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Thioperamide reduces intracellular calcium in mouse brain synaptosomes
European Neuropsychopharmacology (2007) 17, 375–376
w w w. e l s e v i e r. c o m / l o c a t e / e u r o n e u r o
SHORT COMMUNICATION
Thioperamide reduces intracellular calcium in mouse brain synaptosomes Divya Vohora a,⁎, Shanthi N. Pal b , K.K. Pillai a a b
Department of Pharmacology, Faculty of Pharmacy, Hamdard University, New Delhi 110062, India Health Technology & Pharmaceuticals, World Health Organization, Geneva -27, Switzerland
Received 12 April 2006; received in revised form 13 June 2006; accepted 8 August 2006 KEYWORDS Thioperamide; R(α)methylhistamine; H3 receptors; Intracellular calcium; Brain synaptosomes;
Abstract Intracellular calcium regulation is vital for cells, especially for neurons; raised levels are associated with cytotoxicity and neuronal death. In this report, we present the first experimental evidence showing a concentration-dependent reduction of free calcium in the mouse brain synaptosomes by thioperamide (THP), an H3 receptor antagonist. This is interesting in view of the recent reports on the anticonvulsant and cognition facilitating effects of THP. A neuroprotective potential of THP is suggested. © 2006 Elsevier B.V. and ECNP. All rights reserved.
Regulation of intracellular calcium (Ca2+) concentrations is vital for cells, especially for neurons; raised levels are associated with cytotoxicity and neuronal death. Here we show a reduction of free Ca2+ in mouse brain synaptosomes following treatment with thioperamide (THP). This is the first report for such an action by any H3 receptor antagonist. It is quite interesting and points to the probable mechanisms involved in the reported anticonvulsant and procognitive actions of THP. It also opens up exciting possibilities for potential utility of H3 receptor antagonists as neuroprotective agents in varied neurodegenerative disorders in which intracellular Ca2+ accumulation is known to play a key role. The discovery of presynaptic autoreceptors (modulating release and synthesis of histamine) and highly selective H3 receptor ligands by Arrang et al. (1987) generated considerable research interest with spurt in activity in this area. These agents proved to be useful investigative tools and provided ⁎ Corresponding author. Tel.: +91 11 2605 9688/5657; fax: +91 11 2605 9663. E-mail addresses: [email protected], [email protected] (D. Vohora).
evidence for protective action in convulsive and neurodegenerative disorders (Akhtar et al., 2005; Alguacil and PerezGarcia, 2003; Vohora, 2004; Vohora et al., 2004). Calcium ions are known to play a critical role in signal transduction, neurotransmitter release and, therefore, in the physiopathology of the nervous system. Large differences in the intra- and extra-cellular Ca2+ concentration highlight the vital role of the mechanisms controlling their influx and efflux across the cell membrane (Decoster, 1995). Dysfunction or loss of such regulatory mechanisms is known to result in hazardously elevated intracellular Ca2+ levels. The latter may initiate a cascade of events culminating in cytotoxicity and neuronal death (Jimenez-Jimenez et al., 1996). Analysis for Ca 2+ concentrations was done using a Luminescence Spectrometer (Perkin-Elmer, LS-50B) following the method of Martin et al. (1991). The synaptosomes were prepared from the whole brains of Swiss strain male albino mice and loaded with acetoxymethyl ester of the dye fura-2 (fura-2AM) (Komulainen and Bondy, 1987). The Grynkiewicz equation (Grynkiewicz et al. 1985) was used for calculation of results: ½Ca2þ i ¼ fR−Rmin =Rmax −Rg Kd Sf 2 =Sb2 ;
0924-977X/$ - see front matter © 2006 Elsevier B.V. and ECNP. All rights reserved. doi:10.1016/j.euroneuro.2006.08.002
376
D. Vohora et al.
Table 1 Effect of selective histamine H3 receptor ligands on the resting intrasynaptosomal calcium concentrations in the mouse brain Treatments
Control Thioperamide 1 μM 100 μM 1 mM R(α) methylhistamine 100 μM 1 mM
n
Resting [Ca2+]i (nM) 15 s
120 s
7
280.54 ± 27.56
268.51 ± 23.93
6 5 3
211.90 ± 47.57 119.50 ± 30.55⁎ 65.91 ± 23.77⁎⁎
196.58 ± 45.10 113.78 ± 29.59⁎ 64.88 ± 24.99⁎⁎
3 3
266.91 ± 21.51 293.93 ± 22.59
267.52 ± 18.96 289.05 ± 23.31
n: number of samples used per group. ⁎p b 0.05, ⁎⁎p b 0.01 vs. control. Significant by one-way ANOVA followed by Dunnett's t test.
where R, Rmax and Rmin represented the ratio of fluorescence (F340/F380) of the sample, maximum dye response in presence of saturating concentration of calcium and minimal dye response in presence of excess ethyleneglycol-bis-(β aminoethyl ether)-N,N′-tetra acetic acid (EGTA), respectively; Kd is the dissociation constant, and Sf2 and Sb2 denote the fluorescence of fura-2AM at zero calcium and full calcium saturation, respectively, at the excitation wavelength of 380 nm. We show a significant and concentration-dependent reduction in intracellular Ca2+ concentrations in the mouse brain synaptosomes by THP, a selective H3 receptor antagonist. R-(α)-methylhistamine (RAMH), a selective agonist for this receptor, exhibited no appreciable alterations in brain cell Ca2+ concentrations (Table 1). Lowering of intracellular Ca2+ levels by THP is interesting in view of some previous studies by us (Vohora et al., 2000, 2001) and some recent work (Harada et al., 2004) reporting its anticonvulsant effects. This is because reduced intracellular Ca2+ is now considered to be an important mechanism for most conventional and some of the newer antiepileptic drugs (AEDs) (Stefani et al., 1997). Besides epilepsy, elevated intracellular Ca2+ is linked to functional degeneration of aged neurons with impaired cognition (Ouanounou et al., 1999). While lack of effect on cell Ca2+ levels by RAMH is not clear, two possibilities may be considered for such an observation: i) the effect of THP on intracellular Ca2+ may be independent of its action on H3 receptors, and ii) two different receptor sites may be responsible. West et al. (1990) identified a high affinity and a low affinity site for THP. Ample evidence exists for functional distinctions between H3 receptor subtypes. While our findings also attribute differential effects of agonist and antagonist to the presence of distinct subtypes, more investigations are warranted to exclude alternative possibilities. Our results suggest a neuroprotective potential for THP. A modulatory effect on intracellular Ca2+ in brain could be a major target for its reported anticonvulsant and procognitive actions. However, this is only speculative and deserves to be followed up by more experimental studies to explore the full therapeutic potential of THP and other H3 receptor antago-
nists in varied clinical conditions especially those associated with intracellular Ca2+ accumulation (Decoster, 1995; Jimenez-Jimenez et al., 1996) e.g. epilepsy, Alzheimer's disease, Parkinson's disease, ischemic brain injury, etc. As some newer AEDs have been designed on this premise (Stefani et al., 1997), such an optimistic speculation does not appear to be irrational.
Acknowledgement We gratefully acknowledge the financial support from UGC, New Delhi.
References Akhtar, M., Pillai, K.K., Vohora, D., 2005. Effect of thioperamide on modified forced swimming test induced oxidative stress in mice. Basic Clin. Pharmacol. Toxicol. 97, 218-221. Alguacil, L.F., Perez-Garcia, C., 2003. Histamine H3 receptors: a potential drug target for the treatment of central nervous system disorders. Curr. Drug Targets CNS Neurol. Disord. 2, 303-315. Arrang, J.M., Garbarg, M., Lancelot, J.C., Lecomte, J.M., Pollard, H., Robba, M., Schunack, W., Schwartz, J.C., 1987. Highly potent and selective ligands for histamine H3 receptors. Nature 327, 117-123. Decoster, M.A., 1995. Calcium dynamics in the central nervous system. Adv. Neuroimmunol. 5, 233-239. Grynkiewicz, G., Poenie, M., Tsein, R.Y., 1985. A new generation of calcium indicators with greatly improved fluorescent properties. J. Biol. Chem. 260, 3440-3450. Harada, C., Hirai, T., Fujii, Y., Harusawa, S., Kurihara, T., Kamei, C., 2004. Intracerebroventricular administration of histamine H3 receptor antagonists decreases seizures in rat models of epilepsies. Methods Find. Exp. Clin. Pharmacol. 26, 263-270. Jimenez-Jimenez, F.J., Garcia-Ruiz, P.J., de Bustos, F., 1996. Calcium, neuronal death and neurological disease. Rev. Neurol. 134, 1199-1209. Komulainen, H., Bondy, S.C., 1987. The estimation of free calcium within synaptosomes and mitochondria with fura-2; comparison to quin-2. Neurochem. Int. 10, 55-64. Martin, J.V., Keir, D.R., Lee, H., 1991. Diazepam enhances intrasynaptosomal free calcium concentrations. Brain Res. 549, 222-227. Ouanounou, A., Zhang, L., Charlton, M.P., Carlen, P.L., 1999. Differential modulation of synaptic transmission by calcium chelators in young and aged hippocampal CA 1 neurons: evidence for altered calcium homeostasis in ageing. J. Neurosci. 19, 906-915. Stefani, A., Spadoni, F., Bernardi, G., 1997. Voltage-activated calcium channels: targets of antiepileptic drug therapy. Epilepsia 38, 950-965. Vohora, D., 2004. Histamine, selective H3 receptor ligands and cognitive functions: an overview. Investig. Drugs 7, 667-673. Vohora, D., Pal, S.N., Pillai, K.K., 2000. Thioperamide, a selective histamine H3-receptor antagonist, protects against PTZ-induced seizures in mice. Life Sci. 66, 297-301. Vohora, D., Pal, S.N., Pillai, K.K., 2001. Histamine and selective H3receptor ligands: a possible role in the mechanism and management of epilepsy. Pharmacol. Biochem. Behav. 68, 735-741. Vohora, D., Pal, S.N., Pillai, K.K., 2004. Histamine as an anticonvulsant inhibitory neurotransmitter. Curr. Neuropharmacol. 2, 419-425. West, R.E., Zweig, A., Shih, N.Y., Siegel, M.I., Egan, R.W., Clark, M.A., 1990. Identification of two H 3-histamine receptor subtypes. Mol. Pharmacol. 38, 610-613.
w w w. e l s e v i e r. c o m / l o c a t e / e u r o n e u r o
SHORT COMMUNICATION
Thioperamide reduces intracellular calcium in mouse brain synaptosomes Divya Vohora a,⁎, Shanthi N. Pal b , K.K. Pillai a a b
Department of Pharmacology, Faculty of Pharmacy, Hamdard University, New Delhi 110062, India Health Technology & Pharmaceuticals, World Health Organization, Geneva -27, Switzerland
Received 12 April 2006; received in revised form 13 June 2006; accepted 8 August 2006 KEYWORDS Thioperamide; R(α)methylhistamine; H3 receptors; Intracellular calcium; Brain synaptosomes;
Abstract Intracellular calcium regulation is vital for cells, especially for neurons; raised levels are associated with cytotoxicity and neuronal death. In this report, we present the first experimental evidence showing a concentration-dependent reduction of free calcium in the mouse brain synaptosomes by thioperamide (THP), an H3 receptor antagonist. This is interesting in view of the recent reports on the anticonvulsant and cognition facilitating effects of THP. A neuroprotective potential of THP is suggested. © 2006 Elsevier B.V. and ECNP. All rights reserved.
Regulation of intracellular calcium (Ca2+) concentrations is vital for cells, especially for neurons; raised levels are associated with cytotoxicity and neuronal death. Here we show a reduction of free Ca2+ in mouse brain synaptosomes following treatment with thioperamide (THP). This is the first report for such an action by any H3 receptor antagonist. It is quite interesting and points to the probable mechanisms involved in the reported anticonvulsant and procognitive actions of THP. It also opens up exciting possibilities for potential utility of H3 receptor antagonists as neuroprotective agents in varied neurodegenerative disorders in which intracellular Ca2+ accumulation is known to play a key role. The discovery of presynaptic autoreceptors (modulating release and synthesis of histamine) and highly selective H3 receptor ligands by Arrang et al. (1987) generated considerable research interest with spurt in activity in this area. These agents proved to be useful investigative tools and provided ⁎ Corresponding author. Tel.: +91 11 2605 9688/5657; fax: +91 11 2605 9663. E-mail addresses: [email protected], [email protected] (D. Vohora).
evidence for protective action in convulsive and neurodegenerative disorders (Akhtar et al., 2005; Alguacil and PerezGarcia, 2003; Vohora, 2004; Vohora et al., 2004). Calcium ions are known to play a critical role in signal transduction, neurotransmitter release and, therefore, in the physiopathology of the nervous system. Large differences in the intra- and extra-cellular Ca2+ concentration highlight the vital role of the mechanisms controlling their influx and efflux across the cell membrane (Decoster, 1995). Dysfunction or loss of such regulatory mechanisms is known to result in hazardously elevated intracellular Ca2+ levels. The latter may initiate a cascade of events culminating in cytotoxicity and neuronal death (Jimenez-Jimenez et al., 1996). Analysis for Ca 2+ concentrations was done using a Luminescence Spectrometer (Perkin-Elmer, LS-50B) following the method of Martin et al. (1991). The synaptosomes were prepared from the whole brains of Swiss strain male albino mice and loaded with acetoxymethyl ester of the dye fura-2 (fura-2AM) (Komulainen and Bondy, 1987). The Grynkiewicz equation (Grynkiewicz et al. 1985) was used for calculation of results: ½Ca2þ i ¼ fR−Rmin =Rmax −Rg Kd Sf 2 =Sb2 ;
0924-977X/$ - see front matter © 2006 Elsevier B.V. and ECNP. All rights reserved. doi:10.1016/j.euroneuro.2006.08.002
376
D. Vohora et al.
Table 1 Effect of selective histamine H3 receptor ligands on the resting intrasynaptosomal calcium concentrations in the mouse brain Treatments
Control Thioperamide 1 μM 100 μM 1 mM R(α) methylhistamine 100 μM 1 mM
n
Resting [Ca2+]i (nM) 15 s
120 s
7
280.54 ± 27.56
268.51 ± 23.93
6 5 3
211.90 ± 47.57 119.50 ± 30.55⁎ 65.91 ± 23.77⁎⁎
196.58 ± 45.10 113.78 ± 29.59⁎ 64.88 ± 24.99⁎⁎
3 3
266.91 ± 21.51 293.93 ± 22.59
267.52 ± 18.96 289.05 ± 23.31
n: number of samples used per group. ⁎p b 0.05, ⁎⁎p b 0.01 vs. control. Significant by one-way ANOVA followed by Dunnett's t test.
where R, Rmax and Rmin represented the ratio of fluorescence (F340/F380) of the sample, maximum dye response in presence of saturating concentration of calcium and minimal dye response in presence of excess ethyleneglycol-bis-(β aminoethyl ether)-N,N′-tetra acetic acid (EGTA), respectively; Kd is the dissociation constant, and Sf2 and Sb2 denote the fluorescence of fura-2AM at zero calcium and full calcium saturation, respectively, at the excitation wavelength of 380 nm. We show a significant and concentration-dependent reduction in intracellular Ca2+ concentrations in the mouse brain synaptosomes by THP, a selective H3 receptor antagonist. R-(α)-methylhistamine (RAMH), a selective agonist for this receptor, exhibited no appreciable alterations in brain cell Ca2+ concentrations (Table 1). Lowering of intracellular Ca2+ levels by THP is interesting in view of some previous studies by us (Vohora et al., 2000, 2001) and some recent work (Harada et al., 2004) reporting its anticonvulsant effects. This is because reduced intracellular Ca2+ is now considered to be an important mechanism for most conventional and some of the newer antiepileptic drugs (AEDs) (Stefani et al., 1997). Besides epilepsy, elevated intracellular Ca2+ is linked to functional degeneration of aged neurons with impaired cognition (Ouanounou et al., 1999). While lack of effect on cell Ca2+ levels by RAMH is not clear, two possibilities may be considered for such an observation: i) the effect of THP on intracellular Ca2+ may be independent of its action on H3 receptors, and ii) two different receptor sites may be responsible. West et al. (1990) identified a high affinity and a low affinity site for THP. Ample evidence exists for functional distinctions between H3 receptor subtypes. While our findings also attribute differential effects of agonist and antagonist to the presence of distinct subtypes, more investigations are warranted to exclude alternative possibilities. Our results suggest a neuroprotective potential for THP. A modulatory effect on intracellular Ca2+ in brain could be a major target for its reported anticonvulsant and procognitive actions. However, this is only speculative and deserves to be followed up by more experimental studies to explore the full therapeutic potential of THP and other H3 receptor antago-
nists in varied clinical conditions especially those associated with intracellular Ca2+ accumulation (Decoster, 1995; Jimenez-Jimenez et al., 1996) e.g. epilepsy, Alzheimer's disease, Parkinson's disease, ischemic brain injury, etc. As some newer AEDs have been designed on this premise (Stefani et al., 1997), such an optimistic speculation does not appear to be irrational.
Acknowledgement We gratefully acknowledge the financial support from UGC, New Delhi.
References Akhtar, M., Pillai, K.K., Vohora, D., 2005. Effect of thioperamide on modified forced swimming test induced oxidative stress in mice. Basic Clin. Pharmacol. Toxicol. 97, 218-221. Alguacil, L.F., Perez-Garcia, C., 2003. Histamine H3 receptors: a potential drug target for the treatment of central nervous system disorders. Curr. Drug Targets CNS Neurol. Disord. 2, 303-315. Arrang, J.M., Garbarg, M., Lancelot, J.C., Lecomte, J.M., Pollard, H., Robba, M., Schunack, W., Schwartz, J.C., 1987. Highly potent and selective ligands for histamine H3 receptors. Nature 327, 117-123. Decoster, M.A., 1995. Calcium dynamics in the central nervous system. Adv. Neuroimmunol. 5, 233-239. Grynkiewicz, G., Poenie, M., Tsein, R.Y., 1985. A new generation of calcium indicators with greatly improved fluorescent properties. J. Biol. Chem. 260, 3440-3450. Harada, C., Hirai, T., Fujii, Y., Harusawa, S., Kurihara, T., Kamei, C., 2004. Intracerebroventricular administration of histamine H3 receptor antagonists decreases seizures in rat models of epilepsies. Methods Find. Exp. Clin. Pharmacol. 26, 263-270. Jimenez-Jimenez, F.J., Garcia-Ruiz, P.J., de Bustos, F., 1996. Calcium, neuronal death and neurological disease. Rev. Neurol. 134, 1199-1209. Komulainen, H., Bondy, S.C., 1987. The estimation of free calcium within synaptosomes and mitochondria with fura-2; comparison to quin-2. Neurochem. Int. 10, 55-64. Martin, J.V., Keir, D.R., Lee, H., 1991. Diazepam enhances intrasynaptosomal free calcium concentrations. Brain Res. 549, 222-227. Ouanounou, A., Zhang, L., Charlton, M.P., Carlen, P.L., 1999. Differential modulation of synaptic transmission by calcium chelators in young and aged hippocampal CA 1 neurons: evidence for altered calcium homeostasis in ageing. J. Neurosci. 19, 906-915. Stefani, A., Spadoni, F., Bernardi, G., 1997. Voltage-activated calcium channels: targets of antiepileptic drug therapy. Epilepsia 38, 950-965. Vohora, D., 2004. Histamine, selective H3 receptor ligands and cognitive functions: an overview. Investig. Drugs 7, 667-673. Vohora, D., Pal, S.N., Pillai, K.K., 2000. Thioperamide, a selective histamine H3-receptor antagonist, protects against PTZ-induced seizures in mice. Life Sci. 66, 297-301. Vohora, D., Pal, S.N., Pillai, K.K., 2001. Histamine and selective H3receptor ligands: a possible role in the mechanism and management of epilepsy. Pharmacol. Biochem. Behav. 68, 735-741. Vohora, D., Pal, S.N., Pillai, K.K., 2004. Histamine as an anticonvulsant inhibitory neurotransmitter. Curr. Neuropharmacol. 2, 419-425. West, R.E., Zweig, A., Shih, N.Y., Siegel, M.I., Egan, R.W., Clark, M.A., 1990. Identification of two H 3-histamine receptor subtypes. Mol. Pharmacol. 38, 610-613.