British Journal of Pharmacology (2005) 144, 52–58 & 2005 Nature Publishing Group All rights reserved 0007–1188/05 $30.00 www.nature.com/bjp Modulation of voltage-gated channel currents by harmaline and harmane 1Frank Splettstoesser, 2Udo Bonnet, 1Martin Wiemann, 1Dieter Bingmann & *,1Dietrich Bu¨ sselberg 1Institut fu¨ r Physiologie, Universita¨ tsklinikum Essen, Universita¨ t-Duisburg Essen, Hufelandstrasse 55, 45122 Essen, Germany and 2Klinik fu¨ r Psychiatrie und Psychotherapie, Universita¨ tsklinikum Essen, Universita¨ t Duisburg Essen, Hufelandstrasse 55, 45122 Essen, Germany 1 Harmala alkaloids are endogenous substances, which are involved in neurodegenerative disorders such as M. Parkinson, but some of them also have neuroprotective effects in the nervous system. 2 While several sites of action at the cellular level (e.g. benzodiazepine receptors, 5-HT and GABAA receptors) have been identified, there is no report on how harmala alkaloids interact with voltage- gated membrane channels. 3 The aim of this study was to investigate the effects of harmaline and harmane on voltage-activated calcium- (ICa(V)), sodium- (INa(V)) and potassium (IK(V))-channel currents, using the whole-cell patch- clamp method with cultured dorsal root ganglion neurones of 3-week-old rats. Currents were elicited by voltage steps from the holding potential to different command potentials. 4 Harmaline and harmane reduced ICa(V), INa(V) and IK(V) concentration-dependent (10–500 mM) over the voltage range tested. ICa(V) was reduced with an IC50 of 100.6 mM for harmaline and by a significantly lower concentration of 75.8 mM (Po0.001, t-test) for harmane. The Hill coefficient was close to 1. Threshold concentration was around 10 mM for both substances. 5 The steady state of inhibition of ICa(V) by harmaline or harmane was reached within several minutes. The action was not use dependent and at least partly reversible. 6 It was mainly due to a reduction in the sustained calcium channel current (ICa(L þ N)), while the transient voltage-gated calcium channel current (ICa(T)) was only partially affected. 7 We conclude that harmaline and harmane are modulators of ICa(V) in vitro. This might be related to their neuroprotective effects. British Journal of Pharmacology (2005) 144, 52–58. doi:10.1038/sj.bjp.0706024 Keywords: Voltage-gated calcium currents; voltage-gated sodium currents; voltage-gated potassium currents; harmane; harmaline; dorsal root ganglion neurones; rat Abbreviations: BMI, Biomedical Instruments; CNS, central nervous system; DRG, dorsal root ganglion; F-12, Hams F-12 medium; GABAA, g-aminobutyric acid a receptor subtype; GHB, g-hydroxybutyrate; harmaline, 1-methyl-b- carboline; harmane, 1-methyl-7-methoxy-3,4-dihydro-b-carboline; 5-HT, 5-hydroxytryptamin; 5-HT2A, 5-hydro- xytryptamin 2A receptor; 5-HT2C, 5-hydroxytryptamin 2C receptor; ICa(L þ N), sustained voltage-gated calcium channel current; ICa(T), transient voltage-gated calcium channel current; ICa(V), voltage-gated calcium channel current; IK(V), voltage-gated potassium channel current; INa(V), voltage-gated sodium channel current; IC50, inhibitory concentration reducing 50%; I–V curve, current–voltage relation curve; MAO, monoaminoxidase; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridin; NHE, sodium–hydrogen exchange; PC-12, rat adrenal pheochromocytoma Introduction The b-carbolines 1-methyl-b-carboline (harmane), 1-methyl-7- heroin-dependent humans (Stohler et al., 1996), patients with methoxy-3,4-dihydro-b-carboline (harmaline) and 9H-pyri- essential tremor (Louis et al., 2002) or Parkinson’s disease do[3,4-b]indole (norharmane) are naturally present in the (Kuhn et al., 1996), they are assumed to have a crucial role in human food chain and supposed to occur endogenously in the pathophysiology of various disorders of the CNS. normal body constituents. They are found in the blood plasma, Speculations on the biological significance of b-carbolines heart, kidney, liver and also in brain tissue, where they have comprised cytotoxic as well as neuroprotective properties. On been proposed to be endogeneous ligands for benzodiazepine the one hand, b-carbolines have been postulated to act as and imidazoline receptors (Rommelspacher et al., 1980; May endogeneous neurotoxins because of their structural similarity to et al., 1994; Hudson et al., 1999). Since high plasma levels of 1-methyl-4-phenyl-1-1,2,3,6-tetrahydropyridine (MPTP) (Albores these compounds have been found in heavy smokers (Spijker- et al., 1990), a compound which induced a parkinsonian-like man et al., 2002), alcoholics (Rommelspacher et al., 1991), syndrome in animals (Przedborski & Jackson-Lewis, 1998). Furthermore, harmaline has been shown to have a cytotoxic effect on PC-12cells (Cobuzzi et al., 1994) and to cause *Author for correspondence; E-mail: [email protected] degeneration of Purkinje cells in rat cerebellum. The latter finding Advance online publication: 8 November 2004 may explain its tremor-evoking action (O’hearn & Molliver, 1993). F. Splettstoesser et al Harmala alkaloids modulate voltage-gated currents 53 On the other hand, and, more recently, b-carbolines have Spinal cord was removed and dorsal root ganglia (DRG) were been shown to protect neurones against the excitotoxic effects collected by fine forceps from both sides of the spinal column of dopamine and glutamate (Maher & Davis, 1996). Accord- and transferred into ice-cooled F-12medium (Sigma, Tauf- ingly, a protective role of b-carbolines in the pathophysiology kirchen, Germany). Spinal nerves were cut off under optical of Parkinson’s disease has been suggested. Increasing evidence control using fine scissors. Then, the ganglia were transferred also hints to a protective effect of elevated endogeneous into a mixture of 0.9 ml F12medium plus 0.1 ml collagenase b-carbolines on oxidative neuronal damage. For example, medium (2612.5 U mlÀ1, Sigma Type II) and digested in a b-carbolines were shown to depress the dopamine- or humidified atmosphere (5% CO2)at371C for 45–55 min. In 6-hydroxydopamine-induced brain mitochondrial and synap- the next step, the collagenase was removed by washing the tosomal dysfunctions, and also the loss of viability in PC-12 ganglia with F-12medium for at least three times. Trypsina- cells through a scavenging action on reactive oxygen species tion (2525 U trypsin per ml F-12 medium, Sigma Type IX) was and maintenance of reduced thiols (Kim et al., 2001). carried out for another 2–3 min under the same conditions. Apart from actions on benzodiazepine and imidazoline After adding F-12medium (final volume 0.7 ml), the DRG receptors, other effects of b-carbolines have been identified at were titurated with a fire-polished Pasteur pipette (tip diameter the cellular level. These include an activation of 5-HT2A and 150 mM) until the ganglion capsules were opened, and the 5-HT2C receptors, a potent inhibition of synaptosomal g- neurones were released from the ganglia. A portion of 50 mlof hydroxybutyrate (GHB) re-uptake (McCormick & Tunnicliff, the resulting suspension was placed in the middle of small petri 1998), and an impairment of monoamine oxidase enzymes dishes (3 cm; Falcon ‘Easy Grip’) and incubated for 2–4 h, (MAO) and of sodium–hydrogen exchange (NHE) (Glennon allowing cells to adhere. Thereafter, 1 ml F-12(with horse et al., 2000; Anderson et al., 2003). serum) was added to each petri dish. Cultures were used for It appears surprising that, despite these manifold effects of electrophysiological recordings within the next 2days. harmale alkaloids on various targets, little information is available about their action on neuronal sodium, potassium Recording techniques and isolation of the different and calcium channel currents and calcium homeostasis (Shi channel currents et al., 2001). Especially calcium – entering through membrane channels – acts as a major second messenger, whose intracellular With the whole-cell patch-clamp technique, membrane cur- concentration is tightly regulated. De-regulation results in rents of DRG neurones were recorded using a HEKA EPC 9 reduced functionality of neurones, and a permanently increased amplifier and EPC screen software (HEKA). Microelectrodes calcium concentration within the cell could also result in were pulled from borosilicate glass with filament (o.d.: 1.5 mm malfunction and even cell death. Therefore, it might be possible and i.d.: 0.86 mm; Biomedical Instruments (BMI)) with a that the opposing effects of harmala alkaloids described above Sutter electrode puller (model P-87). Electrodes were fire may involve Ca2 þ -homeostasis disturbances. In neurones, i polished with a Narashige micro forge (MF-830) to a final calcium enters the cell through different types of voltage-gated resistance of 3–4 MO and filled with the adequate internal channels. The three main types of voltage-gated calcium solution (see Table 1; all chemicals for these solutions were channels which have originally been described and studied in obtained from Sigma-Aldrich, Taufkirchen, Germany). Special dorsal root ganglion (DRG) neurones are L-, N- and T-type bath solutions were prepared to isolate the different types of channels (Fox et al., 1987). They differ with respect to their voltage-activated channel currents (see Table 1). After a giga- voltage dependency and activation and inactivation properties ohm seal had been obtained and a stable access to the neurones (for details, see Hille, 1992). Most important for Ca2 þ home- i had been established, membrane potential was routinely ostasis are the noninactivating L-type channels, whose activa- clamped at À80 mV. Voltage-activated channel currents were tion starts at a membrane potential of around À30 mV, and elicited by depolarising command pulses to 0 mV for 120 ms transient T-type channels, which are fast inactivating and open (80 ms when measuring current–voltage (I–V) relations) for upon ‘small’ depolarisations (in the range of À50 to À10 mV). In I , 15 ms for I and 200 ms for I . Data were sampled this study, we investigated the effects of harmane and harmaline Ca(V) Na(V) K(V) at 10 kHz and stored on hard disc.