Transglutaminase activity in reversible cerebral ischemia in the rat

Neuroscience Letters, 110 (1990) 232 236 Elsevier Scientific Publishers Ireland Ltd.

232

NSL 06698

Transglutaminase activity in reversible cerebral ischemia in the rat W u l f Paschen, Gabriele R6hn and Rainald Schmidt-Kastner Max-Planek-Institute.[or Neurological Researeh, Department c~["Experimental Neurology. Cologne (F.R.G.) (Received 15 August 1989; Revised version received 26 October 1989; Accepted 8 November 1989)

Key wordsv Cerebral ischemia; Hippocampus; Putrescine; Rat; Striatum; Transglutaminase activity Transglutaminase (TG, EC 2.3.2.13) activity and levels of putrescine (a natural acyl-acceptor in the transglutaminase reaction) were measured in rat brains after 30 min ischemia and 8 or 24 h recirculation, TG activity was significantly increased in the striatum and hippocampus already during cerebral ischemia and, more pronounced, after 8 and 24 h recirculation. In the cortex, in contrast, TG activity did not change during ischemia and 8 h recirculation but was significantly increased after 24 h recirculation. Putrescine levels were sharply increased after 8 h recirculation and even further after 24h recirculation. It is suggested that in vivo during ischemia and early recirculation, when cells are overloaded with calcium ions, a pathological increase in the TG-catalyzed cross-linking of proteins may be apparent especially in the nerve endings of the hippocampus where the intrinsic concentration of the acyl-donor (protein-bound glutamyl-moiely) has been shown to be high.

Several different mechanisms are suspected to participate in the pathological process of ischemic cell damage, including cytosolic calcium overload, release of excitatory amino acids, damage of membranes by free radicals or as a result of lipid degradation, disturbances in protein synthesis or polyamine metabolism (for review see refs. 3, 5, 15, 23, 24). One of the main primary events during cerebral ischemia is the intracellular overload of neurons with calcium ions as a result of the depletion of cellular high energy phosphates. It has been suggested that these disturbances in calcium homeostasis play an important role in the development of ischemic neuronal necrosis because several calcium-dependent reactions are initiated causing changes which are finally lethal for neurons [23]. Interestingly, the enzyme transglutaminase (TG, EC 2.3.2.13) which is strictly calcium dependent has to our knowledge never been measured in any tissue during conditions of disturbed energy metabolism and calcium homeostasis such as ischemia, hypoxia or anoxia. In the present series of experiments regional changes in TG activity were studied in reversible cerebral ischemia in the rat. Correspondence: W. Paschen, Max-Planck-Institute for Neurological Research, Department of Experimcntal Neurology, Ostmerheimer Stral3e 200, 5000 Cologne 91, F.R.G. 0304-3940/90/$ 03.50 4:1990 Elsevier Scientific Publishers Ireland Ltd.

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Reversible forebrain ischemia was produced in rats anaesthetized with 1.2% halothane in 70% N20, 30% 02 using the two-stage technique described elsewhere [18] with minor modifications [22]. After 30 min ischemia and 8 or 24 b recirculation animals were reanaesthetized and brains frozen in situ with liquid nitrogen [17]. Tissue samples were taken from the cerebral cortex, striatum and hippocampus, weighed and homogenized in Tris/HC1 buffer (100 raM, 5 mM DTT, pH 8.3). TG activity was assayed by measuring the incorporation of [1,4-14C]putrescine into the protein dimethylcasein as described elsewhere [10]. In short: the test system was composed of Tris/HCl-buffer (100 mM, pH 8.3), supplemented with NaC1 (15 raM), DTT (5 raM), CaC12 (2.5 raM), putrescine (0.25 mM, 12 mCi/mmol), dimethylcasein (5 rag/ ml) in the presence or absence of EGTA (3 mM) and about 2 mg tissue. The test mix was incubated for 30 min at 37°C and stopped with TCA (10%). After extensive washing with 5% TCA the radioactivity incorporated into proteins was quantified using a liquid scintillation counter. Under these assay conditions the reaction was linear with time and weight of tissue used. In addition, neither methylglyoxalbis(guanylhydrazone) nor aminoguanidine had any effect on the reaction indicating the absence of hypusine production [14] or aldehyde formation due to oxidation of putrescine [12]. TG activity was calculated by substracting the activity measured in the presence of EGTA from that measured in the absence of EGTA. Putrescine levels were quantified using HPLC and a fluorescence detector after extraction from the tissue with perchloric acid and derivatization with o-phthalaldehyde, as described recently [6]. Statistical analysis was performed using t-statistics with the Bonferroni correction for multiple comparisons [28]. The results are summarized in Tables I and II. Reversible cerebral ischemia produced a significant increase in TG activity in all brain structures studied (Table I). The temporal profiles of these changes varied however considerably in the different brain structures: in the cerebral cortex in which cell damage has been shown to be moderate after 30 min ischemia (only certain cell layers are affected [19]) TG activity did not change during ischemia and early recirculation but was significantly increased TABLE I R E G I O N A L T R A N S G L U T A M I N A S E ACTIVITY IN REVERSIBLE CEREBRAL ISCHEMIA OF RAT Transglutaminase activity is given in nmol. g ~ • h i (means _+ S.E.M.). Control

Cortex Striatum Hippocampus

93.8+5.7 53.0+2.7 61.34-6.4

Ischemia

88.0+ 4.8 70.2+ 2.7*** 142.1 4- 10.1"**

Recirculation 8h

24h

104.9_+ 6.1 91.5+11.9"* 123.4+ 16.9"

163.7+12.7"** 112.9__+ 7.9*** 163.2+ 9.2***

*P ~<0.05; **P ~<0.01; ***P ~<0.001 (t-test with the Bonferroni correction for multiple comparisons); significantly different from control.

234 TABLE II REGIONAL PUTRESCINE LEVELS IN REVERSIBLE CEREBRAL ISCHEMIA OF RAT Putrescine content is given in nmol.g ~(means ± S.E.M.). Control

Cortex Striatum Hippocampus

7,6_+ 1.0 7.6+_0.2 6,1 &0.9

lschemia

7.4_+ 1.2 7.7_+ 1.5 9.6+0.9

Recirculation 8h

24h

54.7_+5.5** 66.6+4.6*** 54.5 ±6.8"**

116.3±36.0"** 180.2± 24,2"** 133.9_+23.9"**

*P~<0,05; **P~<0.01:***P~<0.001 (t-test with the Bonferroni correction for multiple comparisons), significantly different from control.

after 24 h recirculation. In contrast, regions known to be severely damaged after 30 rain ischemia, namely the striatum and hippocampus [19], exhibited a significant increase in TG activity already during ischemia (Table I). Thus, in the striatum and hippocampus the increase in TG activity clearly preceded the development of neuronal necrosis, since neurons have been shown not to be morphologically altered during ischemia [16, 19]. When recirculation was extended from 8 to 24h recirculation TG activity increased further in the striatum and hippocampus. Putrescine levels were markedly increased after 8 h recirculation and even more after 24 h recirculation (Table II, 15-, 25- and 22-fold in the cortex, striatum and hippocampus, respectively, P~0.001). TGs are a class of strictly calcium-dependent enzymes catalyzing the formation of ~-(7-glutamyl)lysine cross links between proteins (for review see refs. 7, 12). Since polyamines have free amino groups they are also used as acyl acceptors resulting in cross-linking of proteins via 7-glutamyl-polyamine bonds [7]. In nervous tissue the expression of TG is activated during development [8, 10] and it is induced during nerve regeneration [4, 10, 26]. TG activity displays similar temporal changes as found for the enzyme ornithine decarboxylase [8, 25, 26]. The reason for the increase in TG activity in the striatum and hippocampus during ischemia has yet to be established. Activation of TG may be the result of a calpainmediated limited proteolysis, since this calcium-dependent proteinase has been shown to produce a sharp increase in TG activity [2]. It is interesting to note that calpain has been shown recently to play a pivotal role in the hippocampal damage induced by excitatory amino acids [21]. The changes in TG activity and putrescine levels observed in the present study in reversible cerebral ischemia may be of importance for two different reasons: Firstly, because this is a strictly calcium dependent enzyme and cerebral ischemia is known to cause a sharp decrease in the extracellular and thus increase in the intracellular calcium activity [11, 27]. It is, therefore, conceivable that the TG-catalysed reaction is markedly increased during ischemia and early recirculation when the intracellular

235 calcium activity is high. Secondly, because the concentration of putrescine, one of the natural substrates of TGs, is sharply increased after cerebral ischemia (see above). In the brain the KM of T G for putrescine is in the range of 100/tM [9]. Since putrescine content amounted to about 5-7 n m o l . g - l in the brain of control rats (see above) the increase of putrescine levels found after cerebral ischemia can thus be expected to activate the TG-catalyzed incorporation of this diamine into proteins considerably. In the present study T G activity was measured in an in vitro system under optimal conditions. This is the standard procedure for analyzing enzymatic activities quantitatively. However, it might be argued that the values for T G activity given in Table I have no relation to the actual activity of the enzyme in vivo because this is a strictly calcium-dependent enzyme and in the present study T G activity was measured in the presence of 2.5 m M calcium, a concentration that is never reached in vivo during ischemia. It cannot be excluded, therefore, that the observed changes in T G activity and putrescine levels do not have any significance for the cell. It is, however, important to note that in the brain putrescine has in fact been shown to be incorporated into proteins by T G even under control conditions during which the intracellular calcium activity is far below that found during ischemia [1, 13]. Further, at low alcium concentrations T G activity is sharply increased in the presence of calmodvlin [20]. Thus, it can be supposed that the TG-catalyzed cross-linking of proteins is considerably activated in reversible cerebral ischemia when the intracellular calcium activity is sharply increased, especially in the hippocampus where the overall T G activity is doubled already during ischemia and the content of the intrinsic protein, which is accepted as T G substrate, has been shown to be high, particularly in the synaptosoreal fraction [9].

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