Elsevier

Neuropharmacology

Volume 59, Issues 7–8, December 2010, Pages 567-572
Neuropharmacology

Tetrahydroberberine blocks ATP-sensitive potassium channels in dopamine neurons acutely-dissociated from rat substantia nigra pars compacta

https://doi.org/10.1016/j.neuropharm.2010.08.018Get rights and content

Abstract

Tetrahydroberberine (THB) exhibits neuroprotective effects but its targets and underlying mechanisms are largely unknown. Emerging evidence indicates that ATP-sensitive potassium (KATP) channels in the substantia nigra pars compacta (SNc) promote Parkinson disease (PD) pathogenesis, thus blocking KATP channels may protect neurons against neuronal degeneration. In the present study, we tested a hypothesis that THB blocks KATP channels in dopaminergic (DA) neurons acutely dissociated from rat SNc. Using perforated patch-clamp recording in current-clamp mode, the functional KATP channels can be opened by persistent perfusion of rotenone, an inhibitor of complex I of the mitochondrial respiratory chain. Bath-application of THB reversibly blocks opened KATP channels in a concentration-dependent manner, which is comparable to a classical KATP channel blocker, Tol. Compared to THB analogs, l-stepholidine (l-SPD) or l-tetrahydropalmatine (l-THP), THB exhibits more profound blockade in KATP channels. In addition, exposure of THB alone to the recorded neuron increases action potential firing, and THB also restores rotenone-induced membrane hyperpolarization in the presence of dopamine D2 receptor antagonist (sulpiride), suggesting that THB exhibits an excitatory effect on SNc DA neurons through the block of KATP channels. Collectively, the blockade of neuronal KATP channels by THB in SNc DA neurons is a novel pharmacological mechanism of THB, which may contribute to its neuroprotective effects in PD.

Introduction

Tetrahydroberberine (THB) is a compound isolated from the Chinese herb. l-tetrahydropalmatine (l-THP) and l-stepholidine (l-SPD) are the homologues analogs of THB, and they are called terahydroprotoberberine analogs (THPBs). Accumulating lines of evidence indicate that THPBs exhibit dopamine receptor antagonistic effects on sedation, hypnosis, antinociception, anti-schizophrenia, anti-hypertension, as well as the prevention of drug addiction (Bian et al., 1986, Chu et al., 2008, Xiong et al., 1987, Zhang et al., 1986). In addition, the morphological and biochemical experiments have demonstrated that THPBs also have neuroprotective effects (Tang et al., 1999). For instance, in transient ischemic rat models, l-SPD antagonized ischemic injury by eliminating the activation of calcium/calmodulin-dependent protein kinase II (CCDPKII) (Tang et al., 1999), which has been reported to be involved in the mechanism of neuronal protection against ischemia (Uno et al., 1999). Furthermore, l-SPD also inhibited the release of lactate dehydrogenase (LDH), an indicator of injury, from neurons following ischemia, suggesting that l-SPD is able to decrease neuronal injury induced by hypoxia. Histological examination confirmed that l-SPD protects striatal cells against transient cerebral ischemic injury (Tang et al., 1999). Furthermore, the neuroprotective effects of l-SPD may be related to its ability to scavenge hydroxyl free radicals (Jin et al., 2000). However, the targets and underlying mechanisms of THPB-induced neuroprotection still remain elusive.

Although extensive works have indicated that DA receptors (D1 and D2) are the main targets that mediate pharmacological effects of THPBs (Chen et al., 1986, Chen et al., 1985, Fu et al., 2004, Hu et al., 1992, Jin et al., 1992, Shi et al., 1984, Sun and Jin, 1992, Tang et al., 1999, Zhang et al., 1999, Zhang et al., 1998, Zhu et al., 2000) other targets also have been reported to mediate THPBs’ effects including α-adrenergic receptor (Liu et al., 1989), 5-TH receptor (Miao et al., 1991), Ca2+ channels (Li et al., 1995, Miao et al., 1991, Shen et al., 1991) and K+ channels (Wu et al., 1996, Wu and Jin, 1997a, Wu and Jin, 1997b). These lines of evidence suggest that THPBs may act on multiple targets to exert their pharmacological roles in the CNS. Emerging evidence indicates that ATP-sensitive potassium (KATP) channels in the midbrain substantia nigra compacta (SNc) DA neurons promote pathogenesis in Parkinson disease (PD) animal models, which suggests that the blockade of KATP channels may protect neurons against MPTP neurotoxicity (Liss et al., 2005).

In the present study, we tested whether or not THB, l-SPD or l-THP blocks KATP channels in DA neurons acutely dissociated from rat SNc. Our results demonstrate that THB is a potent KATP channel blocker.

Section snippets

Single DA neuron dissociation from rat SNc

The protocol for preparation of single neurons from the rat SNc was approved by the Institutional Animal Care and Use Committee of the Barrow Neurological Institute.

Single DA neurons were acutely dissociated from the SNc of 2–3-week-old Wistar rats following the protocol as previously described (Wu et al., 2004, Wu et al., 2006, Yang et al., 2009). Briefly, rats were anesthetized with isoflurane, and brain tissue was rapidly removed and immersed in cold (2–4 °C) dissection solution which

Identification of dissociated SNc DA neurons

TH staining showed that the dissociated neurons from SNc exhibited TH positive (Fig. 1Ab, d) and negative (Fig. 1Ad*) reactions. For patch-clamp recording, DA neurons were identified early in the recording session based on previously described criteria (Lacey et al., 1989): (1) 1–3 Hz spontaneous action potential firing (Fig. 1Ba), (2) the duration of action potential is longer than 2.5 ms (Fig. 1Bb), (3) spontaneous action potential firing is eliminated by 10 μM DA (Fig. 1Ba), and (4)

Discussion

In the present study, we provide direct electrophysiological evidence that the KATP channels in SNc DA neurons are novel targets to mediate THB pharmacological effects. We show that THB exhibits the most profound block of KATP channels compared to its analogs l-SPD and l-THP. We also demonstrate that THB inhibits KATP channels in a concentration-dependent manner, and its inhibitory effect is comparable to the classical KATP channel blocker, Tol. Considering the roles of KATP channels in PD

Acknowledgements

We thank Dr. Denis Lecavalier for his assistance to edit the manuscript. This work was supported by CHW Seed Fund (J.W.), and the part of work in this study was also supported by Chinese government grants (973-project: 2009CB52200; CNSF: 30825042; International Collaboration Grant).

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