TRANSITION METAL IONS SIGNIFICANTLY DECREASE ...
Copyright © 2003 by Institute of Pharmacology Polish Academy of Sciences
Polish Journal of Pharmacology
Pol. J. Pharmacol., 2003, 55, 915917 ISSN 1230-6002
PRELIMINARY COMMUNICATION
TRANSITION METAL IONS SIGNIFICANTLY DECREASE PHOSPHOLIPASE C ACTIVITY DEGRADING PHOSPHATIDYLINOSITOL-4,5-BISPHOSPHATE IN THE BRAIN CORTEX Agata Zambrzycka, Magdalena C¹ka³a, Monika Kamiñska Department of Cellular Signaling, Medical Research Center, Polish Academy of Sciences, Pawiñskiego 5, PL 02-106 Warszawa, Poland Transition metal ions significantly decrease phospholipase C activity degrading phosphatidylinositol-4,5-bisphosphate in the brain cortex. A. ZAMBRZYCKA, M. CAKALA, M. KAMIÑSKA. Pol. J. Pharmacol., 2003, 55, 915–917. Highly reactive transition metals, such as copper and iron play an obligatory role in generating of reactive oxygen species (ROS). Many neurodegenerative diseases including Alzheimer’s disease (AD) and Parkinson’s disease (PD) show increased accumulation of these metals. Phosphoinositide metabolism is altered in neurodegenerative diseases. In the present study, we examined the effect of CuSO4 and FeCl2 on phospholipase C (PLC) activity degrading phosphatidylinositol-4,5-bisphosphate (PIP2) and phosphatidylinositol (PI) in synaptic plasma membranes (SPM) from the rat brain cortex. We report that 25 mM CuSO4 and FeCl2 decreased PIP2-PLC activity by 60% and 75%, respectively. However, both compounds had no effect on PI-PLC activity. These data indicated that exclusively PIP2-PLC is sensitive to transition metal ions. We suggest that chelators of these metals may protect brain against alteration of phosphoinositide metabolism and might be beneficial in the treatment of neurodegenerative diseases. Key words: phospholipase C, phosphatidylinositol-4,5-bisphosphate, copper, iron
correspondence; e-mail: [email protected]
A. Zambrzycka, M. Cakala, M. Kamiñska
Increased brain metal levels have been associated with normal aging and a variety of diseases, including Alzheimer’s (AD) and Parkinson’s (PD) diseases. Copper and iron have been found in amyloid plaques at » 0.4 and 1 mM concentration, re-
Hipp
Ctx
Ctx
spectively [4]. These redox active metal ions play an obligatory role in the formation of H2O2 and reactive oxygen species (ROS), such as superoxide anion (O2) and hydroxyl radical (HO) and in mediating ROS-induced damage. Hydroxyl radical, produced when transition metals react with H2O2 in a “Fenton-type” reaction, is the most powerful oxidizing species among several reactive-oxygen radicals [1]. Through ROS formation and oxidation of biomolecules, Cu2+ and Fe3+ may lead to cell damage and necrotic or apoptotic cell death [6]. The phosphoinositide second messenger system is a major signal transduction pathway in the brain. There are three inositol-containing phospholipids (phosphoinositides) in this system, phosphatidylinositol (PI), phosphatidylinositol-4-monophosphate (PIP), and phosphatidylinositol-4,5-bisphosphate (PIP2). There is evidence that each of the three phosphoinositides can be hydrolyzed by phospholipase C (PLC). Degradation of PIP2 generating the intracellular second messengers: diacylglycerol (DAG) and inositol(1,4,5)-trisphosphate (IP3) [5]. PLCb is the key enzyme in PIP2 hydrolysis [2]. It is well documented that phosphoinositide metabolism is impaired in AD [3]. Our previous results indicated that Ab peptide significantly decreased PIP2 degradation by PLC and that neurotoxic fragment
PLC activity [nmol x mg of protein-1 x min -1 ]
2.0
Cd
Pir Fig. 1. PLCb antisense probe hybridization to coronal sections of the rat brain. Cd – caudate nucleus, Ctx – cerebral cortex, Hipp – hippocampus, Pir – pyriform cortex (Bar = 5 mm)
916
PIP2-PLC
1.8
PI-PLC
1.6 1.4 1.2 1.0 0.8 0.6
*
*
FeCl2
CuSO4
0.4 0.2 0.0
control
Fig. 2. The effect of FeCl and CuSO" on PIP -PLC and PI-PLC activity in SPM from the rat brain cortex. SPM were preincubated for 30 min at 37°C in the presence of FeCl and CuSO" at 25 mM concentration or without these compounds (control). Following the preincubation, PIP -PLC and PI-PLC activity was assayed. Each value is the mean ± SD from 3 experiments carried out in triplicate. Statistically significant differences were calculated based on paired Student’s t-test, * p < 0.05 compared to the control
Pol. J. Pharmacol., 2003, 55, 915–917
TRANSITION METAL IONS DECREASE PLC ACTIVITY of Ab peptide, Ab 25-35, had no effect on PI hydrolysis [8]. In the present study, we investigated the distribution of PLCb mRNA in the rat brain and the effect of copper and iron on PIP2 and PI degradation by PLC. In situ hybridization was performed using of the procedure of Young et al. [7]. PIP2- and PI-PLC activities were assayed in synaptic plasma membranes (SPM) from the rat brain cortex prepared as described previously [8]. SPM were incubated in the absence (control) and in the presence of 25 mM CuSO4 and FeCl2 for 30 min at 37°C. Then PIP2-PLC and PI-PLC activity was assayed as described previously [8] with exogenous phosphatidyl[2-3H]inositol-4,5-bisphosphate (sp. act., 1.00 Ci/mmol) and phosphatidyl[3H]inositol (sp. act., 16.3 Ci/mmol) as substrates, respectively. The results obtained by in situ hybridization indicated that PLCb degrading PIP2 is widespread in the brain, mainly in the cerebral cortex and hippocampus (Fig. 1). Exposure of SPM to 25 mM FeCl2 and CuSO4 resulted in a 75% and 60% inhibition of PIP2-PLC, respectively, but they had no effect on PI-PLC activity (Fig. 2). In this work, we report that copper and iron significantly decreased PIP2-PLC activity and that PI-PLC was not sensitive to these metals. These results corresponded with our previous data indicating that exclusively PIP2-PLC was inhibited by Ab 25-35 and that this toxic fragment of Ab had no effect on PI-PLC activity [8]. Mechanism of iron and copper action on PIP2-PLC is still unclear and needs future investigation. However, on the basis of data that copper and iron activated ROS formation, it is possible to suggest that free radicals may be involved in PIP2-PLC inhibition evoked by these metals. In summary, the data reported here demonstrate that accumulation of transition metal ions in neurodegenerative diseases may be respon-
ISSN 1230-6002
sible for the alteration of PIP2 derived lipid messengers formation and their function. We suggest that specific copper and iron chelators could protect brain against Cu and Fe-evoked phosphoinositide metabolism alteration. REFERENCES 1. Gutteridge J.M., Wilkins S.: Copper salt-dependent hydroxyl radical formation. Damage to proteins acting as antioxidants. Biochim. Biophys. Acta, 1983, 759, 38–41. 2. Jope R.S.: Cholinergic muscarinic receptor signaling by the phosphoinositide signal transduction system in Alzheimer’s disease. Alzh. Dis. Rev., 1996, 1, 2–14. 3. Jope R.S., Song L., Powers R.E.: Cholinergic activation of phosphoinositide signaling is impaired in Alzheimer’s disease brain. Neurobiol. Aging, 1997, 18, 111–120. 4. Lovell M.A., Robertson J.D., Teesdale W.J., Campbell J.L., Markesbery W.R.: Copper, iron and zinc in Alzheimer’s disease senile plaques. J. Neurol. Sci., 1998, 158, 47–52. 5. Martin T.F.J.: Phosphoinositide lipids as signaling molecules: common themes for signal transduction, cytoskeletal regulation, and membrane traffiking. Annu. Rev. Cell Biol., 1998, 14, 231–264. 6. Pourahmad J., O’Brien P.J.: A comparison of hepatocyte cytotoxic mechanisms for Cu2+ and Cd2+. Toxicology, 2000, 143, 263–273. 7. Young W.S., Mezey E., Siegel R.E.: Quantitative in situ hybridization histochemistry reveals increased levels of corticotropin-releasing factor mRNA after adrenalectomy in rats. Neurosci. Lett., 1986, 70, 198–203. 8. Zambrzycka A., Strosznajder R.P., Strosznajder J.B.: Aggregated beta amyloid peptide 1-40 decreases Ca2+and cholinergic receptor-mediated phosphoinositide degradation by alteration of membrane and cytosolic phospholipase C in brain cortex. Neurochem. Res., 2000, 25, 189–196. Received: October 13, 2003; in revised form: November 3, 2003.
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Polish Journal of Pharmacology
Pol. J. Pharmacol., 2003, 55, 915917 ISSN 1230-6002
PRELIMINARY COMMUNICATION
TRANSITION METAL IONS SIGNIFICANTLY DECREASE PHOSPHOLIPASE C ACTIVITY DEGRADING PHOSPHATIDYLINOSITOL-4,5-BISPHOSPHATE IN THE BRAIN CORTEX Agata Zambrzycka, Magdalena C¹ka³a, Monika Kamiñska Department of Cellular Signaling, Medical Research Center, Polish Academy of Sciences, Pawiñskiego 5, PL 02-106 Warszawa, Poland Transition metal ions significantly decrease phospholipase C activity degrading phosphatidylinositol-4,5-bisphosphate in the brain cortex. A. ZAMBRZYCKA, M. CAKALA, M. KAMIÑSKA. Pol. J. Pharmacol., 2003, 55, 915–917. Highly reactive transition metals, such as copper and iron play an obligatory role in generating of reactive oxygen species (ROS). Many neurodegenerative diseases including Alzheimer’s disease (AD) and Parkinson’s disease (PD) show increased accumulation of these metals. Phosphoinositide metabolism is altered in neurodegenerative diseases. In the present study, we examined the effect of CuSO4 and FeCl2 on phospholipase C (PLC) activity degrading phosphatidylinositol-4,5-bisphosphate (PIP2) and phosphatidylinositol (PI) in synaptic plasma membranes (SPM) from the rat brain cortex. We report that 25 mM CuSO4 and FeCl2 decreased PIP2-PLC activity by 60% and 75%, respectively. However, both compounds had no effect on PI-PLC activity. These data indicated that exclusively PIP2-PLC is sensitive to transition metal ions. We suggest that chelators of these metals may protect brain against alteration of phosphoinositide metabolism and might be beneficial in the treatment of neurodegenerative diseases. Key words: phospholipase C, phosphatidylinositol-4,5-bisphosphate, copper, iron
correspondence; e-mail: [email protected]
A. Zambrzycka, M. Cakala, M. Kamiñska
Increased brain metal levels have been associated with normal aging and a variety of diseases, including Alzheimer’s (AD) and Parkinson’s (PD) diseases. Copper and iron have been found in amyloid plaques at » 0.4 and 1 mM concentration, re-
Hipp
Ctx
Ctx
spectively [4]. These redox active metal ions play an obligatory role in the formation of H2O2 and reactive oxygen species (ROS), such as superoxide anion (O2) and hydroxyl radical (HO) and in mediating ROS-induced damage. Hydroxyl radical, produced when transition metals react with H2O2 in a “Fenton-type” reaction, is the most powerful oxidizing species among several reactive-oxygen radicals [1]. Through ROS formation and oxidation of biomolecules, Cu2+ and Fe3+ may lead to cell damage and necrotic or apoptotic cell death [6]. The phosphoinositide second messenger system is a major signal transduction pathway in the brain. There are three inositol-containing phospholipids (phosphoinositides) in this system, phosphatidylinositol (PI), phosphatidylinositol-4-monophosphate (PIP), and phosphatidylinositol-4,5-bisphosphate (PIP2). There is evidence that each of the three phosphoinositides can be hydrolyzed by phospholipase C (PLC). Degradation of PIP2 generating the intracellular second messengers: diacylglycerol (DAG) and inositol(1,4,5)-trisphosphate (IP3) [5]. PLCb is the key enzyme in PIP2 hydrolysis [2]. It is well documented that phosphoinositide metabolism is impaired in AD [3]. Our previous results indicated that Ab peptide significantly decreased PIP2 degradation by PLC and that neurotoxic fragment
PLC activity [nmol x mg of protein-1 x min -1 ]
2.0
Cd
Pir Fig. 1. PLCb antisense probe hybridization to coronal sections of the rat brain. Cd – caudate nucleus, Ctx – cerebral cortex, Hipp – hippocampus, Pir – pyriform cortex (Bar = 5 mm)
916
PIP2-PLC
1.8
PI-PLC
1.6 1.4 1.2 1.0 0.8 0.6
*
*
FeCl2
CuSO4
0.4 0.2 0.0
control
Fig. 2. The effect of FeCl and CuSO" on PIP -PLC and PI-PLC activity in SPM from the rat brain cortex. SPM were preincubated for 30 min at 37°C in the presence of FeCl and CuSO" at 25 mM concentration or without these compounds (control). Following the preincubation, PIP -PLC and PI-PLC activity was assayed. Each value is the mean ± SD from 3 experiments carried out in triplicate. Statistically significant differences were calculated based on paired Student’s t-test, * p < 0.05 compared to the control
Pol. J. Pharmacol., 2003, 55, 915–917
TRANSITION METAL IONS DECREASE PLC ACTIVITY of Ab peptide, Ab 25-35, had no effect on PI hydrolysis [8]. In the present study, we investigated the distribution of PLCb mRNA in the rat brain and the effect of copper and iron on PIP2 and PI degradation by PLC. In situ hybridization was performed using of the procedure of Young et al. [7]. PIP2- and PI-PLC activities were assayed in synaptic plasma membranes (SPM) from the rat brain cortex prepared as described previously [8]. SPM were incubated in the absence (control) and in the presence of 25 mM CuSO4 and FeCl2 for 30 min at 37°C. Then PIP2-PLC and PI-PLC activity was assayed as described previously [8] with exogenous phosphatidyl[2-3H]inositol-4,5-bisphosphate (sp. act., 1.00 Ci/mmol) and phosphatidyl[3H]inositol (sp. act., 16.3 Ci/mmol) as substrates, respectively. The results obtained by in situ hybridization indicated that PLCb degrading PIP2 is widespread in the brain, mainly in the cerebral cortex and hippocampus (Fig. 1). Exposure of SPM to 25 mM FeCl2 and CuSO4 resulted in a 75% and 60% inhibition of PIP2-PLC, respectively, but they had no effect on PI-PLC activity (Fig. 2). In this work, we report that copper and iron significantly decreased PIP2-PLC activity and that PI-PLC was not sensitive to these metals. These results corresponded with our previous data indicating that exclusively PIP2-PLC was inhibited by Ab 25-35 and that this toxic fragment of Ab had no effect on PI-PLC activity [8]. Mechanism of iron and copper action on PIP2-PLC is still unclear and needs future investigation. However, on the basis of data that copper and iron activated ROS formation, it is possible to suggest that free radicals may be involved in PIP2-PLC inhibition evoked by these metals. In summary, the data reported here demonstrate that accumulation of transition metal ions in neurodegenerative diseases may be respon-
ISSN 1230-6002
sible for the alteration of PIP2 derived lipid messengers formation and their function. We suggest that specific copper and iron chelators could protect brain against Cu and Fe-evoked phosphoinositide metabolism alteration. REFERENCES 1. Gutteridge J.M., Wilkins S.: Copper salt-dependent hydroxyl radical formation. Damage to proteins acting as antioxidants. Biochim. Biophys. Acta, 1983, 759, 38–41. 2. Jope R.S.: Cholinergic muscarinic receptor signaling by the phosphoinositide signal transduction system in Alzheimer’s disease. Alzh. Dis. Rev., 1996, 1, 2–14. 3. Jope R.S., Song L., Powers R.E.: Cholinergic activation of phosphoinositide signaling is impaired in Alzheimer’s disease brain. Neurobiol. Aging, 1997, 18, 111–120. 4. Lovell M.A., Robertson J.D., Teesdale W.J., Campbell J.L., Markesbery W.R.: Copper, iron and zinc in Alzheimer’s disease senile plaques. J. Neurol. Sci., 1998, 158, 47–52. 5. Martin T.F.J.: Phosphoinositide lipids as signaling molecules: common themes for signal transduction, cytoskeletal regulation, and membrane traffiking. Annu. Rev. Cell Biol., 1998, 14, 231–264. 6. Pourahmad J., O’Brien P.J.: A comparison of hepatocyte cytotoxic mechanisms for Cu2+ and Cd2+. Toxicology, 2000, 143, 263–273. 7. Young W.S., Mezey E., Siegel R.E.: Quantitative in situ hybridization histochemistry reveals increased levels of corticotropin-releasing factor mRNA after adrenalectomy in rats. Neurosci. Lett., 1986, 70, 198–203. 8. Zambrzycka A., Strosznajder R.P., Strosznajder J.B.: Aggregated beta amyloid peptide 1-40 decreases Ca2+and cholinergic receptor-mediated phosphoinositide degradation by alteration of membrane and cytosolic phospholipase C in brain cortex. Neurochem. Res., 2000, 25, 189–196. Received: October 13, 2003; in revised form: November 3, 2003.
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