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Picrotoxin-induced convulsions alters rat brain microsomal membrane structural properties
Neuroscience Letters 394 (2006) 9–12
Picrotoxin-induced convulsions alters rat brain microsomal membrane structural properties Munjal M. Acharya a,∗ , Surendra S. Katyare b a
Department of Biochemistry and Biotechnology, Institute of Science, Nirma University of Science and Technology, Sarkhej-Gandhinagar Highway, Ahmedabad 382481, Gujarat, India b Department of Biochemistry, Faculty of Science, M.S. University of Baroda, Sayajigunj, Vadodara 390002, Gujarat, India Received 14 June 2005; received in revised form 23 August 2005; accepted 24 August 2005
Abstract Cerebral microsomal membrane properties were assessed in the chronic condition of generalized seizure induced by picrotoxin (PTX) in rats. PTX-induced seizures resulted in increased lysophosphatidyl glycerol, phosphatidylcholine and phosphatidic acid components, while phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol were significantly reduced by 19–73%. The cholesterol (CHL) content increased considerably by 25% without alteration in total phospholipids content. Microsomal membrane was more fluidized in the epileptic condition. Possible consequences of microsomal membrane alterations are discussed in terms of deregulation of Ca2+ homeostasis. In conclusion, alterations in the microsomal membrane properties may have a significant influence on the cerebral function in the chronic epileptic condition. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Picrotoxin; Epilepsy; Microsome; Membrane fluidity; Phospholipids profile
Microsomes are the major site of biosynthetic and xenobiotic metabolism processes in the cell [9,24]. During resting condition, low-cytosolic free-calcium concentration is maintained, in part, by microsomal membrane-bound Mg2+ , Ca2+ -ATPase [38]. Intense neuronal activity may cause structural and functional changes in plasma and subcellular membranes. Brain damage during seizure episodes is related to increased intracellular Ca2+ levels [Ca2+ ]i , which has an important role in the regulation of diverse spectrum of cellular events as well as in epileptogenesis [19]. Additionally, intense seizure activity is reported to be associated with the inhibition of microsomal Mg2+ , Ca2+ -ATPasemediated Ca2+ uptake and deregulation of Ca2+ homeostasis [7,25,27]. Repeated seizure episodes lead to membrane degradation and accumulation of bioactive lipids [5]. Evidences also suggest the possible role of mitochondria and microsomes in the activation of programmed cell death (PCD) pathways during seizures [16,28,34]. Earlier studies in our laboratory demonstrated altered rat brain lysosomal and mitochondrial membrane function in the chronic epileptic condition [1,2].
∗
Corresponding author. Tel.: +91 2717 241900/01/02/03/04x627; fax: +91 2717 241916. E-mail address: [email protected] (M.M. Acharya). 0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.08.069
In the light of above, it may be anticipated that some epileptoform activity could be directly related to the alterations in crucial neuronal structures. It is therefore of interest to find whether chronic epileptic condition can also influence the structural properties of brain microsomes. Present studies report these changes in picrotoxin (PTX)-induced chronic epileptic condition. Male albino rats of Charles–Foster strain (200–250 g) were used. The animals had free access to food and water. PTX solution was prepared freshly in saline and was injected intraperitoneally (i.p.) at the dose of 1.5 mg/kg body weight for 20 consecutive days [21]. Initially, animals developed seizures within 30 min of PTX administration. Tonic–clonic convulsions were well established within 8–10 days of treatment. At the later stages, seizures developed within 10–20 min of PTX administration. The controls were given equivalent volume of saline. There was no mortality of rats. The animals were kept in individual cages and observed for incidences, character and intensity of epileptic manifestations. The animals were scored according to the scale described by Kubova et al. [15] as indicated below: 0: No changes 1: Uneasiness, scratching tremor, single myoclonic jerks 2: Atypical minimal seizures
10
M.M. Acharya, S.S. Katyare / Neuroscience Letters 394 (2006) 9–12
3: Minimal seizures consisting of clonic convulsions involving the head and forelimb muscles and leaving righting reflexes intact 4: Major seizures without the tonic phase 5: Complete major seizures, i.e. generalized tonic–clonic convulsions with loss of righting reflexes. The animals were killed by decapitation on day 21 of PTX treatment, and the brains were quickly removed and placed in beakers containing chilled (0–4 ◦ C) isolation medium (0.25 M sucrose containing 10 mM Tris–HCl pH 7.4 and 1 mM EDTA). Microsomes were isolated from post-mitochondrial supernatant as described earlier [12,26], washed thrice to give a protein concentration of 8–10 mg/ml. Microsomal lipids were extracted essentially according to the procedure described earlier [3,8]. Briefly, samples of microsomal suspension containing a total of 8–10 mg protein were extracted twice with chloroform: methanol (2:1, v/v) mixture, and pooled extracts were washed with 0.1 volume of 0.017% MgCl2 . Measured aliquots of final extracts were removed for total phospholipid (TPL) and CHL [41] estimation. TLC for separation of individual phospholipid classes was carried out according to the procedure of Skipiski et al. [35] using Silica gel G. Individual phospholipid spots from TLC plates and TPL samples were subjected to digestion with H2 SO4 , and complete oxidation was insured by treatment with perchloric acid. Liberated phosphorous was estimated by the method of Katewa and Katyare [13]. The measurements on membrane fluidity was carried out with 1,6-diphenyl-1,3,5 hexatriene (DPH) as the probe following the procedures described earlier in details [3,22]. Fluorescence polarization (P) was measured at 25 ◦ C in a Shimadzu RF-5000 spectrophotofluorimeter containing an inbuilt program for calculating and printing out fluorescence polarization (P) values. Calculations for fluorescence anisotropy (r), limited hindered anisotropy (r∞ ) of the fluorophore and order parameter (S) was done as described by van Blitterswijk et al. [40]. Protein estimation was done by the method of Lowry et al. [17] with BSA as the standard. Statistical evaluation of the data was performed using the Students’ t-test. Results on the effect of PTX-induced epileptic condition on the structural properties of microsomal membrane in terms of phospholipid (PL) composition, TPL and CHL contents and
Table 1 Effect of PTX-induced seizures on phospholipid composition of rat brain microsomes Phospholipid class
Phospholipid composition (nmol/mg protein) Control
Lyso CerPCho ChoGpl PtdSer PtdIns EtnGpl PtdOH
0.030 0.064 0.225 0.029 0.063 0.106 0.028
± ± ± ± ± ± ±
PTX 0.0036 0.0032 0.0132 0.0034 0.0046 0.0052 0.0023
0.052 0.064 0.295 0.015 0.017 0.086 0.035
± ± ± ± ± ± ±
0.0037**** 0.0031 0.0059**** 0.0011*** 0.0011**** 0.0041** 0.0020*
Change (%) +73 – +31 −48 −73 −19 +25
The experimental details are as described in the text. Results are given as mean ± S.E.M. of eight independent observations. Lyso, lysophosphatidyl glycerol; CerPCho, sphingomyelin; ChoGpl, phosphatidylcholine; PtdSer, phosphatidylserine; PtdIns, phosphatidylinositol; EtnGpl, phosphatidylethanolamine; and PtdOH, phosphatidic acid. * p < 0.05. ** p < 0.01. *** p < 0.002. ****p < 0.001.
membrane fluidity are shown in Tables 1 and 2. It is evident that the CHL content increased significantly by 25% without alteration in TPL content. Therefore a decrease in TPL:CHL (mole:mole) ratio was observed. When analyses were carried out to find out the PL contents, it was noted that lysophosphatidyl glycerol (Lyso), phosphatidylcholine (ChoGpl) and phosphatidic acid (PtdOH) components increased significantly by 1.25 to 1.7-fold (Table 1). While phosphatidylethanolamine (EtnGpl), phosphatidylserine (PtdSer) and phosphatidylinositol (PtdIns) were reduced drastically by 19–73% with maximum lowering observed for PtdIns (73% decrease). In the view of changes in PL composition, measurements were extended on the fluidity of the microsomal membranes as represented by the reciprocal of lipid structural order parameter, S [40]. It is evident from Table 2 that the microsomal membrane was more fluidized in PTX-induced epileptic animals. Endoplasmic reticulum (ER)-mediated sequestration of Ca2+ is crucial for proper neuronal functioning and maintenance of basal [Ca2+ ]i levels [6,23]. Activation of excitatory neurotransmitter receptor leads to Ca2+ influx into the neurons and increasing free [Ca2+ ]i [18], which has been implicated in the patho-
Table 2 Effect of PTX-induced seizures on total phospholipids, cholesterol content and membrane fluidity of rat brain microsomes Parameter
Control
PTX
Total phospholipid Cholesterol TPL:CHL
TPL (nmol/mg protein) CHL (nmol/mg protein) Molar ratio
0.571 ± 0.027 (8) 0.589 ± 0.024 (8) 0.974 ± 0.046 (8)
0.564 ± 0.016 (8) 0.738 ± 0.040 (8)* 0.772 ± 0.027 (8)**
Membrane fluidity
Fluorescence polarization (P) Fluorescence anisotropy (r) Limited hindered anisotropy (r∞ ) Order parameter (S)
0.206 0.147 0.097 0.494
± ± ± ±
0.007 (8) 0.005 (8) 0.005 (8) 0.017 (8)
0.133 0.093 0.024 0.242
± ± ± ±
The experimental details are as described in the text. Results are given as mean ± S.E.M., the number of observation indicated in the parentheses. * p < 0.01. ** p < 0.002. *** p < 0.001.
0.0016 (12)*** 0.0011 (12)*** 0.0016 (12)*** 0.0086 (12)***
M.M. Acharya, S.S. Katyare / Neuroscience Letters 394 (2006) 9–12
physiology of epilepsy [31,39]. Our results on PTX-induced epileptic condition on the microsomal membrane properties clearly indicated drastic alterations in the membrane composition. These compositional changes could lead to distorted charge distribution in the membrane microenvironment. Maintenance of Ca2+ homeostasis is regulated by proper functioning of Mg2+ , Ca2+ -ATPase. However, ATPases activity is known to be dependent on acidic phospholipids, in particular PtdSer and PtdIns [32]. PTX-induced convulsions resulted in 48–73% reduction in PtdSer and PtdIns components (Table 1). Significant inhibition of cortical Mg2+ , Ca2+ -ATPase-mediated Ca2+ uptake and Na+ , K+ -ATPase activity is reported in epileptic humans and animals [10,11,27]. Hence, it may be suggested that the changes in the microsomal ATPases could be attributed to decreased PtdSer and PtdIns components and altered membrane charge distribution. A decreased efficacy of ER means that neurons are less able to sequester Ca2+ influxes and re-establish the resting [Ca2+ ]i . These higher Ca2+ influxes may maintain further spontaneous recurrent seizures, characteristic of epileptic phenotype through buildup of [Ca2+ ]i and persistent increased basal Ca2+ levels. Substantial increase in Lyso (Table 1) may lead to fluidization of microsomal membrane, which is evident here (Table 2) since lysophosphoglycerides can exert detergent like action on the membranes [33]. Increased lysophosphoglycerides in microsomes (Table 1) and mitochondria [1] also indicates activation of phosopholipases, that are known to be activated by Ca2+ [5,37]. Significant alterations of [Ca2+ ]i has been observed in several models of epilepsy [20,29,33]. Disruption of ER activity and Ca2+ homeostasis has been implicated in apoptosis, excitotoxic disorders such as seizures and ischemia, neurodegenerative diseases such as Alzheimer’s, Huntington’s and Parkinson’s disease and uncontrolled cell growth [20,29,28]. PtdOH is an important precursor in phospholipid synthesis. Thus, increased PtdOH component (Table 1) is an indicator of altered phospholipid metabolism. In the present study, we did not explore fatty acid compositional changes but decreased EtnGpl would be expected to be associated with decrease in the unsaturation index [22]. Decreased PtdIns (by 73%; Table 1) might be related to membrane degradation, as PtdIns hydrolysis is often a feature of stimulated phospholipid turnover [30]. This observation is supported by earlier reports that decreased PtdIns could also be associated with seizure-induced excessive release of excitatory neurotransmitters and activation of phospholipases (PLA2 and PLC)-mediated signaling pathway [5]. Arachidonate is released from SN 2 position of glycerol backbone, which is implicated in neuronal plasticity, ischemic brain damage and epilepsy [4]. Earlier we have noted similar kind of drastic alterations in the structural and functional properties of mitochondrial membrane in PTX-induced chronic epileptic condition [1]. Participation of microsomal and mitochondrial function in Ca2+ handling [36] is a crucial task important for excitability and viability of neurons. In fact earlier reports also suggest the possible role of mitochondria and microsomes in the activation of PCD pathways during seizures [14,28,34]. Thus, intracellular membrane dysfunction may be implicated in the process of epileptogenesis.
11
In conclusion, our results on the alterations in microsomal membrane properties are coherent with earlier reports on the microsomal membrane dysfunction in Ca2+ homeostasis that may have a significant impact on the neuronal operation in the chronic epileptic condition. References [1] M.M. Acharya, S.S. Katyare, Structural and functional alterations in mitochondrial membrane in picrotoxin-induced epileptic rat brain, Exp. Neurol. 192 (2005) 79–88. [2] M.M. Acharya, S.H. Khamesra, S.S. Katyare, Picrotoxin-induced convulsions and lysosomal function in rat brain, Ind. J. Clin. Biochem. 20 (2005) 56–60. [3] C.S. Bangur, J.L. Howland, S.S. Katyare, Thyroid hormone treatment alters phospholipid composition and membrane fluidity of the rat brain mitochondria, Biochem. J. 305 (1995) 29–32. [4] N.G. Bazan, Arachidonic acid in the modulation of excitable membrane function and at the onset of brain damage, Ann. N.Y. Acad. Sci. 559 (1989) 1–16. [5] N.G. Bazan, B. Tu, E.B. Rodriguez de Turco, What synaptic lipid signaling tell us about seizure-induced damage and epileptogenesis, Prog. Brain Res. 135 (2002) 175–185. [6] E. Carafoli, Intracellular calcium homeostasis, Annu. Rev. Biochem. 56 (1987) 395–433. [7] M.S. Costa, M.G. Funke, L. de Meis, E.A. Cavalheiro, M.da G. NaffahMazzacoratti, Status epilepticus induced by pilocarpine and Ca2+ transport by microsome in the hippocampus of rats, Neurosci. Lett. 366 (2004) 292–296. [8] J. Folch, M. Less, G.H. Sloane-Stanley, A simple method for isolation and purification of total phospholipids from animal tissues, J. Biol. Chem. 226 (1957) 497–509. [9] L. Gerace, R. Gilmore, A. Johnson, P. Lazarow, W. Neupert, E. O’Shea, K. Weis, Intracellular compartments and protein sorting: the endoplasmic reticulum, in: B. Alberts, A. Johnson, J. Lewis, M. Raff, K. Roberts, P. Walter (Eds.), Molecular Biology of the Cell, fourth ed., Garland Science, New York, 2002, pp. 689–708. [10] D. Guillaume, Brain cortical (Na+ , K+ )-ATPase in epilepsy. A biochemical study in animals and humans, Acta Neurol. Belg. 88 (1988) 257– 280. [11] D. Guillaume, T. Grisar, A.V. Delgado-Escueta, J. Laschet, M. BureauHeeren, Phosphorylation of brain (Na+ , K+ )-ATPase alpha catalytic subunits in normal and epileptic control cerebral cortex: II. Partial seizures in human epilepsy, J. Neurosci. Res. 29 (1991) 218–224. [12] S.S. Katayre, P. Fatterpaker, A. Sreenivasan, Heterogenicity of rat liver mitochondrial fractions and the effect of tri-iodothyronine on their protein turnover, Biochem. J. 118 (1970) 111. [13] S.D. Katewa, S.S. Katyare, A simplified method for inorganic phosphate determination and its application for phosphate analysis in enzyme assays, Anal. Biochem. 323 (2003) 180–187. [14] G. Kromer, J.C. Reed, Mitochondrial control of cell death, Nat. Med. 6 (2000) 513–519. [15] M. Kubova, T. Bohuslav, P. Mares, Effects of clonazepam on picrotoxin induced convulsions, Physiol. Res. 41 (1992) 167–169. [16] M. Lam, G. Dubyak, L. Chen, G. Nunez, R.L. Miesfeld, C.W. Distelhorst, Evidence that Bcl-2 represses apoptosis by regulating endoplasmic reticulum-associated Ca2+ fluxes, Proc. Natl. Acad. Sci. U.S.A. 91 (1994) 6569–6573. [17] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with Folin phenol reagent, J. Biol. Chem. 193 (1951) 265–275. [18] A. MacDermott, G.L. Mayer, G.L. WestBrook, S.J. Smith, J.C. Barker, NMDA-receptor activation increases cytoplasmic calcium concentration in cultured spinal cord neurons, Nature (Lond.) 321 (1986) 519–522. [19] J.W. MacDonald, E.A. Garofalo, T. Hood, J.C. Sackellares, S. Gliman, P.C. McKeever, J.C. Troncoso, M.W. Johnston, Altered excitatory and inhibitory amino acid receptor binding in hippocampus of patients with temporal lobe epilepsy, Ann. Neurol. 29 (1991) 529–541.
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[31] M. Raza, S. Pal, A. Rafiq, R.J. DeLorenzo, Long-term alterations of calcium homeostatic mechanism in the pilocarpine model of temporal lobe epilepsy, Brain Res. 903 (2001) 1–12. [32] J.D. Robinson, M.A. Flashner, The (Na+ , K+ )-activated ATPase. Enzymatic and transport properties, Biochim. Biophys. Acta 549 (1979) 145–176. [33] R.J. Rossiter, K.P. Strickland, The metabolism and function of phosphatides, in: K. Bloch (Ed.), Lipid Metabolism, John Wiley, 1960, p. 69. [34] C.K. Schindler, S. Shinoda, R.P. Simon, D.C. Henshall, Subcellular distribution of Bcl-2 family proteins and 14-3-3 within the hippocampus during seizure-induced neuronal death in the rat, Neurosci. Lett. 356 (2004) 163–166. [35] V.P. Skipiski, M. Barelay, R.K. Barelay, V.A. Fetzer, J.J. Good, F.M. Archibald, Lipid composition of human serum lipoprotein, Biochem. J. 104 (1967) 340–361. [36] G. Szabadkai, R. Rizzuto, Participation of endoplasmic reticulum and mitochondrial calcium handling in apoptosis: more than just neighborhood? FEBS Lett. 567 (2004) 111–115. [37] M.A. Trimenstein, S.D. Nelson, Subcellular binding and effects on calcium homeostasis produced by acetaminophen and non-hepatotoxic regioisomer, 3 hydroxyacetanilide, in mouse liver, J. Biol. Chem. 264 (1989) 1814. [38] E.E. Trotta, L. de Meis, ATP-dependent calcium accumulation in brain microsomes enhancement by phosphate and oxalate, Biochim. Biophys. Acta 394 (1975) 239–247. [39] J.A. Ure, M. Perassolo, Update on the pathophysiology of the epilepsy, J. Neurol. Sci. 177 (2000) 1–17. [40] W.S. van Blitterswijk, R.P. van Hoeven, B.W. van der Meer, Lipid structural order parameters (reciprocal of fluidity) in biomembranes derived from steady state fluorescence polarization measurements, Biochim. Biophys. Acta 644 (1981) 323–332. [41] A. Zlatkis, B. Zak, J.A. Boyle, A new method for determination of serum cholesterol, J. Lab. Clin. Med. 41 (1953) 486–492.
Picrotoxin-induced convulsions alters rat brain microsomal membrane structural properties Munjal M. Acharya a,∗ , Surendra S. Katyare b a
Department of Biochemistry and Biotechnology, Institute of Science, Nirma University of Science and Technology, Sarkhej-Gandhinagar Highway, Ahmedabad 382481, Gujarat, India b Department of Biochemistry, Faculty of Science, M.S. University of Baroda, Sayajigunj, Vadodara 390002, Gujarat, India Received 14 June 2005; received in revised form 23 August 2005; accepted 24 August 2005
Abstract Cerebral microsomal membrane properties were assessed in the chronic condition of generalized seizure induced by picrotoxin (PTX) in rats. PTX-induced seizures resulted in increased lysophosphatidyl glycerol, phosphatidylcholine and phosphatidic acid components, while phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol were significantly reduced by 19–73%. The cholesterol (CHL) content increased considerably by 25% without alteration in total phospholipids content. Microsomal membrane was more fluidized in the epileptic condition. Possible consequences of microsomal membrane alterations are discussed in terms of deregulation of Ca2+ homeostasis. In conclusion, alterations in the microsomal membrane properties may have a significant influence on the cerebral function in the chronic epileptic condition. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Picrotoxin; Epilepsy; Microsome; Membrane fluidity; Phospholipids profile
Microsomes are the major site of biosynthetic and xenobiotic metabolism processes in the cell [9,24]. During resting condition, low-cytosolic free-calcium concentration is maintained, in part, by microsomal membrane-bound Mg2+ , Ca2+ -ATPase [38]. Intense neuronal activity may cause structural and functional changes in plasma and subcellular membranes. Brain damage during seizure episodes is related to increased intracellular Ca2+ levels [Ca2+ ]i , which has an important role in the regulation of diverse spectrum of cellular events as well as in epileptogenesis [19]. Additionally, intense seizure activity is reported to be associated with the inhibition of microsomal Mg2+ , Ca2+ -ATPasemediated Ca2+ uptake and deregulation of Ca2+ homeostasis [7,25,27]. Repeated seizure episodes lead to membrane degradation and accumulation of bioactive lipids [5]. Evidences also suggest the possible role of mitochondria and microsomes in the activation of programmed cell death (PCD) pathways during seizures [16,28,34]. Earlier studies in our laboratory demonstrated altered rat brain lysosomal and mitochondrial membrane function in the chronic epileptic condition [1,2].
∗
Corresponding author. Tel.: +91 2717 241900/01/02/03/04x627; fax: +91 2717 241916. E-mail address: [email protected] (M.M. Acharya). 0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.08.069
In the light of above, it may be anticipated that some epileptoform activity could be directly related to the alterations in crucial neuronal structures. It is therefore of interest to find whether chronic epileptic condition can also influence the structural properties of brain microsomes. Present studies report these changes in picrotoxin (PTX)-induced chronic epileptic condition. Male albino rats of Charles–Foster strain (200–250 g) were used. The animals had free access to food and water. PTX solution was prepared freshly in saline and was injected intraperitoneally (i.p.) at the dose of 1.5 mg/kg body weight for 20 consecutive days [21]. Initially, animals developed seizures within 30 min of PTX administration. Tonic–clonic convulsions were well established within 8–10 days of treatment. At the later stages, seizures developed within 10–20 min of PTX administration. The controls were given equivalent volume of saline. There was no mortality of rats. The animals were kept in individual cages and observed for incidences, character and intensity of epileptic manifestations. The animals were scored according to the scale described by Kubova et al. [15] as indicated below: 0: No changes 1: Uneasiness, scratching tremor, single myoclonic jerks 2: Atypical minimal seizures
10
M.M. Acharya, S.S. Katyare / Neuroscience Letters 394 (2006) 9–12
3: Minimal seizures consisting of clonic convulsions involving the head and forelimb muscles and leaving righting reflexes intact 4: Major seizures without the tonic phase 5: Complete major seizures, i.e. generalized tonic–clonic convulsions with loss of righting reflexes. The animals were killed by decapitation on day 21 of PTX treatment, and the brains were quickly removed and placed in beakers containing chilled (0–4 ◦ C) isolation medium (0.25 M sucrose containing 10 mM Tris–HCl pH 7.4 and 1 mM EDTA). Microsomes were isolated from post-mitochondrial supernatant as described earlier [12,26], washed thrice to give a protein concentration of 8–10 mg/ml. Microsomal lipids were extracted essentially according to the procedure described earlier [3,8]. Briefly, samples of microsomal suspension containing a total of 8–10 mg protein were extracted twice with chloroform: methanol (2:1, v/v) mixture, and pooled extracts were washed with 0.1 volume of 0.017% MgCl2 . Measured aliquots of final extracts were removed for total phospholipid (TPL) and CHL [41] estimation. TLC for separation of individual phospholipid classes was carried out according to the procedure of Skipiski et al. [35] using Silica gel G. Individual phospholipid spots from TLC plates and TPL samples were subjected to digestion with H2 SO4 , and complete oxidation was insured by treatment with perchloric acid. Liberated phosphorous was estimated by the method of Katewa and Katyare [13]. The measurements on membrane fluidity was carried out with 1,6-diphenyl-1,3,5 hexatriene (DPH) as the probe following the procedures described earlier in details [3,22]. Fluorescence polarization (P) was measured at 25 ◦ C in a Shimadzu RF-5000 spectrophotofluorimeter containing an inbuilt program for calculating and printing out fluorescence polarization (P) values. Calculations for fluorescence anisotropy (r), limited hindered anisotropy (r∞ ) of the fluorophore and order parameter (S) was done as described by van Blitterswijk et al. [40]. Protein estimation was done by the method of Lowry et al. [17] with BSA as the standard. Statistical evaluation of the data was performed using the Students’ t-test. Results on the effect of PTX-induced epileptic condition on the structural properties of microsomal membrane in terms of phospholipid (PL) composition, TPL and CHL contents and
Table 1 Effect of PTX-induced seizures on phospholipid composition of rat brain microsomes Phospholipid class
Phospholipid composition (nmol/mg protein) Control
Lyso CerPCho ChoGpl PtdSer PtdIns EtnGpl PtdOH
0.030 0.064 0.225 0.029 0.063 0.106 0.028
± ± ± ± ± ± ±
PTX 0.0036 0.0032 0.0132 0.0034 0.0046 0.0052 0.0023
0.052 0.064 0.295 0.015 0.017 0.086 0.035
± ± ± ± ± ± ±
0.0037**** 0.0031 0.0059**** 0.0011*** 0.0011**** 0.0041** 0.0020*
Change (%) +73 – +31 −48 −73 −19 +25
The experimental details are as described in the text. Results are given as mean ± S.E.M. of eight independent observations. Lyso, lysophosphatidyl glycerol; CerPCho, sphingomyelin; ChoGpl, phosphatidylcholine; PtdSer, phosphatidylserine; PtdIns, phosphatidylinositol; EtnGpl, phosphatidylethanolamine; and PtdOH, phosphatidic acid. * p < 0.05. ** p < 0.01. *** p < 0.002. ****p < 0.001.
membrane fluidity are shown in Tables 1 and 2. It is evident that the CHL content increased significantly by 25% without alteration in TPL content. Therefore a decrease in TPL:CHL (mole:mole) ratio was observed. When analyses were carried out to find out the PL contents, it was noted that lysophosphatidyl glycerol (Lyso), phosphatidylcholine (ChoGpl) and phosphatidic acid (PtdOH) components increased significantly by 1.25 to 1.7-fold (Table 1). While phosphatidylethanolamine (EtnGpl), phosphatidylserine (PtdSer) and phosphatidylinositol (PtdIns) were reduced drastically by 19–73% with maximum lowering observed for PtdIns (73% decrease). In the view of changes in PL composition, measurements were extended on the fluidity of the microsomal membranes as represented by the reciprocal of lipid structural order parameter, S [40]. It is evident from Table 2 that the microsomal membrane was more fluidized in PTX-induced epileptic animals. Endoplasmic reticulum (ER)-mediated sequestration of Ca2+ is crucial for proper neuronal functioning and maintenance of basal [Ca2+ ]i levels [6,23]. Activation of excitatory neurotransmitter receptor leads to Ca2+ influx into the neurons and increasing free [Ca2+ ]i [18], which has been implicated in the patho-
Table 2 Effect of PTX-induced seizures on total phospholipids, cholesterol content and membrane fluidity of rat brain microsomes Parameter
Control
PTX
Total phospholipid Cholesterol TPL:CHL
TPL (nmol/mg protein) CHL (nmol/mg protein) Molar ratio
0.571 ± 0.027 (8) 0.589 ± 0.024 (8) 0.974 ± 0.046 (8)
0.564 ± 0.016 (8) 0.738 ± 0.040 (8)* 0.772 ± 0.027 (8)**
Membrane fluidity
Fluorescence polarization (P) Fluorescence anisotropy (r) Limited hindered anisotropy (r∞ ) Order parameter (S)
0.206 0.147 0.097 0.494
± ± ± ±
0.007 (8) 0.005 (8) 0.005 (8) 0.017 (8)
0.133 0.093 0.024 0.242
± ± ± ±
The experimental details are as described in the text. Results are given as mean ± S.E.M., the number of observation indicated in the parentheses. * p < 0.01. ** p < 0.002. *** p < 0.001.
0.0016 (12)*** 0.0011 (12)*** 0.0016 (12)*** 0.0086 (12)***
M.M. Acharya, S.S. Katyare / Neuroscience Letters 394 (2006) 9–12
physiology of epilepsy [31,39]. Our results on PTX-induced epileptic condition on the microsomal membrane properties clearly indicated drastic alterations in the membrane composition. These compositional changes could lead to distorted charge distribution in the membrane microenvironment. Maintenance of Ca2+ homeostasis is regulated by proper functioning of Mg2+ , Ca2+ -ATPase. However, ATPases activity is known to be dependent on acidic phospholipids, in particular PtdSer and PtdIns [32]. PTX-induced convulsions resulted in 48–73% reduction in PtdSer and PtdIns components (Table 1). Significant inhibition of cortical Mg2+ , Ca2+ -ATPase-mediated Ca2+ uptake and Na+ , K+ -ATPase activity is reported in epileptic humans and animals [10,11,27]. Hence, it may be suggested that the changes in the microsomal ATPases could be attributed to decreased PtdSer and PtdIns components and altered membrane charge distribution. A decreased efficacy of ER means that neurons are less able to sequester Ca2+ influxes and re-establish the resting [Ca2+ ]i . These higher Ca2+ influxes may maintain further spontaneous recurrent seizures, characteristic of epileptic phenotype through buildup of [Ca2+ ]i and persistent increased basal Ca2+ levels. Substantial increase in Lyso (Table 1) may lead to fluidization of microsomal membrane, which is evident here (Table 2) since lysophosphoglycerides can exert detergent like action on the membranes [33]. Increased lysophosphoglycerides in microsomes (Table 1) and mitochondria [1] also indicates activation of phosopholipases, that are known to be activated by Ca2+ [5,37]. Significant alterations of [Ca2+ ]i has been observed in several models of epilepsy [20,29,33]. Disruption of ER activity and Ca2+ homeostasis has been implicated in apoptosis, excitotoxic disorders such as seizures and ischemia, neurodegenerative diseases such as Alzheimer’s, Huntington’s and Parkinson’s disease and uncontrolled cell growth [20,29,28]. PtdOH is an important precursor in phospholipid synthesis. Thus, increased PtdOH component (Table 1) is an indicator of altered phospholipid metabolism. In the present study, we did not explore fatty acid compositional changes but decreased EtnGpl would be expected to be associated with decrease in the unsaturation index [22]. Decreased PtdIns (by 73%; Table 1) might be related to membrane degradation, as PtdIns hydrolysis is often a feature of stimulated phospholipid turnover [30]. This observation is supported by earlier reports that decreased PtdIns could also be associated with seizure-induced excessive release of excitatory neurotransmitters and activation of phospholipases (PLA2 and PLC)-mediated signaling pathway [5]. Arachidonate is released from SN 2 position of glycerol backbone, which is implicated in neuronal plasticity, ischemic brain damage and epilepsy [4]. Earlier we have noted similar kind of drastic alterations in the structural and functional properties of mitochondrial membrane in PTX-induced chronic epileptic condition [1]. Participation of microsomal and mitochondrial function in Ca2+ handling [36] is a crucial task important for excitability and viability of neurons. In fact earlier reports also suggest the possible role of mitochondria and microsomes in the activation of PCD pathways during seizures [14,28,34]. Thus, intracellular membrane dysfunction may be implicated in the process of epileptogenesis.
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