The endogenous alkaloid harmane: Acidifying and activity-reducing effects on hippocampal neurons in vitro

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Progress in Neuro-Psychopharmacology & Biological Psychiatry 32 (2008) 362 – 367 www.elsevier.com/locate/pnpbp

The endogenous alkaloid harmane: Acidifying and activity-reducing effects on hippocampal neurons in vitro Udo Bonnet a,b,⁎, Norbert Scherbaum a , Martin Wiemann c a

Department of Addictive Behaviour and Addiction Medicine, University of Duisburg/Essen, Virchowstr. 174, D-45147 Essen, Germany b Department of Psychiatry und Psychotherapy, University of Duisburg/Essen, Virchowstr. 174, D-45147 Essen, Germany c Department of Physiology, University of Duisburg/Essen, Virchowstr. 174, D-45147 Essen, Germany Received 31 March 2007; received in revised form 28 August 2007; accepted 31 August 2007 Available online 7 September 2007

Abstract Rationale: The endogenous alkaloid harmane is enriched in plasma of patients with neurodegenerative or addictive disorders. As harmane affects neuronal activity and viability and because both parameters are strongly influenced by intracellular pH (pHi), we tested whether effects of harmane are correlated with altered pHi regulation. Methods and results: Pyramidal neurons in the CA3 field of hippocampal slices were investigated under bicarbonate-buffered conditions. Harmane (50 and 100 μM) reversibly decreased spontaneous firing of action potentials and caffeine-induced bursting of CA3 neurons. In parallel experiments, 50 and 100 μM harmane evoked a neuronal acidification of 0.12 ± 0.08 and 0.18 ± 0.07 pH units, respectively. Recovery from intracellular acidification subsequent to an ammonium prepulse was also impaired, suggesting an inhibition of transmembrane acid extrusion by harmane. Conclusion: Harmane may modulate neuronal functions via altered pHi-regulation. Implications of these findings for neuronal survival are discussed. © 2007 Elsevier Inc. All rights reserved. Keywords: Addiction; Harmane; Intracellular pH; MAO; Neuroprotection; pH regulation

1. Introduction Harmane is an alkaloid found to be elevated in blood plasma of patients suffering from Parkinson's disease (Kuhn et al., 1995) or addictive disorders (Stohler et al., 1993; Rommelspacher et al., 1996; Spijkerman et al., 2002). The molecule (1-methyl-9H-pyridol[3,4-b]indole, C12H10N2) is formed in peripheral and brain tissue by the Pictet–Spengler condensation reaction between an indoleamine and acetaldehyde Abbreviations: AP, action potential; MAO, monoamine oxidase; BCECF AM, 2′,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-acetoxymetyl ester; CA3, cornu ammonum 3; 5-HT, 5-hydroxytryptamine; MAO, monoamine oxidase; NHE, Na+/H+-exchanger; NH4Cl, ammonium chloride; pHi, intracellular pH. ⁎ Corresponding author. Rheinische Kliniken Essen, Klinik für abhängiges Verhalten und Suchtmedizin & Klinik für Psychiatrie und Psychotherapie der Universität Duisburg/Essen, Virchowstr. 174, D-45147 Essen, Germany. E-mail address: [email protected] (U. Bonnet). 0278-5846/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2007.08.043

(Susilo et al., 1987). However, exogenous harmane from coffee or cigarette smoke may also reach the brain (Herraiz and Chaparro, 2006; Rommelspacher et al., 2002). Up to now, the effective concentration of harmane around neurons is not known and effective levels range from 10− 10– 10− 4 M (Ergene and Schoener, 1993; Adell and Myers, 1995; Adell et al., 1996). In brain lysates a half maximal inhibition of monoamine-oxidase A (MAO-A) occurred at 0.3 μM (Herraiz and Chaparro, 2006). However, concentrations of harmane increasing the level of monoamines in vivo are in the upper micromolar range: A local intracerebral infusion of 200 μM harmane elevated extracellular levels of 5-HT and noradrenalin in the hippocampus (Adell and Myers, 1995; Adell et al., 1996). An increase of 5-HT upon systemic application also demanded 5–20 mg/kg harmane, which roughly corresponds to 25–100 μM (Adell et al., 1996). A largely uniform depression of neuronal activity in nucleus accumbens neurons was observed at 0.01 to 1 μM harmane, but not with lower

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concentrations (Ergene and Schoener, 1993). Based on these previous studies we used micromolar concentrations of harmane in the present study on hippocampal slices. Harmane has been suggested to act as an endogenous antidepressant and/or anxiolytic compound (Rommelspacher et al., 1994), or as a neuroprotectant (Kim et al., 2001; Park et al., 2003). The beneficial effects of harmane have mainly been attributed to MAO-A inhibition (Rommelspacher et al., 1994). In previous studies (Bonnet et al., 2000a) we found that various synthetic MAO-A inhibitors such as moclobemide and clorgyline reduced intracellular steady state pH (pHi). A moderate intracellular acidification is known to reduce bioelectric activity of hippocampal neurons (Bonnet et al., 1998, 2000c) and has strong neuroprotective properties (Tombaugh, 1994; Vornov et al., 1996; Simon et al., 1993). In fact, moclobemide also reduced the bioelectric activity in this model (Bonnet et al., 2000a). In the present investigation we hypothesized that harmane – in analogy to other MAO-A inhibitors – could reduce both, pHi and neuronal activity. To this aim we used BCECF AM loaded CA3 neurons in the hippocampal slice preparation (Bonnet and Wiemann, 1999) and measured the effect of harmane on steady state pHi and pHi regulation subsequent to ammonium prepulse. In parallel, effects of harmane on spontaneous and caffeinestimulated bioelectric activity of CA3 neurons (Moraidis et al., 1991) were tested via intracellular recording. 2. Material and methods 2.1. Tissue preparation Transverse slices (about 500 μm thick) were cut with a guided razor blade from hippocampi of ether anaesthetised adult guinea pigs (300–400 g). Slices were pre-incubated in 28 °C warm saline equilibrated with 5% CO2 in O2 for 2 h. This saline contained in mM: NaCl 124, KCl 3, CaCl2 0.75, MgSO4 1.3, KH2PO4 1.25, NaHCO3 26 and glucose 10. After pre-incubation, slices were transferred to a perspex recording chamber (volume 4 ml) mounted on an inverted microscope. The submerged slices were superfused with 32 °C warm saline (rate: 4.5 ml/min). The composition of this control solution was the same as during the pre-incubation period except for the calcium concentration which was raised to 1.75 mM. Harmane (Sigma) and caffeine (Sigma) were added to this control saline. The pH of all solutions was 7.35–7.4 and was kept constant in each experiment. 2.2. Analysis of pHi changes Hippocampal slices were stained with 0.5–1 μM 2′,7-bis(2carboxyethyl)-5(6)-carboxyfluorescein-acetoxymetyl ester (BCECF-AM, Molecular Probes) for 3–5 min in the preincubation saline. Free dye was removed by superfusing slices for at least 30 min with control saline. Superficially located neurons of the stratum pyramidale of the CA3 region were identified by stained apical dendrites using a 40× or 60× water immersion objective (Olympus) (Bonnet and Wiemann, 1999). Neurons selected for measurements had a stable BCECF

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Fig. 1. Effects of harmane (100 μM) on CA3 neurons (hippocampus slice, guinea-pig). A: Steady pHi of a BCECF-AM loaded neuron is reversibly reduced by harmane. B: pHi regulation subsequent to an ammonium prepulse is inhibited by harmane. In this experiment the BCECF AM loaded slice was preincubated for 20 min with harmane before acid extrusion was challenged twice by repetitive ammonium prepulses (20 mM NH4Cl, 3 min). Note that pHi recovery re-appeared after wash out of harmane. Dotted lines superimposed to both curves reflect the slopes of pHi recovery. C: Intracellular recording of membrane potential. Harmane reversibly reduced the frequency of caffeineinduced bursting. Single bursts are shown above at an expanded time scale.

fluorescence (ratio imaging with alternating excitation 440 nm/ 490 nm, emission wavelength N520 nm) with loss of fluorescence intensity (at 440 nm) being b1% per min, indicating that cells under investigation were in good condition (Bevensee et al., 1995). Changes in pHi were measured as described (Bonnet et al., 2002; Hentschke et al., 2006). In brief, background-corrected fluorescence image pairs were captured every 20 s by an intensified CCD camera (PTI, Surbiton Surrey, England), ratioed, and converted into pHi changes using a standard curve (Bonnet and Wiemann, 1999). Harmane, in contrast to harmaline (Bonnet et al., 2000b) had no autofluorescence when excited at 440 nm or 490 nm and, hence, did not change the BCECF fluorescent signal. pHi curves shown in Fig. 1 were smoothed by calculating sliding averaging from 3–6 values, to remove noise and/or smaller pHi deflections due to neuronal activity. To study pHi regulation, the ammonium-prepulse technique (Bevensee and Boron, 1998) adapted to hippocampal slice preparations (Bonnet and Wiemann, 1999) was used. NH4Cl (20 mM) applied for 3 min acidified neurons by ∼0.3 pH units. Recovery of pHi (ΔpHi/min) was calculated from the negative slope by linear regression as described (Bonnet et al., 2000a). 2.3. Intracellular recordings Membrane potential was intracellularly recorded from CA3 neurons of stratum pyramidale using sharp glass microelectrodes

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filled with 2 M potassium methylsulfate (120–180 MΩ). To estimate input resistance, current pulses (0.1 nA, 100 ms) were injected into neurons through the recording electrode by means of a bridge circuit. 2.4. Statistics All data are given as mean ± standard deviation of at least three independent experiments on different slices. The t-test for paired samples was used to compare pHi values prior to and after application of harmane. Differences were considered significant for P ≤ 0.05. 3. Results 3.1. Effect of harmane on steady state pHi and pHi-regulation of CA3-neurons To investigate effects of harmane on pHi and pHi regulation, 13 measurements were carried out on slices from 12 different animals. During superfusion with control solution CA3 neurons had a steady state pHi of 6.92 ± 0.18 (n = 13). Addition of either 50 or 100 μM harmane resulted in an intracellular acidification of 0.12 ± 0.08 (n = 6, P = 0.015) and 0.18 ± 0.07 pH units (n = 7, P b 0.01), respectively. There were no significant differences between both groups (P = 0.36). Steady state pHi values upon harmane application were reached within 5–20 min (Fig. 1A). In no case did we find signs of pHi regulation, such as a gradual return to baseline value pH in the presence of harmane, or an alkaline overshoot upon washout of the drug. The harmanemediated acidification was partially reversible upon washout within 30 min, as was observed earlier for other non-alkaloid MAO-inhibitors (Bonnet et al., 2000a). The influence of harmane on pHi regulation was studied in five CA3 neurons after acid loading using the ammonium prepulse technique. In these experiments slices were pre-treated with harmane for 15–20 min before 20 mM ammonium chloride was applied for 3 min. Harmane (100 μM) attenuated the re-increase of pHi (Fig. 1B) such that ΔpHi/min decayed from 0.017 ± 0.003 (control) to 0.0063 ± 0.008 (n = 5, P b 0.05). Together, these findings point to an impairment of cytoplasmic acid extrusion (Bevensee and Boron, 1998; Bonnet et al., 2000a; b). 3.2. Effect of harmane on bioelectric activity of CA3-neurons To investigate effects of harmane on bioelectric activity, a total of 10 CA3 neurons (each from a different guinea pig) were intracellularly recorded. All neurons had a resting membrane potential more negative than − 50 mV and action potential (AP) amplitudes N 50 mV. Spontaneous activity of CA3 neurons (n = 5) consisted of postsynaptic potential-like fluctuations of the membrane potential, single AP, and occasional burst activity. In five recordings caffeine (1 mM) was added to the control solution to test for effects of harmane while the neuron had adopted a stable state of hyperexcitability (Fig. 1C), which was characterized by a regular periodic burst activity (Moraidis et al., 1991; Bonnet and Wiemann, 1999; Bonnet et al., 2000a).

Harmane (50 and 100 μM) induced a continuous and partially reversible suppression of both spontaneous bioelectric activity (n = 5) and caffeine-induced epileptiform activity (n = 5): Application of 50 μM (n = 2) or 100 μM harmane (n = 3) for 20 min reduced action potential (AP) frequency from 17.7 ± 4.6 min− 1 or 13.8 ± 4.6 min− 1 to 2.5 ± 1.4 min− 1 and 1.2 ± 0.9 min− 1, respectively. Twenty min after return to nominally drug-free conditions, AP frequency partially recovered and reached 5.7 ± 3.9 min− 1 and 5.5 ± 2.3 min− 1, respectively. Similarly, 50 μM (n = 3) or 100 μM harmane (n = 2) given for 20 min lowered the frequency of caffeine-induced bursts from 7.3 ± 2.4 min− 1 or 10.2 ± 3.2 min− 1 to 0.7 ± 0.7 min− 1 and 0.3 ± 0.2 min− 1, respectively. Burst frequency re-increased upon a 30 minute lasting wash period to 2.8 ± 0.8 min− 1 and 1.8 ± 1.8 min− 1, respectively. A typical example is shown in Fig. 1C. In both groups neither membrane potential nor input resistance of neurons were altered by harmane. These results are in line with our previous studies with MAO-inhibitors (Bonnet et al., 2000a; Buesselberg et al., 2002) and inhibitors of transmembrane acid extrusion (Bonnet et al., 2000b; c). 4. Discussion The main findings of this investigation were that micromolar concentrations of harmane reversibly reduced the steady state pHi of hippocampal CA3-neurons by less than 0.2 pH units and that this moderate acidification developed along with a reduction in neuronal activity. Since harmane inhibited recovery from an intracellular acid load subsequent to an ammonium prepulse, the effect on steady state pHi may be caused by an inhibition of transmembrane acid extrusion. 4.1. pHi-effects of MAO-inhibitors It has been shown that pHi regulation of hippocampal neurons depends on the activity of Na+/H+-exchanger (NHE) and the Na+-dependent HCO3−/Cl− exchange (Bevensee and Boron, 1998; Bonnet et al., 2000b). With respect to the various subtypes of NHE (reviewed by Orlowski and Grinstein, 2004), preliminary data showed that 50–100 μM harmane can inhibit the activity of NHE1, but not of NHE2 or NHE3 (W. Jansen, personal communication). NHE1 as well as other NHE isoforms are expressed in hippocampal neurons (Ma and Haddad, 1997) but the contribution of single NHE isotypes to pHi regulation of pyramidal neurons is not yet clear. At least, amiloride, which also inhibits NHE1, was able to acidify neurons under the same experimental conditions (Bonnet et al., 2000b). Provided that NHE1 makes a major contribution to pHi regulation, this could explain effects of harmane on pHi and pHi regulation seen in this investigation. However, as intracellular H+-activity is determined by cellular H+ production and by the sum of all transmembrane fluxes of H+ or HCO3− (reviewed by Chesler, 2003) we cannot exclude that harmane alters net influx of H+ (e.g. by stimulating acid loaders) or influences H+-production. For example, concentrations of harmane used in this investigation most likely inhibited MAO-A and MAO-B (Rommelspacher et al., 1994), and the harmane-mediated acidification

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may be partially linked to this effect. This is because desamination catalyzed by MAO produces NH3, whose pK value is larger than that of the previous amino group (9.2 vs 8.6). Lowering the rate of desamination could, therefore, increase activity of free protons. It is in line with these considerations that the related compound harmaline, which has long been recognized as a MAO-A inhibitor (Udenfriend et al., 1958; Pletscher et al., 1959), also lowers steady state pHi (Raley-Susman et al., 1991; Bonnet et al., 2000b), as did MAOA inhibitors moclobemide and clorgyline (Bonnet et al., 2000a), as well as the MAO-B inhibitor selegeline (Buesselberg et al., 2002). However, in case of moclobemide and harmaline we also observed an inhibition of pHi recovery (Bonnet et al., 2000a; b). Together these findings suggest that inhibition of the MAO reaction lowers steady state pHi per se, and/or that at least some MAO inhibitors, when provided at micromolar concentrations, interfere with acid extruders. As in other pharmacological studies (c.f. Adell et al., 1996) the concentration of harmane used here were comparatively high, and because the tissue concentration of harmane in vivo is unknown, we are unable to address the question in as much our findings really reflect the situation in vivo. Certainly, the mechanism by which harmane and other MAO inhibitors acidify neurons needs further investigation. 4.2. pHi and bioelectric activity The electrophysiological recordings showed that harmane suppressed AP- and burst firing of caffeine-treated hippocampal neurons while membrane potential and input resistance were not changed. Recordings strongly resembled previous neurograms in which a similar inhibition of burst frequency was obtained upon physicochemical acidification, e.g. by NH4Cl or bicarbonate withdrawal (Bonnet et al., 1998; Bonnet and Wiemann, 1999). However, the effect of harmane may be aided by some inhibitory action on voltage-gated calcium channel currents, which were observed in a similar concentration range (IC50: 75.8 μM, Splettstoesser et al., 2005) and which might inhibit caffeine-induced epileptiform activity (Moraidis et al., 1991). On the other hand, some of these calcium-fluxes could have also been impaired by a decrease in pHi (Tombaugh, 1998; Bonnet et al., 1998). The increase in H+-activity necessary to lower membrane excitability in the hippocampus and also in human cortical neurons is quite low (Bonnet and Wiemann, 1999; Bonnet et al., 2000a, b, c; Xiong et al., 2000; Wiemann et al., 2006) and may be mediated by an interplay of numerous ion channels (summarized by Bonnet et al., 1998). In case of the hippocampal zero calcium epileptic model system, a superficially recorded acidification of only 0.03 pH units lowered the frequency of epileptiform bursting (Xiong et al., 2000). Similarly, we found that a drop of less than 0.1 pH unit upon bath application of e.g. propionate had suppressive effects on epileptiform discharges elicited by bicuculline, penicilline, caffeine, or 4-aminopyridine in the hippocampus (Bonnet et al., 2000c). Therefore, the pHi changes of 0.12–0.18 pH units evoked by 50–100 μM harmane appear sufficient to inhibit epileptiform burst firing at least in

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part. The fact that full blown inhibition of neuronal activity was somewhat delayed when compared to the onset of superficial acidification seems to contradict this interpretation. Taking into account that in hippocampal slices intracellularly recorded neurons are typically at a depth of more than 100 μm (Moraidis et al., 1991), a delayed response to harmane could be based on limitations set by diffusion. It has also been shown that a moderate intracellular acidification of central neurons influences the release and uptake of monoamines, and the binding to or activity of monoamine receptors (Hartly and Dubinsky, 1993; Kaila, 1994; Cao et al., 1998; Trudeau et al., 1999; Smith et al., 1998). This might explain accessory effects of harmane observed at the level of complex neuronal networks (Airaksinen and Mikkonen, 1980; Ergene and Schoener, 1993; Adell and Myers, 1995; Baum et al., 1996; RuizDurantez et al., 2001; Anderson et al., 2005; Touiki et al., 2005). For example, the seizure-threshold which is typically lowered during alcohol-withdrawal, might be raised upon endogenous synthesis of harmane, and this may be regarded a counteradaptive process in alcoholics. In line with this, the urinary harmane excretion is negatively correlated with the severity of alcohol withdrawal (Wodarz et al., 1996). The fact that harmane increases the threshold for convulsions (Aricioglu et al., 2003) is important in this context and is reflected by the anticonvulsive effect of harmane on caffeine-treated neurons in the present study. These examples substantiate the view that pHi effects should be considered when effects of harmane on the nervous system are discussed. 4.3. Impact on cellular viability Although harmane can induce toxic or apoptotic effects (Uezono et al., 2001; Hans et al., 2005) results from this study are more suited to explain the beneficial or neuroprotective side of harmane. In fact, especially higher concentrations have been found to decrease firing rate of most though not all neurons (Ergene and Schoener, 1993). The question arising from our findings is: Does neuronal acidification fit into the concept of harmane-induced effects? In general, a moderate decrease in pHi may help to limit excitotoxicity (Tombaugh and Sapolsky, 1993; Tombaugh, 1994; Saybasili, 1998). It will also lower energy expenditure in vulnerable neurons and this could finally result in neuroprotection (Simon et al., 1993; Tombaugh and Sapolsky, 1993; Tombaugh, 1994). Moderate intracellular acidosis may especially be helpful for neutral or slightly alkalotic neurons which may become protected (Simon et al., 1993; Tombaugh and Sapolsky, 1993; Tombaugh, 1994; Vornov et al., 1996; Almaas et al., 2003). On the other hand, stressed and already acidic neurons may respond to harmane with apoptosis or necrosis (Siesjö et al., 1993; Ying et al., 1999; Ding et al., 2000), especially when mechanisms of acid extrusion are further inhibited. In addition to effects on intracellular pH, neuroprotection is achieved if the concentration of reactive oxygen species generated by mitochondria is reduced (Kuhn et al., 1995; Stern, 1998). Although MAO inhibitors have no effect on mitochondrial respiration (Cohen and Kesler, 1999) they restrict

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