is the slope. During control conditions, average V1/2 for activation of IA in control and berberine 30 AM were 4.59 F 1.52 and 4.44 F 1.37 mV (n = 8, P < 0.05 vs. control), with k of 23.89 F 2.10 and 24.65 F 2.71 mV (n = 8, P>0.05 vs. control), respectively. And V1/2 for activation of IK in control and berberine 30 AM were 7.10 F 2.91 and 24.65 F 6.33 mV (n = 8, P < 0.01 vs. control), with k of
3.2. Effects of berberine on I –V relationship of IA and IK Fig. 2 showed current – voltage curves of IA and IK generated by applying nine depolarizing pulses from 50 to + 30 mV for 160 ms with a 10 mV increment from a holding potential 100 mV. In the presence of berberine 30 AM, the amplitude of IA was significantly reduced at potential + 10 through + 30 mV (n = 8, P < 0.05 vs. control). Berberine 30 AM also reduced the amplitudes of IK significantly at potential 0 through + 30 mV (n = 8, P < 0.05 or P < 0.01 vs. control). 3.3. Effects of berberine on IK activated by the protocol of HP -50 mV To quantitate reliably blocking action of berberine on IK, we separated IK from total outward currents by a procedure taking advantage of the kinetic difference between these two currents, which based on the protocol described by Ficker and Heinemann [6]. Previous studies have demonstrated that IA undergoes steady-state inactivation at HP positive than 50 mV, therefore, IK could be activated nearly uncontaminated with IA by holding at 50 mV and stepping to more positive potentials. With this protocol the IK were activated slowly to a plateau with minimal time-dependent inactivation. As shown in Fig. 3, berberine 30 AM dramatically inhibited IK amplitude about 48.16 F 5.76%. The blocking action is also concentrationdependent and similar to the inhibition on IK activated by the protocol with HP 100 mV.
Fig. 4. Effects of berberine on the steady-state activation kinetics of (A) IA and (B) IK in hippocampal neurons of rats. The effect was examined in the absence (closed circles) and presence of berberine 30 AM (open circles). n = 8. x¯ F s.
96
F. Wang et al. / Brain Research 999 (2004) 91–97
Fig. 5. Effects of berberine on the steady-state inactivation of IA in rat CA1 hippocampal neurons. The ordinate is a measure of normalized peak current for different amplitudes of conditioning prepulses in the absence (closed circles) or presence of berberine 30 AM (open circles). n = 8. x¯ F s.
24.65 F 5.76 and 25.76 F 6.04 mV (n = 8, P>0.05 vs. control), respectively. Berberine negatively shifted the voltage-dependence of the activation of IA, and shifted the activation curve of IK to more positive potential with no change in slope factor. 3.5. Effects of berberine on the inactivation kinetics of IA Berberine effects on IA could reflect actions to shift the voltage-dependence of inactivation. The steady-state inactivation was examined by changing the prepulse potentials at levels between 120 to 10 mV (80 ms duration) before depolarization to a test pulse of + 50 mV (duration of 120 ms). The inactivation curves shown in Fig. 5 were obtained by normalizing the test current amplitudes by taking the maximum value under each condition as unity. The curves were fit to the Boltzmann equation: IA/IA V1/2)/k)), where V is the magnitude max = 1/(1 + exp ((V of the conditioning pulse potential, V1/2 is the potential where normalized IA was reduced to one-half and k is the slope factor. During control conditions, average V1/2 was 73.58 F 4.59 mV and k was 13.77 F 3.15 mV. After the addition of berberine 30 AM, the inactivation curve was shifted to more negative potentials: V1/2 was 87.85 F 4.55 mV (n = 8, P < 0.01 vs. control) and k was 13.03 F 2.00 mV (n = 8, P>0.05 vs. control). The shift in V1/2 of steady-state inactivation might account for the reduced peak IA and the shift in the I– V curve to the downward in the presence of berberine.
4. Discussion In this study, for the first time, we characterized the effects of berberine on voltage-dependent potassium currents by patch-clamp techniques and demonstrated that
berberine effectively inhibited IA and IK in a concentration-dependent manner in rat CA1 hippocampal neurons. The EC50 values of berberine for inhibition of IA and IK were 22.94 and 10.86 AM, respectively. This result suggested that although berberine exhibited qualitatively comparable blocking effects on these two currents, the blocking potency was much stronger on IK than IA. It appears to have different sensitivity for IK and IA. Berberine shifted the activation curves of IA to negative potential; however it shifted the activation curves of IK to positive potential. The significantly negative shift in the steadystate inactivation of IA (about 15 mV) is more than the negative shift in the steady-state activation of IA (about 8 mV), which suggested that berberine mainly affected the voltage-dependent inactivation kinetics of IA. So the effect of berberine on IA is inhibitory. We also noticed that the maximum inhibition of berberine on IK only reached about 70% even at 300 AM. Because voltage-activated potassium channels in rat pyramidal neurons are composed of various potassium channel subtypes such as Kv1, Kv2, Kv3 and Kv4 [19,23], berberine is probably selective for some delayed rectifier K+ channel subtypes in hippocampal neurons. It needs further studies to understand the channel selectivity of berberine. K+ is the predominant cation in the cytosol. Maintenance of a high [K+] in the cytoplasm (140 – 150 mM) is essential for (1) governing cell excitability [20], (2) setting resting Em [24], (3) regulating apoptotic enzyme activity [33], and (4) controlling cell volume [22]. It reinforces the notion that K+ acts as an endogenous modulator of several checkpoints (e.g., cytochrome c release, caspase cleavage, and endonuclease activation) in the apoptotic cascade [10]. Under physiological conditions, potassium currents are important for the regulation of neuronal excitability and the maintenance of baseline membrane potential [27]. Regulation of potassium channel activity is believed to have a major impact on the overall neuronal response and adaptation to O2 deprivation [9]. Activation of K+ channels induces hyperpolarization, decreases membrane excitability and reduces O2 consumption [8]. Traditionally, ischemic neuronal death is considered to be a consequence of necrosis [3,26]. However, in recent years, accumulating evidence has indicated that many neurons undergo apoptosis after global or focal ischemia [11,21]. Studies have shown that increased K+ efflux might be a primary step leading to apoptosis. Cytoplasmic K+ at normal concentration (~140 mM) decreases apoptotic DNA fragmentation and caspase-3-like protease activation [28]. Decrease in [K+]c, due to elevated K+ efflux through opened K+ channels, results in cell shrinkage [7,16,35] and reduces the inhibitory effect of cytoplasmic K+ on caspase-3-like protease and the internucleosomal DNA cleavage nuclease [29], which contributed to ischemia-triggered apoptosis. Thus, the increment in potassium currents in CA1 neurons may contribute not only to excitability changes but also to cell death after ischemia.
F. Wang et al. / Brain Research 999 (2004) 91–97
Berberine blocks potassium channels of hippocampal CA1 neurons, which is beneficial to cation balance of neurons under anoxic/ischemic injury, and leads to the suppression of apoptosis and a substantial increase in the rate of cell survival. Therefore, our results demonstrate that the blocking actions of berberine on K+ currents should be considered as a protective mechanism against ischemic brain damage.
References [1] S. Bova, R. Padrini, W.F. Goldman, D.M. Berman, G. Cargnelli, On the mechanism of vasodilating action of berberine: possible role of inositol lipid signaling system, J. Pharmacol. Exp. Ther. 261 (1992) 318 – 323. [2] J.S. Chapula, Increase in action potential duration and inhibition of the delayed rectifier outward current IK by berberine in cat ventricular myocytes, Br. J. Pharmacol. 117 (1996) 1427 – 1437. [3] X.X. Chi, Z.C. Xu, Differential changes of potassium currents in CA1 pyramidal neurons after transient forebrain ischemia, J. Neurophysiol. 84 (2000) 2834 – 2843. [4] W.F. Chiou, M.H. Yen, C.F. Chen, Mechanism of vasodilatory effect of berberine in rat mesenteric artery, Eur. J. Pharmacol. 204 (1991) 35 – 40. [5] D.Z. Dai, Vulnerable substrate and multiple ion channel disorder in a diseased heart will be new targets for antiarrhythmic therapy, Acta Pharm. Sin. 21 (2000) 289 – 295. [6] E. Ficker, U. Heinemann, Slow and fast transient potassium currents in cultured rat hippocampal cells, J. Physiol. 445 (1992) 431 – 455. [7] I.J. Furlong, C. Lopez-Mediavilla, R. Ascaso, A. Lopez-Rivas, M.K. Collins, Induction of apoptosis by valinomycin: mitochondrial permeability transition causes intracellular acidification, Cell Death Differ. 5 (1998) 214 – 221. [8] T.M. Gao, W.A. Pulsinelli, Z.C. Xu, Changes in membrane properties of CA1 pyramidal neurons after transient forebrain ischemia in vivo, Neuroscience 90 (1999) 771 – 780. [9] E.V. Golanov, J.D. Christensen, D.J. Reis, Role of potassium channels in the central neurogenic neuroprotection elicited by cerebellar stimulation in rat, Brain Res. 842 (1999) 496 – 500. [10] M. Gomez-Angelats, J.A. Cidlowski, Cell volume and ion changes during apoptotic cell death, Adv. Cancer Res. 85 (2002) 175 – 201. [11] X. Hu, I.M. Johansson, T. Brannstrom, T. Olsson, P. Wester, Longlasting neuronal apoptotic cell death in regions with severe ischemia after photothrombotic ring stroke in rats, Acta Neuropathol. 104 (2002) 462 – 470(Berl.). [12] Z. Hua, X.L. Wang, Inhibitory effect of berberine on potassium channels in guinea pig ventricular myocytes, Acta Pharm. Sin. 29 (1994) 576 – 580. [13] W. Huang, Ventricular tachyarrhythmias treated with berberine, Zhonghua Xinxue Guanbing. Zazhi 18 (1990) 155 – 156. [14] A. Kathleen, Berberine, Altern. Med. Rev. 5 (2000) 175 – 177. [15] A.R. Kay, R.K. Wong, Isolation of neurons suitable for patch-clamping from adult mammalian central nervous systems, J. Neurosci. Methods 16 (1986) 227 – 238. [16] S. Krick, O. Platoshyn, M. Sweeney, H. Kim, J.X. Yuan, Activation of K+ channels induces apoptosis in vascular smooth muscle cells, Am. J. Physiol., Cell Physiol. 280 (2001) C970 – C979.
97
[17] O. Krishtal, Y. Kirichok, T. Tsintsadze, N. Lozovaya, W. Loesel, D. Arndts, New channel blocker BIIA388CL blocks delayed rectifier, but not A-type potassium current in central neurons, Neuropharmacology 40 (2001) 233 – 241. [18] C.W. Lau, X.Q. Yao, Z.Y. Chen, W.H. Ko, Y. Huang, Cardiovascular actions of berberine, Cardiovasc. Drug Rev. 19 (2001) 234 – 244. [19] M. Martina, J.H. Schultz, H. Ehmke, H. Monyer, P. Jonas, Functional and molecular differences between voltage-gated K+ channels of fastspiking interneurons and pyramidal neurons of rat hippocampus, J. Neurosci. 18 (1998) 8111 – 8125. [20] M.T. Nelson, J.M. Quayle, Physiological roles and properties of potassium channels in arterial smooth muscle, Am. J. Physiol., Cell Physiol. 268 (1995) C799 – C822. [21] R.W. Neumar, Molecular mechanisms of ischemic neuronal injury, Ann. Emerg. Med. 36 (2000) 483 – 506. [22] N. O’Reilly, Z. Xia, H. Fiander, J. Tauskela, D.L. Small, Disparity between ionic mediators of volume regulation and apoptosis in N1E 115 mouse neuroblastoma cells, Brain Res. 943 (2002) 245 – 256. [23] Y.P. Pan, X.H. Xu, X.L. Wang, Galantamine blocks delayed rectifier, but not transient outward potassium current in rat dissociated hippocampal pyramidal neurons, Neurosci. Lett. 336 (2002) 37 – 40. [24] R.L. Parsons, K.L. Barstow, F.S. Scornik, Spontaneous miniature hyperpolarizations affect threshold for action potential generation in mudpuppy cardiac neurons, J. Neurophysiol. 88 (2002) 1119 – 1127. [25] N.F. Ricciopo, Electropharmacological effects of berberine on canine cardiac Purkinje fibers and ventricular muscle and atrial muscle of the rabbit, Br. J. Pharmacol. 108 (1993) 534 – 537. [26] B.J. Snider, F.J. Gottron, D.W. Choi, Apoptosis and necrosis in cerebrovascular disease, Ann. N.Y. Acad. Sci. 893 (1999) 243 – 253. [27] J.F. Storm, Potassium currents in hippocampal pyramidal cells, Prog. Brain Res. 83 (1990) 161 – 187. [28] S. Uryu, J. Harada, M. Hisamoto, T. Oda, Troglitazone inhibits both post-glutamate neurotoxicity and low-potassium-induced apoptosis in cerebellar granule neurons, Brain Res. 924 (2002) 229 – 236. [29] X. Wang, A.Y. Xiao, T. Ichinose, S.P. Yu, Effects of tetraethylammonium analogs on apoptosis and membrane currents in cultured cortical neurons, J. Pharmacol. Exp. Ther. 295 (2000) 524 – 530. [30] Y.Z. Wei, Z.B. Yao, Q.F. Yuan, Y.C. Chen, Protective effect of berberine on rat hippocampus against cerebral ischemia, Chinese, J. Chem. Neuroanat. 11 (1995) 315 – 321. [31] L. Wei, S.P. Yu, F. Gottron, B.J. Snider, G.J. Zipfel, D.W. Choi, Potassium channel blockers attenuate hypoxia- and ischemia-induced neuronal death in vitro and in vivo, Stroke 34 (2003) 1281 – 1286. [32] J.F. Wu, Y.J. Shi, T.P. Liu, Protective effects of berberine on cerebral ischemia in mice and rats, Chinese, J. Pharmacol. Toxicol. Methods 9 (1995) 100 – 103. [33] A.Y. Xiao, L. Wei, S.L. Xia, S. Rothman, S.P. Yu, Ionic mechanism of ouabain-induced concurrent apoptosis and necrosis in individual cultured cortical neurons, J. Neurosci. 15 (2002) 1350 – 1362. [34] Z. Xu, H.Y. Cao, Q. Li, Protective effects of berberine on spontaneous ventricular fibrillation in dogs after myocardial infarction, Zhongguo Yaoli Xuebao 10 (1989) 320 – 324. [35] S.P. Yu, C.H. Yeh, F. Gottron, X. Wang, M.C. Grabb, D.W. Choi, Role of the outward delayed rectifier K+ current in ceramide-induced caspase activation and apoptosis in cultured cortical neurons, J. Neurochem. 73 (1999) 933 – 941. [36] T.S. Zetterstrom, R.D. Vaughan-Jones, D.G. Grahame-Smith, A short period of hypoxia produces a rapid and transient rise in [K+] e in rat hippocampus in vivo which is inhibited by certain K+-channel blocking agents, Neuroscience 67 (1995) 815 – 821.
Research report
Effects of berberine on potassium currents in acutely isolated CA1 pyramidal neurons of rat hippocampus Fang Wang a,*, Gang Zhao b, Lan Cheng a, Hong-Yi Zhou a, Li-Ying Fu a, Wei-Xing Yao a a
Department of Pharmacology, Tongji Medical College, Huazhong University of Science and Technology, Hong Kong Road 13, Wuhan, Hubei 430030, PR China b Pancreatic Surgery Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, PR China Accepted 24 November 2003
Abstract The effects of berberine, an isoquinoline alkaloid with antiarrhythmic action, on voltage-dependent potassium currents were studied in acutely isolated CA1 pyramidal neurons of rat hippocampus by using the whole-cell patch-clamp techniques. Berberine blocked transient outward potassium current (IA) and delayed rectifier potassium current (IK) in a concentration-dependent manner with EC50 of 22.94 F 4.96 AM and 10.86 F 1.06 AM, Emax of 67.47 F 4.00% and 67.14 F 1.79%, n of 0.77 F 0.08 and 0.96 F 0.07, respectively. Berberine 30 AM shifted the steady-state activation curve and inactivation curve of IA to more negative potentials, but mainly affected the inactivation kinetics. Berberine 30 AM positively shifted the steady-state activation curve of IK. These results suggested that blockades on K+ currents by berberine are preferential for IK, and contribute to its protective action against ischemic brain damage. D 2003 Elsevier B.V. All rights reserved. Theme: Excitable membranes and synaptic transmission Topic: Potassium channel physiology, pharmacology, and modulation Keywords: Berberine; Hippocampus; CA1 neuron; Patch-clamp technique; Potassium current
1. Introduction Berberine is an isoquinoline alkaloid with a long history of medicinal used in both Ayurvedic and Chinese medicine. It presents in Hydrastis canadensis (goldenseal), Coptis chinensis (Coptis or goldenthread), Berberis aquifolium (Oregon grape), Berberis vulgaris (barberry), and Berberis aristata (tree turmeric). The berberine alkaloid can be found in the roots, rhizomes, and stem bark of the plants [14]. These medicinal plants have been used as folk medicine in treatment of jaundice, dysentery, hypertension, inflammation and liver diseases [1,4]. Berberine possesses potent antiarrhythmic activities in animal models [13,34]. Electrophysiological studies have demonstrated that berberine prolonged the action potential duration (APD) and effective refractory period (ERP) in Purkinje fibers and ventricular muscle [5,25]. The previous studies also have shown that berberine could block the delayed rectifier potassium current (IK),
* Corresponding author. Tel.: +86-27-8369-2033. E-mail address: [email protected] (F. Wang). 0006-8993/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2003.11.036
inward rectifier potassium current (IK1) and L-type calcium current (ICa,L) in guinea pig ventricular myocytes [2,12,18]. Recently, Wu et al. [32] reported that berberine prevented ischemic brain damage induced by carotid ligation. Wei et al. [30] also reported berberine prolonged the survival time of mice under the condition of closed normobaric hypoxia. Elevations of [K+]0 have been observed in pathological states of the central nervous system, such as hypoxia, ischemia, and so on. It has been shown recently that the early increase in [K+]0 in the hippocampus of rats which accompanies short periods of hypoxia, results from the opening of voltagedependent potassium currents, rather than from the lack of energy provision for the ATP-dependent Na+ – K+-pump [17,36]. Yu et al. have recently demonstrated that the apoptosis of mouse neocortical neurons induced by serum deprivation or by staurosporine was associated with the early enhancement of the delayed rectifier potassium current and loss of total intracellular K+ [31]. However, to date, the neuroprotective mechanism of berberine still remains unknown. And there are no data available to the relationship between ion currents in CNS neurons and the neuroprotective effect of berberine. In the
92
F. Wang et al. / Brain Research 999 (2004) 91–97
present study, the effects of berberine on whole-cell potassium currents recorded from acutely isolated rat hippocampal CA1 neurons were investigated.
2. Materials and methods 2.1. Cell preparation All experiments were performed on hippocampal CA1 pyramidal cells which were acutely isolated from 14 –20 days old Sprague – Dawley (SD) rats according to the method
described by Kay and Wong [15]. Briefly, hippocampal CA1 region was cut into 300 – 500 Am thick slices and incubated for 40 –60 min at 30 jC in artificial cerebrospinal solution (ACS). Slices were transferred into ACS containing 1.5 mg/ml protease E at 30 jC for 20 min. Throughout the entire procedure the media were continuously saturated with a 95% O2 and 5% CO2 gas mixture to maintain a pH of 7.4. Single cells were isolated by successive trituration of the tissue pieces in this solution through several fire polished glass pipettes with opening diameters from 0.1 to 0.5 mm. The pyramidal neurons with a diameter of 15– 30 Am were identified by their characteristic bright pyramidal-shaped
Fig. 1. Effects of berberine on IA and IK in rat hippocampal neurons. (A) Represent tracings in rat CA1 hippocampal neurons illustrate a typical example of IA and IK at + 30 mV from a holding potential of 100 mV during the control condition and after berberine 10, 30 AM. (B) Dose – response curve for the effects of berberine on IA. (C) Dose – response curve for the effects of berberine on IK. Number in parentheses indicates the number of cells used. x¯ F s.
F. Wang et al. / Brain Research 999 (2004) 91–97
soma under a phase contrast microscope and two or three short branched dendrites and a long axon. Neurons with bright and smooth appearance and no visible organelles were selected for recording. 2.2. Patch-clamp recording A programmable vertical puller (pp-83, Narishige, Japan) was used to pull the electrodes. The resistance of the
93
capillary glass electrode (GC150TF-10, England) used was 2– 4 MV when filled with internal solution. A patchclamp amplifier (EPC-9, Germany) was used to record whole-cell currents with four-pole Bessel filter set at 1 kHz, digitized at 5 kHz. The protocols for patch-clamp and data analysis were established with routines using pClamp 6.0 software (Axon Instruments, USA), and data were stored on computer for subsequent analysis. Drug actions were measured only after steady-state conditions
Fig. 2. Effects of berberine on I – V relationship of IA and IK in rat CA1 hippocampal neurons. (A) Families of IA and IK recorded with changes in the absence or presence of berberine 30 A`M. The voltage steps used to elicit IA and IK were shown in the inset. (B) I – V relations of the control IA (closed circles) and IA in the presence of berberine 30 A`M (open circles) constructed from original currents presented in (A). (C) I – V relations of the control IK (closed circles) and IK in the presence of berberine 30 AM. n = 8. x¯ F s. *P < 0.05, **P < 0.01 vs. control.
94
F. Wang et al. / Brain Research 999 (2004) 91–97
reached, which were judged by the amplitudes and time courses of currents remaining constant with further perfusion of drug.
cal significance of differences between means. A value of P < 0.05 was considered to be statistically significant.
2.3. Drugs and solutions
3. Results
Berberine hydrochloride was obtained from Yichang Pharmaceutical Company of China as base powders and dissolved in distilled water. Berberine was added to bath solution for extracellular application. All drugs were from Sigma unless otherwise indicated. ACS contained (in mM): NaCl 124, KCl 5, NaHCO3 26, NaH2PO4 1.25, CaCl2 2.4, MgSO4 1, and Glucose 10 (pH 7.4). The bath solution contained (in mM): NaCl 144, KCl 4.0, CaCl2 1.8, MgCl2 0.53, Na2HPO4 0.33, HEPES 10 and Glucose 10 (pH 7.3). The patch pipette solution contained (in mM): KCl 130, K2ATP 5.0, creatine phosphate 5.0 and HEPES 5.0 (pH 7.4). To record potassium current, 1 AM TTX and 0.2 mM CdCl2 were added to extracellular solution.
3.1. Effects of berberine on IA and IK
2.4. Data analysis All values were presented as mean F S.E.M. and error bars were plotted as S.E.M. All data were analyzed by the use of Sigmaplot 6.0 (Jandel Scientific, San Rafael, CA) software. Student’s t-test was used to evaluate the statisti-
The traces shown in Fig. 1A were obtained when the cell was generated by depolarizing pulse to + 30 mV for 160 ms from a holding potential 100 mV. Step depolarization to above 50 mV from 100 mV activated two components of outward currents. First, a rapidly activating and inactivating current, sensitive to 4-AP, referred to IA, and a delayed sustained current inactivating minimally during the 160 ms depolarization, sensitive to TEA, named as IK. Therefore, IA was estimated as the peak current, and IK was determined as late current at 158 ms step depolarization. In control experiments, there was a little decrease in the amplitude of both IA and IK in time-dependent, and IA and IK recorded by the end of 20 min were decreased by 6.51 F 4.20% (n = 10), 6.12 F 4.43% (n = 10) at + 30 mV, respectively. The percentage block was defined as (ICon Iberberine)/ICon and plotted as a function of logarithm [berberine] in Fig. 1B,C. Concentration –response curves were fitted by the Hill equation: Inhibition of current (%) = Emax/[1+(EC50/C)nH], Where EC50 is the concentra-
Fig. 3. Effects of berberine on IK with a holding potential 50 mV in rat CA1 hippocampal neurons. (A) Current traces are shown before and after application of berberine 30 AM. The voltage steps used to elicit IK were shown in the inset. (B) The influence on I – V curves for IK by berberine 30 AM. n = 8. x¯ F s. *P < 0.05, **P < 0.01 vs. control.
F. Wang et al. / Brain Research 999 (2004) 91–97
tion of berberine for half-maximum block, C is the concentration of berberine, and nH, the Hill coefficient. Berberine 1– 300 AM exhibited a concentration-dependent blockade on IA and IK. The EC50 values for block of IA and IK were 22.94 F 4.96 and 10.8 F 61.06 AM, respectively. The Emax were 67.47 F 4.00% and 67.14 F 1.79%, with nH of 0.77 F 0.08 and 0.96 F 0.07, respectively. The effect of the drug was poorly reversible after washout. As shown in Fig. 1A, at + 30 mV, berberine 10, 30 AM decreased the amplitudes of IA from 2820.78 F 292.45 to 2181.27 F 212.65 and 1799.44 F 177.21 pA, respectively. And the inhibition rates were 22.65% F 4.30% and 36.19% F 5.40%, respectively. The inhibitory effects at the same concentrations on IK were from 1866.72 F 148.96 to 1297.43 F 121.48 and 930.57 F 96.71 pA, respectively. And the inhibition rates were 30.47 F 6.42% and 50.13 F 6.88%, respectively. Consistent observations represented by the examples in Fig. 1 revealed that the following features of the action of berberine: the initial phase of the total outward current (including its peak value) is less affected by the drug as compared to the steady-state correspondingly. These observations suggested that berberine could elicit different effects on the early and delayed potassium current components.
95
3.4. Effects of berberine on the activation kinetics of IA and IK The steady-state activation curves for IA and IK under control (closed circles) and after exposure to berberine 30 AM (open circles) were shown in Fig. 4. The membrane was held at 100 mV and depolarized from 80 to + 90 mV for 160 ms with a 10-mV increment. The normalized currents were fitted by the Boltzmann function as follows: I/Imax = 1/ (1 + exp((V1/2 V)/k)), where V is the conditioning voltage, V1/2 is the voltage at which half-maximal effect is obtained, and k is the slope. During control conditions, average V1/2 for activation of IA in control and berberine 30 AM were 4.59 F 1.52 and 4.44 F 1.37 mV (n = 8, P < 0.05 vs. control), with k of 23.89 F 2.10 and 24.65 F 2.71 mV (n = 8, P>0.05 vs. control), respectively. And V1/2 for activation of IK in control and berberine 30 AM were 7.10 F 2.91 and 24.65 F 6.33 mV (n = 8, P < 0.01 vs. control), with k of
3.2. Effects of berberine on I –V relationship of IA and IK Fig. 2 showed current – voltage curves of IA and IK generated by applying nine depolarizing pulses from 50 to + 30 mV for 160 ms with a 10 mV increment from a holding potential 100 mV. In the presence of berberine 30 AM, the amplitude of IA was significantly reduced at potential + 10 through + 30 mV (n = 8, P < 0.05 vs. control). Berberine 30 AM also reduced the amplitudes of IK significantly at potential 0 through + 30 mV (n = 8, P < 0.05 or P < 0.01 vs. control). 3.3. Effects of berberine on IK activated by the protocol of HP -50 mV To quantitate reliably blocking action of berberine on IK, we separated IK from total outward currents by a procedure taking advantage of the kinetic difference between these two currents, which based on the protocol described by Ficker and Heinemann [6]. Previous studies have demonstrated that IA undergoes steady-state inactivation at HP positive than 50 mV, therefore, IK could be activated nearly uncontaminated with IA by holding at 50 mV and stepping to more positive potentials. With this protocol the IK were activated slowly to a plateau with minimal time-dependent inactivation. As shown in Fig. 3, berberine 30 AM dramatically inhibited IK amplitude about 48.16 F 5.76%. The blocking action is also concentrationdependent and similar to the inhibition on IK activated by the protocol with HP 100 mV.
Fig. 4. Effects of berberine on the steady-state activation kinetics of (A) IA and (B) IK in hippocampal neurons of rats. The effect was examined in the absence (closed circles) and presence of berberine 30 AM (open circles). n = 8. x¯ F s.
96
F. Wang et al. / Brain Research 999 (2004) 91–97
Fig. 5. Effects of berberine on the steady-state inactivation of IA in rat CA1 hippocampal neurons. The ordinate is a measure of normalized peak current for different amplitudes of conditioning prepulses in the absence (closed circles) or presence of berberine 30 AM (open circles). n = 8. x¯ F s.
24.65 F 5.76 and 25.76 F 6.04 mV (n = 8, P>0.05 vs. control), respectively. Berberine negatively shifted the voltage-dependence of the activation of IA, and shifted the activation curve of IK to more positive potential with no change in slope factor. 3.5. Effects of berberine on the inactivation kinetics of IA Berberine effects on IA could reflect actions to shift the voltage-dependence of inactivation. The steady-state inactivation was examined by changing the prepulse potentials at levels between 120 to 10 mV (80 ms duration) before depolarization to a test pulse of + 50 mV (duration of 120 ms). The inactivation curves shown in Fig. 5 were obtained by normalizing the test current amplitudes by taking the maximum value under each condition as unity. The curves were fit to the Boltzmann equation: IA/IA V1/2)/k)), where V is the magnitude max = 1/(1 + exp ((V of the conditioning pulse potential, V1/2 is the potential where normalized IA was reduced to one-half and k is the slope factor. During control conditions, average V1/2 was 73.58 F 4.59 mV and k was 13.77 F 3.15 mV. After the addition of berberine 30 AM, the inactivation curve was shifted to more negative potentials: V1/2 was 87.85 F 4.55 mV (n = 8, P < 0.01 vs. control) and k was 13.03 F 2.00 mV (n = 8, P>0.05 vs. control). The shift in V1/2 of steady-state inactivation might account for the reduced peak IA and the shift in the I– V curve to the downward in the presence of berberine.
4. Discussion In this study, for the first time, we characterized the effects of berberine on voltage-dependent potassium currents by patch-clamp techniques and demonstrated that
berberine effectively inhibited IA and IK in a concentration-dependent manner in rat CA1 hippocampal neurons. The EC50 values of berberine for inhibition of IA and IK were 22.94 and 10.86 AM, respectively. This result suggested that although berberine exhibited qualitatively comparable blocking effects on these two currents, the blocking potency was much stronger on IK than IA. It appears to have different sensitivity for IK and IA. Berberine shifted the activation curves of IA to negative potential; however it shifted the activation curves of IK to positive potential. The significantly negative shift in the steadystate inactivation of IA (about 15 mV) is more than the negative shift in the steady-state activation of IA (about 8 mV), which suggested that berberine mainly affected the voltage-dependent inactivation kinetics of IA. So the effect of berberine on IA is inhibitory. We also noticed that the maximum inhibition of berberine on IK only reached about 70% even at 300 AM. Because voltage-activated potassium channels in rat pyramidal neurons are composed of various potassium channel subtypes such as Kv1, Kv2, Kv3 and Kv4 [19,23], berberine is probably selective for some delayed rectifier K+ channel subtypes in hippocampal neurons. It needs further studies to understand the channel selectivity of berberine. K+ is the predominant cation in the cytosol. Maintenance of a high [K+] in the cytoplasm (140 – 150 mM) is essential for (1) governing cell excitability [20], (2) setting resting Em [24], (3) regulating apoptotic enzyme activity [33], and (4) controlling cell volume [22]. It reinforces the notion that K+ acts as an endogenous modulator of several checkpoints (e.g., cytochrome c release, caspase cleavage, and endonuclease activation) in the apoptotic cascade [10]. Under physiological conditions, potassium currents are important for the regulation of neuronal excitability and the maintenance of baseline membrane potential [27]. Regulation of potassium channel activity is believed to have a major impact on the overall neuronal response and adaptation to O2 deprivation [9]. Activation of K+ channels induces hyperpolarization, decreases membrane excitability and reduces O2 consumption [8]. Traditionally, ischemic neuronal death is considered to be a consequence of necrosis [3,26]. However, in recent years, accumulating evidence has indicated that many neurons undergo apoptosis after global or focal ischemia [11,21]. Studies have shown that increased K+ efflux might be a primary step leading to apoptosis. Cytoplasmic K+ at normal concentration (~140 mM) decreases apoptotic DNA fragmentation and caspase-3-like protease activation [28]. Decrease in [K+]c, due to elevated K+ efflux through opened K+ channels, results in cell shrinkage [7,16,35] and reduces the inhibitory effect of cytoplasmic K+ on caspase-3-like protease and the internucleosomal DNA cleavage nuclease [29], which contributed to ischemia-triggered apoptosis. Thus, the increment in potassium currents in CA1 neurons may contribute not only to excitability changes but also to cell death after ischemia.
F. Wang et al. / Brain Research 999 (2004) 91–97
Berberine blocks potassium channels of hippocampal CA1 neurons, which is beneficial to cation balance of neurons under anoxic/ischemic injury, and leads to the suppression of apoptosis and a substantial increase in the rate of cell survival. Therefore, our results demonstrate that the blocking actions of berberine on K+ currents should be considered as a protective mechanism against ischemic brain damage.
References [1] S. Bova, R. Padrini, W.F. Goldman, D.M. Berman, G. Cargnelli, On the mechanism of vasodilating action of berberine: possible role of inositol lipid signaling system, J. Pharmacol. Exp. Ther. 261 (1992) 318 – 323. [2] J.S. Chapula, Increase in action potential duration and inhibition of the delayed rectifier outward current IK by berberine in cat ventricular myocytes, Br. J. Pharmacol. 117 (1996) 1427 – 1437. [3] X.X. Chi, Z.C. Xu, Differential changes of potassium currents in CA1 pyramidal neurons after transient forebrain ischemia, J. Neurophysiol. 84 (2000) 2834 – 2843. [4] W.F. Chiou, M.H. Yen, C.F. Chen, Mechanism of vasodilatory effect of berberine in rat mesenteric artery, Eur. J. Pharmacol. 204 (1991) 35 – 40. [5] D.Z. Dai, Vulnerable substrate and multiple ion channel disorder in a diseased heart will be new targets for antiarrhythmic therapy, Acta Pharm. Sin. 21 (2000) 289 – 295. [6] E. Ficker, U. Heinemann, Slow and fast transient potassium currents in cultured rat hippocampal cells, J. Physiol. 445 (1992) 431 – 455. [7] I.J. Furlong, C. Lopez-Mediavilla, R. Ascaso, A. Lopez-Rivas, M.K. Collins, Induction of apoptosis by valinomycin: mitochondrial permeability transition causes intracellular acidification, Cell Death Differ. 5 (1998) 214 – 221. [8] T.M. Gao, W.A. Pulsinelli, Z.C. Xu, Changes in membrane properties of CA1 pyramidal neurons after transient forebrain ischemia in vivo, Neuroscience 90 (1999) 771 – 780. [9] E.V. Golanov, J.D. Christensen, D.J. Reis, Role of potassium channels in the central neurogenic neuroprotection elicited by cerebellar stimulation in rat, Brain Res. 842 (1999) 496 – 500. [10] M. Gomez-Angelats, J.A. Cidlowski, Cell volume and ion changes during apoptotic cell death, Adv. Cancer Res. 85 (2002) 175 – 201. [11] X. Hu, I.M. Johansson, T. Brannstrom, T. Olsson, P. Wester, Longlasting neuronal apoptotic cell death in regions with severe ischemia after photothrombotic ring stroke in rats, Acta Neuropathol. 104 (2002) 462 – 470(Berl.). [12] Z. Hua, X.L. Wang, Inhibitory effect of berberine on potassium channels in guinea pig ventricular myocytes, Acta Pharm. Sin. 29 (1994) 576 – 580. [13] W. Huang, Ventricular tachyarrhythmias treated with berberine, Zhonghua Xinxue Guanbing. Zazhi 18 (1990) 155 – 156. [14] A. Kathleen, Berberine, Altern. Med. Rev. 5 (2000) 175 – 177. [15] A.R. Kay, R.K. Wong, Isolation of neurons suitable for patch-clamping from adult mammalian central nervous systems, J. Neurosci. Methods 16 (1986) 227 – 238. [16] S. Krick, O. Platoshyn, M. Sweeney, H. Kim, J.X. Yuan, Activation of K+ channels induces apoptosis in vascular smooth muscle cells, Am. J. Physiol., Cell Physiol. 280 (2001) C970 – C979.
97
[17] O. Krishtal, Y. Kirichok, T. Tsintsadze, N. Lozovaya, W. Loesel, D. Arndts, New channel blocker BIIA388CL blocks delayed rectifier, but not A-type potassium current in central neurons, Neuropharmacology 40 (2001) 233 – 241. [18] C.W. Lau, X.Q. Yao, Z.Y. Chen, W.H. Ko, Y. Huang, Cardiovascular actions of berberine, Cardiovasc. Drug Rev. 19 (2001) 234 – 244. [19] M. Martina, J.H. Schultz, H. Ehmke, H. Monyer, P. Jonas, Functional and molecular differences between voltage-gated K+ channels of fastspiking interneurons and pyramidal neurons of rat hippocampus, J. Neurosci. 18 (1998) 8111 – 8125. [20] M.T. Nelson, J.M. Quayle, Physiological roles and properties of potassium channels in arterial smooth muscle, Am. J. Physiol., Cell Physiol. 268 (1995) C799 – C822. [21] R.W. Neumar, Molecular mechanisms of ischemic neuronal injury, Ann. Emerg. Med. 36 (2000) 483 – 506. [22] N. O’Reilly, Z. Xia, H. Fiander, J. Tauskela, D.L. Small, Disparity between ionic mediators of volume regulation and apoptosis in N1E 115 mouse neuroblastoma cells, Brain Res. 943 (2002) 245 – 256. [23] Y.P. Pan, X.H. Xu, X.L. Wang, Galantamine blocks delayed rectifier, but not transient outward potassium current in rat dissociated hippocampal pyramidal neurons, Neurosci. Lett. 336 (2002) 37 – 40. [24] R.L. Parsons, K.L. Barstow, F.S. Scornik, Spontaneous miniature hyperpolarizations affect threshold for action potential generation in mudpuppy cardiac neurons, J. Neurophysiol. 88 (2002) 1119 – 1127. [25] N.F. Ricciopo, Electropharmacological effects of berberine on canine cardiac Purkinje fibers and ventricular muscle and atrial muscle of the rabbit, Br. J. Pharmacol. 108 (1993) 534 – 537. [26] B.J. Snider, F.J. Gottron, D.W. Choi, Apoptosis and necrosis in cerebrovascular disease, Ann. N.Y. Acad. Sci. 893 (1999) 243 – 253. [27] J.F. Storm, Potassium currents in hippocampal pyramidal cells, Prog. Brain Res. 83 (1990) 161 – 187. [28] S. Uryu, J. Harada, M. Hisamoto, T. Oda, Troglitazone inhibits both post-glutamate neurotoxicity and low-potassium-induced apoptosis in cerebellar granule neurons, Brain Res. 924 (2002) 229 – 236. [29] X. Wang, A.Y. Xiao, T. Ichinose, S.P. Yu, Effects of tetraethylammonium analogs on apoptosis and membrane currents in cultured cortical neurons, J. Pharmacol. Exp. Ther. 295 (2000) 524 – 530. [30] Y.Z. Wei, Z.B. Yao, Q.F. Yuan, Y.C. Chen, Protective effect of berberine on rat hippocampus against cerebral ischemia, Chinese, J. Chem. Neuroanat. 11 (1995) 315 – 321. [31] L. Wei, S.P. Yu, F. Gottron, B.J. Snider, G.J. Zipfel, D.W. Choi, Potassium channel blockers attenuate hypoxia- and ischemia-induced neuronal death in vitro and in vivo, Stroke 34 (2003) 1281 – 1286. [32] J.F. Wu, Y.J. Shi, T.P. Liu, Protective effects of berberine on cerebral ischemia in mice and rats, Chinese, J. Pharmacol. Toxicol. Methods 9 (1995) 100 – 103. [33] A.Y. Xiao, L. Wei, S.L. Xia, S. Rothman, S.P. Yu, Ionic mechanism of ouabain-induced concurrent apoptosis and necrosis in individual cultured cortical neurons, J. Neurosci. 15 (2002) 1350 – 1362. [34] Z. Xu, H.Y. Cao, Q. Li, Protective effects of berberine on spontaneous ventricular fibrillation in dogs after myocardial infarction, Zhongguo Yaoli Xuebao 10 (1989) 320 – 324. [35] S.P. Yu, C.H. Yeh, F. Gottron, X. Wang, M.C. Grabb, D.W. Choi, Role of the outward delayed rectifier K+ current in ceramide-induced caspase activation and apoptosis in cultured cortical neurons, J. Neurochem. 73 (1999) 933 – 941. [36] T.S. Zetterstrom, R.D. Vaughan-Jones, D.G. Grahame-Smith, A short period of hypoxia produces a rapid and transient rise in [K+] e in rat hippocampus in vivo which is inhibited by certain K+-channel blocking agents, Neuroscience 67 (1995) 815 – 821.