Protein kinase C as an early and sensitive marker of ischemia-induced progressive neuronal damage in...

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Protein Kinase C as an Early and Sensitive Marker of Ischemia-lnduced Progressive

Neuronal Damage in Gerbil Hippocampus

KRYSTYNA DOMAIqSKA-JANIK AND BARBARA ZABLOCKA* Department of Neurochemistry, Medical Research Centre,

Polish Academy of Sciences, 3 Dworkowa str., 00. 784 Warsaw, Poland

Received October 28, 1992; Accepted January 5, 1993

ABSTRACT

In the model of transient brain ischemia of 6-min duration in gerbils we have estimated:

1. The concentration of brain gangliosides: A significant decrease to about 70% of control was observed selectively in the hippocampus at 3 and 7 d after ischemia.

2. The activity of Na§ The enzyme activity was not affected in either hippocampus nor in cerebral cortex.

3. The malonylaldehyde (MDA) concentration: The levels of MDA had increased at 30 min after ischemia up to 123 and 129% of control in hippocampus and cerebral cortex, respectively.

4. Immunoreactivity of protein kinase C detected by Western blotting: In hippocampus the early translocation toward membranes was followed by a decrease in total enzyme content at 6, 24, 72, and 96 h of postischemic recovery. Also, a sharp increase of 50 kDa isoform (PKM) was noticed immediately and at the early recovery times.

The behavior of these biochemical markers of ischemic brain injury in the hippocampus after the short (6 min) insult was contrasted with their reaction in the cerebral cortex as well as after prolongation of the ischemia to 15 min. These results taken together indicate that an early increase in PKC translocation followed by a decrease is the most symp- tomatic for selective, delayed, postischemic hippocampal injury, resulting from short duration (6 rain) ischemia of the gerbil brain.

*Author to whom all correspondence and reprint requests should be addressed.

Molecular and Chemical Neuropathology 1 1 1 Vol. 20, 1993

W 112 Domanska.danlk and Zabtocka

Index Entries: Ischemia; Na+,K§ malonylaldehyde; hippocampus; lipid peroxidation; ion transport; gangliosides; protein kinase C.

Abbreviations: MDA, malonylaldehyde; PKC, protein kinase C; PKM, protein kinase M; NeuAc, N-acetylneuraminic acid; TCA, tri- chloroacetic acid; Na§ Na§ + adenosine triphosphatase; BHT, 2,6-ditertbutyl-p-cresol; ECL, enchanced chemiluminescence Western blotting detection system; NC, nitrocellulose; NMDA, N-Methyl-D-aspartic acid.

INTRODUCTION

Development of delayed neuronal death in gerbil hippocampus after relatively brief, transient ischemia is a well-documented phenomenon (Kirino and Sano, 1984), with a potential involvement of various patho- logical mechanisms discussed in the past years. Among them was inhibition of Na § § (Palmer et al., 1985) with the secondary impairment of tissue ions. There is also experimental evidence that rapid activation of lipid peroxidation after ischemia could trigger a chain of events leading to delayed neurodegeneration (Chan et al., 1985; McCord, 1985; Cerchiari et al., 1987).

Recently, attention has been focused on the neuroexcitatory mechanism mediated by the postischemic activation of excitatory amino acid receptors of NMDA subtype (Simon et al., 1984b) and the increase of intraceUular calcium (Simon et al., 1984a). In the present study, we have employed the model of transient gerbil brain ischemia of 6-min duration for the induc- tion of selective, delayed neuronal death in the vulnerable hippocampal regions (Kirino and Sano, 1984). The time-course and extent of irreversible cell injury (neuronal death and elimination) has been correlated with the appearance of biochemical markers of the abovementioned hypothetical casual factors. We have estimated the concentration of brain gangliosides as a biochemical measure of neuronal density and postischemic cell loss.

For monitoring calcium-dependent signaling pathways we have estimated, by Western blotting, the concentration and subcellular distribution of the calcium-dependent protein kinase.

The activity of Na § § and malonylaldehyde (MDA) concentration measurements have been used as markers of the ionic transport and lipid peroxidation processes, respectively, in the postischemic brain.

MATERIALS AND METHODS

Animal Model

Ischemia of gerbil hippocampus (Meriones unguiculatus) was produced by bilateral ligation of the common carotid arteries in the midcervical region through a midline incision under light ether anesthesia. The animals were

Molecular and Chemical Neuropathology Vol. 20, 1993

Protein Kinase C in lschemia 1 13

decapitated after 6-rain occlusion, or the clips were removed. The animals were then allowed to recover for the indicated times. Sham-operated animals awakening from anesthesia served as controls. During ischemia and recovery the body temperature of gerbils was maintained at 36.6- 37.5~ (as monitored by means of a rectal probe) with the heating pad and a lamp.

Biochemical Procedures

Measurements of Brain Gangliosides

The excised hippocampi were sonicated in 20 vol of chloroform/meth- anol (C/M) 2:1 (v/v) and reextracted with 10 vol of C/M 1:1, with the subsequent purification on a Unisil column (for details, see Domafiska-Janik, 1988). The total lipid-bound sialic acid (NeuAc) was determined according to Svennerholm (1957) by the resorcinol-colorimetric method. Thin layer chromatography (TLC) analysis was performed according to a previously described procedure (Domaflska-Janik, 1988). The amount of sialic acid was normalized either to protein concentration or to the indicator of cell density measured by DNA extraction procedure (Domaflska-Janik and Zalewska, 1979). Briefly, the residues obtained after lipid extraction were washed twice with ice-cold 10% (w/v) TCA to remove acid-soluble material. The residues were heated in 5% TCA (5 vol of initial tissue weight) for 20 min at 90~ centrifuged, washed again with 5% TCA, and supernatants were collected. This fraction was quantitatively determined by UV absorp- tion at 268.5 nm according to Logan et al. (1969). The extinction equivalent to one hippocampus was used as a cell density indicator. The pellet was washed with 96% ethyl alcohol, acetone, and diethyl ether, then solubil- ized with 0.5M NaOH for protein determination (see below).

Na § ,K § -ATPase Activity Assay

The enzyme activity was determined by measuring the amount of inor- ganic phosphate liberated at ATP hydrolysis in the presence of cations and in the presence or absence of ouabain as previously described (Domafiska- Janik and Bourre, 1990).

MDA Determination

The concentration of thiobarbituric acid-reactive oxidation products was determined as described by Domafiska-Janik and Bourre (1990) with the addition of 2,6-ditertbutyl-p-cresol (BHT) to prevent artifactual oxidation of fatty acids during the assay procedure.

Western Blotting of PKC

After decapitation, the brain was rapidly (< 30 s) removed, cooled in ice-cold physiological saline, then the hippocampi were dissected, frozen on the dry ice, and stored at -80~ until use.

Hippocampi were homogenized in 0.5 mL of ice-cold 5 mM HEPES buffer pH 8.0 as recommended by Cardell et al. (1990), but without sucrose


1 14 Domahska-Janik and Zabtocka

and trypsin inhibitor. The homogenates were centrifuged at 40,000g for 1 h at 4~ The pellets were resuspended in homogenization buffer and, together with collected supernatants, frozen at -80 ~ until analysis. Sam- ples (30 #g protein) were analyzed by SDS-PAGE electrophoresis on 10% polyacrylamide gels according to Laemmli (1970). The proteins were trans- ferred from the gels onto nitrocelulose (NC) paper (Hybond- C extra) by electroblotting overnight.

Western analysis followed the protocol of enhanced chemiluminescence (ELC) detection system provided by the manufacturer (Amersham, Amersham, UK). NC was incubated with monoclonal antiprotein kinase C (clone Mc5) antibodies, which were visualized with peroxidase-linked antimouse IgG. Western blots developed by ELC system were analyzed densitometrically using a LKB Ultroscan XL densitometer. The samples that were on the same NC were directly compared with the control on the same blot--it means that all these samples and controls were incubated and exposed in the identical conditions (an average exposure time was about 30 s to 1.5 min).

Protein Determination Proteins were determined using the method of Bradford (1976). The

samples were dissolved in 0.5% SDS then diluted to 0.1% of SDS before estimation.

Statistical Methods Student's t-test for small samples was used to evaluate significant dif-

ferences (Bailey, 1975).

RESULTS

The ganglioside concentration in gerbil hippocampus was estimated at 24 h, 3 d, and I wk after the ischemic episode. In the first group (24 h after ischemia), no changes in the total ganglioside levels were observed as compared with controls (result not shown). In the group of animals evaluated 3 d after ischemia, a significant reduction to 77+ 2.73% of control values was noticed. This reduction progressed during the next 4 d down to 67% of control (Fig. 1). When ganglioside concentration was normalized to the cell density indicator (see Material and Methods) instead of protein concentration, the deficit was still present but attenuated to 86% of control. Thus, ganglioside diminution could be accounted for not only by the decrease in the neuronal cell number but also by the concomitant changes in the average cell volume (e.g., by occurrence of the reactive gliosis in postischemic tissue). However, analysis of gangliosides separated by TLC did not reveal any changes in the proportion of a particular species after ischemia.


Protein Kinase C in Ischernia 115

r-q I0 OQ -H >4

8 b.O

o

Z

(1) Z

1 2 r -

6

4

2

0 - -

1 2

Fig. 1. Total ganglioside expressed as lipid-bound sialic acid (NeuNAc) concentration in control hippocampus and hippocampi exposed to 6-rain ischemia model followed by 7-d recovery; (1) control, (2) 7 d after 6*min ischemia.

In all experiments, parallel measurements of ganglioside concentration in the cerebral cortex were performed. The basal levels were not signifi- cantly different from those of the hippocampus and amounted to 9.65+ 0.37 ~g NeuAc/mg protein in controls without any significant changes in all investigated postischemic groups.

Figures 2 and 3 demonstrate the negligible effect of 6-min ischemia on Na*,K+-ATPase activity and MDA concentration in gerbil hippocampus.

However, when ischemia was prolonged up to 15 rain, the inhibition of ATPase activity was observed, followed by its transient stimulation at 30-min recovery. In this case also, the tissue concentration of MDA increased to 134 and 143% of control as measured immediately or after 30 min of reperfusion. Moreover, a slight increase in MDA level to 123% of control was also noticed at 30-min recovery after 6-min ischemia. The above parameters measured in parallel in cerebral cortex behaved similarly to those in hippocampus.

The concentration and subcellular distribution of hippocampal PKC in 6-rain gerbil-brain ischemia model were estimated by Western immunoblots analyses (Fig. 4). In controls, over 60% of the total PKC was found in the soluble (cytosolic) fraction. In the cytosol, only one immunoreactive band with an apparent Mr of 80 kDa was observed. By contrast, in the particulate fraction, an additional, thin band of about 50 kDa was noticed in the control preparation, and accounted for about 3% of total immunoreactivity.

Representative densitograms comparing the control membrane-bound enzymes distribution with that found at 15-min recovery after 6-min brain


1 16 Domahska.Janik and Zabtocka

Fig. 2. Na +,K +-ATPase activity in gerbil hippocampi measured immediately and at 30 min of recovery after ischemia of different duration; (1) control, (2) ischemia 6 rain, (3) ischemia 6 min + recovery 30 rain, (4) ischemia 15 rain, (5) ischemia 15 min + recovery 30 min.

Fig. 3. Malonylaldehyde (MDA) level in gerbil hippocampi measured immediately and at 30 rain of recovery after ischemia of different duration; (1) control, (2) ischemia 6 rain, (3) ischemia 6 rain recovery 30 rain, (4) ischemia 15 rain, (5) ischemia 15 min + recovery 30 rain.


Protein Kinase C in lschemia 117

Fig. 4. The enhanced chemiluminescence (ECL) Western blotting detection system was used to detect PKC in subcellular fractions (particulate [P], cytosol [C], and homogenate [H]) separated by SDS-PAGE electrophoresis on 10% gels. The samples of 30 #g protein comprised hippocampi from gerbils exposed to 6-rain ischemia (I) followed by 30 rain, 3 h, 24 h, 72 h, and 96 h of recovery (R), incubated with monoclonal antibodies against PKC, and then incubated with antimouse whole antibody linked with horseradish peroxidase. Sham-operated animals served as a control (C).

ischemia are demonstra ted in Fig. 5. A substantial increase in the membrane-bound forms was followed by an increased contribution of 50-kDa form. This reaction, suggesting proteolytic cleavage of the membrane- translocated enzyme, was highly variable in the other animals. However , in all cases estimated up to 3 h of postischemic recovery, a significant increase of 50-kDa enzyme form was detected, giving the average of about 20% of total immunoreactivity as compared with 3% of the control value.

The combined results of densitometrically quantitated PKC concentration and the related percentages of the subcellular enzyme distribution at different times after ischemia are demonstra ted in Figs. 6 and 7. On


1 1 8 ' �9 Domanska.Janzk and Zabtocka

OD 1"00 t

0~601

0.20-

1.00- .

0.60-

Q20,

50kDa

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1

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80kDa 2

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35 40 4'5 5'0 5'5 6'0 6'5 7'Omm

Fig. 5. Densitometric analysis of ECL-detected Western blots of PKC in particulate fractions separated by SDS-PAGE electrophoresis as described in legend to Fig. 4. The samples represent gerbils exposed to sham operation (1) and 6-min ischemia followed by 15-min recovery (2). Each sample contains 30 #g protein.

140

0

c~

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0

120

100

(3)

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80

60

40 - 2 0 0

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20 40 60 80 100 20

Lime ( h o u r s )

Fig. 6. Total amount of PKC in time-course of recovery following 6-min ischemia model in gerbil hippocampus. Each point represents the mean value from an individual sample, evaluated on three separate blots, obtained from one or more animals (numbers in brackets).


Protein Kinase C in lschemia

1 0 0 q I I I I I

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80

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Fig. 7. Subcellular distribution of PKC in time-course of recovery following 6-min ischemia model in gerbil hippocampus. The values are calculated from the same experiments as described in the legend to Fig. 6. (�9 PKC in cytosol, (O) PKC in particulate fraction, (V) PKM in particulate fraction.

these diagrams, the short-term experiments have been reduced to the representative two: nonrecovery and 3-h-recovery time-points. Similar translocation of the cytosolic enzyme toward membranes with the concomitant increase of 50-kDa form has been observed in all tested animals recovering by 5, 15, and 30 min after ischemia (these experiments are demonstrated only on the immunoblots).

The enzyme translocation into membranes as an early reaction to ischemia was later followed by a sharp decrease of the total detected immunoreactivity. Total PKC (the sum of all immunoreactive bands) remained reduced to about 60% of the initial values at all the time-points studied later than 3 h after ischemia (Fig. 6). However, in contrast to the early postischemic recovery the subcellular distribution of this downregulated enzyme showed steadily increasing contribution of the soluble form (Fig. 7).

DISCUSSION

Transient (6 min), incomplete brain ischemia induces within a few days subacute neuronal damage only in the circumscribed, vulnerable brain areas (Kirino and Sano, 1984). The postischemic cell death and elimination was monitored in this study by the decrease of total ganglioside


t ~

120 Domanska-Jantk and Zabtocka

concentration in the brain regions. Seifert and Fink (1984) and other inves- tigators (Segler-Stahl et al., 1982; Domafiska-Janik, 1988) reported quantitative changes in the gangliosides concentration connected with the neuronal cell loses occurring in various experimental models. In this study, 6 min of brain ischemia induced more than a 30% decrease of the total ganglioside content in gerbil hippocampus at 3 and 7 d after the insult. This reaction was restricted to the hippocampus and contrasted with the lack of changes in the other brain regions, indicating once more its higher vulnerability to ischemia. The concomitant decrease of ganglioside/DNA ratio in postischemic hippocampus, together with unchanged proportion of particular ganglioside subspecies indicates the elimination of the relatively large, probably pyramidal, neurons from vulnerable regions of hippocampus.

It is generally accepted that during ischemia, energy failure results in a series of acute membrane lipid changes that may initiate progressive tissue destruction. The functional consequences of the membrane lipid alterations include changes in the membrane permeability, tissue edema, and inactivation of the membrane-bound ion-pumping enzymes such as Na+,K+-ATPase (Palmer et al. 1985; Pylowa et al. 1989). However, short- term ischemia employed in our study and being sufficient to induce delayed neuronal necrosis did not inhibit Na § ,K§ activity. Upon extending blood flow interruption for 15 min, significant and reversible reduction of the enzyme activity became evident at a very early postischemic period. Perhaps, and in a good agreement with other data (Enseleit et al., 1984), the inhibition of ATPase correlates rather with the more profound, immed- iate ischemic brain damage.

On the other hand, the increase of MDA levels after 30-rain recovery following 6-rain ischemia is consistent with the early activation of lipolytic enzymes resulting in the release and accumulation of free fatty acids (Edgar et al., 1981). Such changes have recently been reported to occur in this ischemic model (Nakano et al., 1990). Free fatty acids (Gaudet et al., 1980; Yoshida et al., 1980; Domafiska-Janik et al., 1985) and stimulation of xanthine oxidase during and after ischemia (Kleihues et al., 1974; McCord, 1985) initiate a chain of peroxidative reactions with MDA accumulation as their end product. Activation of phospholipase C also leads to the accumulation of diacylglycerol (Strosznajder et al., 1987; Wikiel and Strosznajder, 1987), a transient product of inositol phospholipid turnover, which in turn could activate PKC alone or in concert with fatty acids (Nishizuka, 1989).

Signs of PKC activation in our 6-min ischemia model were noticed at very early time-points of postischemic recovery (0-30 min). These consisted of translocation of the cytosolic enzyme toward membranes, a known para- digm of PKC activation (Nishizuka, 1989). Also, a sharp increase of the 50-kDa isoform after ischemia indicates proteolytic cleavage of a part of the translocated membrane-bound enzyme. This form was postulated to be a permanently active species no longer controlled by the presence of specific activators. The occurrence of this form (PKM) after ischemia was



reported by Louise et al. (1988) but denied by others (Crumrine et al., 1990; Domafiska-Janik and Zalewska, 1992). These controversies could perhaps be explained by recently reported very high Mg 2+ requirement for PKM- dependent histone phosphorylation (Hashimoto and Yamamura, 1989) not fulfilled in our previous study.

The protective role of the primary postischemic PKC activation would involve feedback inhibition of the receptor-mediated hydrolysis of inositol phospholipids with the subsequent, temporary attenuation of the Ca 2+- signaling pathway. Later, PKC depletion could result in uncontrolled activation of the lipolytic enzymes and calcium fluxes, leading to progressive cell destruction during the longer postischemic time-course. These sugges- tions are in aggreement with the results of Deshpande et al. (1987) demon- strating that net calcium accumulation in hippocampus did not change earlier after 24 h of recirculation and preceded delayed necrosis of CA1 hippocampal pyramidal cells. As suggested before, postischemic downregulation of PKC could be directly involved in the irreversible brain damage (Crumrine et al., 1990; Zivin et al., 1990; Louis et al., 1991; Domafiska- Janik and Zalewska, 1992).

The PKC purified from brain contains three major isoenzymes, termed type I, II, and Ill. These isoforms account for 90% of the total protein kinase C activity in the brain (Huang et al., 1986). In our study, the monoclonal antibody provided by Amersham recognizes only the II and III PKC subtypes. This could partly account for the substantial discrepancy between the changes in phosphorylating enzyme activity measured in the same ischemic model (Domafiska-Janik and Zalewska, 1992) and the present results using the immunoblotting technique. On the other hand, the previously described early inhibition of the phosphorylating PKC activity could precede protein reduction as recognized by immunoblotting. How- ever, a similar trend toward early translocation/activation of the hippocampal enzyme with its subsequent downregulation was noticed in both studies. In future experiments, probing of the wider spectrum of isoenzymes will be necessary to gain a more complete knowledge about the reactions and involvement of PKC in the postischemic pathology.

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