Nuclear-Envelope Nucleoside Triphosphatase Kinetics and mRNA Transport Following Brain Ischemia and Reperfusion

LABORATORY I N V E S T I G A T I O N

Nuclear-Envelope Nucleoside Triphosphatase Kinetics and mRNA Transport Following Brain Ischemia and Reperfusion From the Department of Emergency Medicine, Wayne State University, Detroit, Michigan.

Brian R Tiffany, MD, PhD Blaine C White, MD Gary S Krause, MD, MS

Receivedfor publication October 6, 1994. Revision received December 5, 1994. Acceptedfor publication December 12, 1994. supported by grant 07G934from the American Heart Association of Michigan. Dr Tiffany was supported by afdIowship grant from the Emergency Medicine Foundation and Eli Lilly & Co.

Study Hypothesis:We attempted to determine whether the reduced egress of mRNA from brain nuclei following in vivo ischemia and reperfusion is caused by direct damage to the nuclear pore-associated NTPasethat impairs the system for nuclear export of polyadenylated,or poly (A)+, mRNA. Design: Prospective animal study.

This work was

Copyright © by the American College of Emergency Physicians.

Interventions: NTPaseactivity and poly(A)+ mRNA transport were studied in nuclear envelope vesicles (NEVs) prepared from canine parietal cortex isolated after 20 minutes of ischemia or 20 minutes of ischemia and 2 or 6 hours of reperfusion. Results: Brain NEV NTPase Michaelis-Menten constant {Km) and maximum uptake velocity (Vma×) and the ATP-stimulated poly(A)+ mRNA egress rates were not significantly affected by ischemia and reperfusion. In vitro exposure of the NEVsto the 0H, radical-generating system completely abolished NTPase activity. Conclusion: We conclude that brain ischemia and reperfusion do not induce direct inhibition of nucleocytoplasmictransport of poly(A)+ mRNA. This suggests that the nuclear membrane is not exposed to significant concentrations of 0H, radical during reperfusion. [Tiffany BR, White BC, Krause GS: Nuclear-envelope nucleoside triphosphatase kinetics and mRNA transport following brain ischemia and reperfusion. Ann EmergMedJune 1995;25:809-817.]

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INTRODUCTION

Protein synthesis in the brain is disrupted as a consequence of ischemia and reperfusion. ~ This response is not uniform throughout the brain; the cortex, hippocampus, and caudate show severe suppression of protein synthesis, whereas the brain stem and midbrain structures are relatively unscathed. 2-5 These areas with suppressed protein synthesis are also the ones most vulnerable to delayed neuronal death during reperfusion 6, and it has been suggested that failure of protein synthesis contributes directly to the observed neuronal l o s s The mechanism involved in suppressed protein synthesis during postischemic brain reperfusion is located between the end of transcription and the beginning of translation. ~ Matsumoto et al demonstrated normal levels of newly synthesized mRNA during brain reperfusion in the nucleus and mitochondria but decreased levels of new mRNA in the microsomal and ribosomal fractions. 8 In gerbils reperfused after 5 minutes of forebrain ischemia, auto radiograms of pulse-labeled RNA showed accumulation of newly synthesized RNA in the nucleus, with little in the cytoplasm. 9 Similarly, Maruno et al, using acridine orange staining, showed apparent retention of RNA in the nucleus during reperfusion, lo These data suggest a block in mRNA transport from nucleus to cytoplasm. Mature polyadenylated, or poly (A)+, mRNA is actively exported through pores in the nuclear membrane 11,t2 by an energy-dependent process requiring hydrolysis of ATP or GTP by a nucleoside-triphosphatase (NTPase) associated with the inner nuclear membrane. 13-~5 Radicalmediated lipid peroxidation, known to damage selectively vulnerable neurons during reperfusion 16, could conceivably damage the nuclear membrane mRNA transport system. Moreover, other membrane-bound ATPases lose activity when their membranes are peromdized. ~r-21 Reperfusion damage to the nuclear pore apparatus or to its associated NTPase would result in nuclear retention of newly synthesized mRNA. Therefore we prepared nuclear envelope vesicles (NEVs) from normal brains and brains subjected to global ischemia and reperfusion to study the effect of an in vitro radical insult on NTPase activity and the effects of cardiac arrest and resuscitation on NTPase activity and mRNA transport. MATERIALS AND METHODS

All experimental protocols were approved by the Wayne State University Animal Investigation Committee. A canine model of cardiac arrest and resuscitation was used to produce global brain ischemia as previously de-

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scribed. 22 In brief, heartworm-free mongrel dogs were divided into four groups of three each: (1) nonischemic controls in which parietal cortex was taken without ischemic insult (NIC); (2) animals subjected to 20 minutes of KCl-induced cardiac arrest without resuscitation (ISCH); (3) animals subjected to 20 minutes of cardiac arrest, resuscitation by internal cardiac massage and defibrillation, and 2 hours of postresuscitation intensive care (R2); and (4) animals subjected to 20 minutes of cardiac arrest, resuscitation, and 6 hours of postresuscitation intensive care (R6). Nuclei were isolated by use of the method of Blobel and Potter 23, modified to include 5 mM 2-mercaptoethanol and protease mhibitors (2 pg/mL leupeptin, 2 pg/mL aprotinin, 1 pg/mL pepstatin A, 1 mM phenylmethylsulfonyl fluoride) in all buffers. A Dounce homogenizer on ice was used to homogenize the cortex in ice-cold .25 M sucrose in 50 mM Tris-HC1 (pH 7.5), 25 mM KC1, and 5 mM MgC12 (TKM), and the homogenate was strained through three layers of cheese cloth. A step gradient was formed by layering 1 volume of homogenate mixed with 2 volumes of 2.3 M sucrose in TKM onto 1 volume of 2.3 M sucrose in TKM, and then centrifuged at 124,000g for 40 minutes. The supernatant was decanted and the nuclear pellet resuspended in 0.25 M sucrose in TKM. The DNA concentration was determined by the diphenylamine reaction. 24 Nuclei (200 pg/mL DNA) were solubilized in lysis buffer (10 mM Tris-HC1 [pH 8.0], 10 mM Na2HPO4, and 300 pg/mL heparin sulfate). 25 For mRNA transport studies, labeled poly(A)+ mRNA was added to the lysis buffer. After the suspension was gently agitated at room temperature for 5 minutes, the NEVs were pelleted by means of centrifugation at 5,000g for 10 min. The NEVs were resealed by washing them twice in .25 M sucrose in 50 mM Tris-HC1 (pH 7.4), 25 mM KC1, 5 mM MgC12, and 3.3 mM CaC12, and protein concentrations were determined using the Lowry assay. NEVs will remain sealed in solution for up to 2 days. 25 The in situ NTPase has been reported to exhibit Michaelis-Menten kinetics at 150 mM KC126 and is stimulated in situ by poly(A) ÷ mRNA, but not by poly (A)mRNA or that without polyadenylation. 2r,2s We assayed NEV-associated NTPase activity by means of a modification of the method of Schr6der. 26 NEVs (1 mg protein/mL) were resuspended in 50 mM HEPES (n-2-hydroxyethylpiperazine-N- 2-ethanesulfonic acid) (pH 8.0) and 150 mM KC1, and incubated at 30°C for 10 minutes. [3H]ATP (8,000 dpm/nM) in concentrations between .25 and 2.0 mM was added with equimolar MgC12 in a final reaction volume of 100 tlL. 29 The reaction was quenched by addi-

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tion of 100 mM ATE 100 mM ADP, and 200 mM AMP (pH 4.0). Reaction samples and ATE ADP, and AMP standards were loaded onto polyethylenimine-cellulose thinlayer chromatography plates (Sigma Chemical Company), which were developed in 1 M KH2PO4 (pH 3.4). The ADP and ATP spots were visualized under UV light, excised, added to scintillation fluid, and counted. Control experiments containing all reagents but NEVs were run to assess autohydrolysis of ATE At each ATP concentration, assays were performed in triplicate and the velocity of ATP hydrolysis was determined from the slope of the linear regression of the means. Michaelis-Menten constant (Kin) and maximum uptake velocity (Vma×) for the hydrolysis of ATP by the nuclear envelope NTPase were derived from the Lineweaver-Burk plot. Labeled mRNA was produced by inoculating Sac&a> omyces cerevisiaeinto PGY medium (1 g peptone, 3 g glucose, and 1 g yeast extract per liter) containing 2.5 mCi of [3H-]uridine/L.3° Yeasts were grown for 4 days at 30°C and harvested by means of centrifugation. To minimize degradation by RNase, RNA isolation solutions were made •1% in diethylpyrocarbonate (DEPC) and autoclaved; Triscontaining buffers were made in DEPC-treated water with freshly opened chemicals. All glassware was baked overnight at 200°C. The yeast pellet was frozen with liquid nitrogen, ground to a powder, dissolved (in 5.5 M guanidinium thiocyanate, .5% SDS, and 25 mM sodium citrate [pH 7.0]), vortexed with acid-washed glass beads, and centrifuged at 5,000g. The supernatant was repeatedly passed through a 16-gauge needle to shear the DNA. The solution was then layered over an equal volume of cesium trifluoroacetate (1.5 Fg/mL) and centrifuged at 125,000g for 16 hours at 15°C. The pellet, containing the RNA, was resuspended in TE buffer (10 mM Tris-HC1 [pH 7.4] and 1 mM A (ethylenediamine tetra acetic acid). The quantity of RNA was determined by means of absorbance at 260 nm. The quality of the mRNA was verified with electrophoresis on a formaldehyde denaturing agarose gel. The ribosomal RNA bands were well preserved, indicating that the RNA was not degraded during isolation. The specific activity was 67.5x103 cpm/pg RNA. Poly(A)+ mRNA was isolated from the labeled total cellular RNA by use of a commercial mRNA purification kit (Pharmacia). In brief, 1 mg RNA dissolved in 1 mL TE was heat-denatured at 65°C for 5 minutes, and an equal volume of 3.0 M NaC1 in TE was added. The sample was then placed on a oligo(dT)-cellulose spin column (preequilibrated with 1.5 M NaC1 in TE buffer) and allowed to soak in under gravity. The column was washed twice with .5 M NaC1 in TE and three times with .1 M NaC1 in TE.

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Poly(A) + mRNA was then eluted from the column by use of four successive .25-mL aliquots of TE warmed to 65°C. The mRNA concentration in the eluant was measured by means of absorbance at 260 nm. The poly(A) + mRNA specific activity was 92. lx103 cpm/gg. Poly(A) + mRNA was trapped inside the NEVs during their preparation by adding 4.0 pg PH-]poly(A)+ mRNA to 100 gL of the heparin-containing lysis buffer. 31 The pelleted vesicles were resealed by washing twice in 10 mM Tris-HC1 (pH 8.0), 100 mM NaC1, 30 mM KC1, 3 mM MgC12, and 0.5 mM CaC12, and then resuspended at 2 mg protein/mL. The reaction mixture was prepared on ice by layering in succession 10 pL 60% HC104, 40 pL silicone oil, and 150 t~L of the NEV suspension. The reaction was initiated by bringing the mixture to room temperature and adding ATP to 2 raM. At the appropriate time, the reaction was terminated by means of centrifugation. The lower layer, containing NEVs, was discarded, and the upper layer, containing exported mRNA, was aspirated and counted in scintillation fluid. 32 NEVs from normal brains were exposed to hydroxyl radical generated by the reaction between ferrous iron and H202 by suspending NEVs at a concentration of 2 mg protein/mL in 10 mM dithiothreitol and between 2 and 30 laM H202. The reaction was started by adding FeSO4, to final concentrations between 6 and 100 I~M. NEVs were incubated for 15 minutes at 37°C, and the reaction stopped by the addition of DETAPAC to 10 raM. After the radical reaction, the NEVs were pelleted, washed, and assayed for NTPase activity as previously described. RESULTS In pilot studies, NEV NTPase isolated from rat liver had a Km of .8 mM with a Vm~x of 27 nmo1 ADP/mg protein/ minute; it was stimulated 115% by poly(A) [data not shown]. NEV NTPase isolated from normal dog brain was found to have a Km of 1.0 mM for ATP and a Vma× of 20 nmol ADWmg protein/minute (Table 1). These values are consistent with findings from other studies. 33 We studied the effect of radical injury on NEV ATP hydrolysis in normal NEVs by subjecting them to the Fenton reaction at various Fe +2 and H202 concentrations. All concentrations of iron and hydrogen peroxide tested resulted in complete loss of NTPase activity in the NEVs (Figure 1). There was no loss of NTPase activity if only Fe +2 or only H202 w a s added (data not shown). Suspending the NEVs in DTT alone caused a 52% loss of NTPase activity. This may have been a consequence of altering the optimum ferric/ferrous ratio of endogenous

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iron to favor lipid peroxidation, s4 Addition of the iron chelator DETAPAC to experiments in which NEVs were incubated with iron and H202 prevented the loss of NTPase activity. These results show that the NEV NTPase is exquisitely sensitive to inactivation by free radical damage and that the NTPase assay method used here could detect damage induced by low concentrations of hydroxyl radical. The NTPase activity in NEVs extracted from brains subjected to either ischemia or ischemia and reperfusion showed no significant change i n Vmax or Km (Table 1). We did not observe the reduction in Vmax that would be expected with loss of catalytic sites. The ability of polyadenosine to stimulate NTPase activity was studied in NEVs isolated from each of the four animal groups. We incorporated poly(A) (molecular weight 175,000; Sigma) into NEVs by including it at a concentration of 600 lag/mL during NEV preparation and resealing. NEVs prepared with and without inclusion of poly(A) were then assayed for hydrolysis of 2 mM ATE ATP hydrolysis by NEVs not loaded internally with poly(A) was also examined in the presence of 600 lag/mL poly(A) externally in the reaction buffer to compare the effects of extra-NEV and intra-NEV poly(A). All four groups demonstrated significant enhancement of NEV ATP hydrolysis by their inclusion of poly(A) within the NEVs (t tests of the regression slopes; Table 2). We observed no evidence of significant inhibition of Figure 1. Effect of in vitro radical damage on NTPase activity. IJMADP/mgprotein

[I.5

t • NIC

~n2

.

-

-

poly(A) + stimulation of NEV NTPase by brain ischemia or reperfusion. Poly(A) stimulation of NTPase activity was observed only when the poly(A) was present internally in the NEVs and not when the poly(A) was only present externally in the reaction medium. These findings show that our preparative technique generated well-sealed NEVs; the poly(A) binding protein is associated with the inner membrane of the nuclear envelope and is therefore inaccessible to poly(A) in the buffer. We further examined this issue by including fluorescein in the NEVs during their preparation. No leakage of fluorescein into the buffer was seen for at least 16 hours (data not shown). Radiolabeled poly(A) + mRNA was incorporated inside the NEVs during their preparation, and mRNA egress was measured as described in Materials and Methods. mRNA export obtained in the absence of ATE the "leak" slope, is about 15% of the total ATP-stimulated poly(A) + egress (Figure 2), in good agreement with findings described in the literature) 5 mRNA egress from normal NEVs demonstrates significant ATP-dependent poly(A) + mRNA egress (Figure 2, t=8.7, P<.001 against the "leak" slope). Ischemia and reperfusion did not induce significant differences (F=I.0~-, P>.60) among any of the experimental animal groups in the ATP-stimulated rates of poly(A) + mRNA egress from the NEVs (Figure 2). DISCUSSION

Although membrane lipids are extensively peroxidized by iron-dependent radical reactions during reperfusion 30-42 and in vitro exposure of NEVs to an iron-dependent radical reaction ablates their NTPase activity, we found no evidence of inhibition of NEV NTPase activity or of inhibition of mRNA egress from NEVs in association with brain Table 1, Michaelis-Menten constants for canine brain NTPase.

• 301~MH20/100 #M Fe

• 15zM H20/50pM Fe • 2 #M H20/6~tMFe

0.2

O.

Group (n=3 each) NIC

i

0

2

4

6 8 Time(min)

1'0

1'2

NEVswere preparedfrom normal brain and subjectedto hydroxyl radicalsgenerated by addition of hydrogen peroxideand ferrous sulfate at the concentrationsshown in the legend. After 15 minutes of incubation, we measuredNTPaseactivity as described in the text. All concentrationsof the hydroxyl radical tested resulted in complete inhibition of the NEV NTPase.

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ISCH R2 R6

K,. (nM ATP)

Vmax (nM ADP/mg protein/min)

1.0 .5 1.0 1.2

20 18 19 24

Lineweaver-Burkplots ware constructedfor the hydrolysis of ATP by NEV NTPaseextractedfrom each group. Linearregression lines were fit (NIC, rt=.72; ISCH,P=.82; R2, r2=.87; RB, r2=.92),and the correspondingvalues of Kmand Vmax were calculated.ANOVAof the regressionslopes showed no significant differences between experimentalgroups(F316=2.28, P>.20),indicating that ischemia and reperfusiondo not affect the activity of the brain nuclear envelope NTPase.

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ischemia and reperfusion. Loss of NEV NTPase activity by way of a radical insult is not surprising; plasmalemmabound ATPase loses activity when its membrane is peroxidized 17-21, and NEV NTPase is also a membrane-bound ATPase. Our observations that (1) low concentrations of hydroxyl radical generated from Fe +2 and H20 2 resulted in complete loss of NTPase activity and (2) neither the Km and Vm~x of the NEV NTPase activity nor the NEV ATPdependent poly(A) + mRNA export rate was significantly affected by ischemia and reperfusion in the experimental groups argue that the nuclear membrane is not exposed in vivo to this level of radicals during reperfusion. Mature poly(A) ÷ mRNA does not directly interact with the NTPase during export through the nuclear pore. 12 Instead, the poly(A) tail first binds to a nuclear membrane-associated protein, which then stimulates ATP hydrolysis by the NTPase. This membrane-associated protein offers another potential site for inhibition of mRNA transport, which would be reflected in loss of poly(A) stimulation of NTPase activity NEVs from normal brains showed a 5305 increase in NTPase activity when poly(A) was sealed within the NEVs during their preparation, but we found no evidence of loss of poly(A) stimulation of NTPase activity caused by ischemia and reperfusion. These results indicate that neither the NTPase nor the poly(A) binding protein is the source of inhibition of mRNA transport during postischemic reperfusion. The lipid peroxidation reactions that extensively damage the plasmalemma membrane evidently do not extend to involve the nuclear membrane.

One might argue that we failed to detect inhibition of NTPase or mRNA transport activity because only a few cells sustained significant reperfusion injury at the times examined. The nuclei used in our experiments were isolated from the parietal cortex, where neuronal nuclei outnumber glial nuclei by 10 to 1.43 Previously we examined the histologic distribution of lipid peroxidation products in the reperfused brain. ~6 We constructed photomontages of the full thickness of the parietal cortex from adjacent sections stained by means of the Nissl technique or with a histochemical technique for lipid peroxidation products. In these photomontages, 25% to 30°5 of total parietal cortex cells had sustained a visible degree of lipid peroxidation by 90 minutes of reperfusion after a 10-minute global ischemic insult in rats, and the only cells positive for lipid peroxidation products were neurons; neither glia nor white matter showed any visible evidence of lipid peroxidation. Thus it is unlikely that our results were negative because an insufficient number of cells was damaged. The lack of disturbance of the mRNA nucleocytoplasmic transport system continues a series of observations that now suggest that the major target of free radical attack after ischemia and reperfusion is the plasmalemma membrane. Most if not all maj or intracellular macromolecules are exquisitely sensitive to damage by free radical attack in vitro. Rhaese et al showed that 10 pM FeC13 and 50 laM H20 2 liberated all four bases from DNA; this was rapidly followed by strand scission. 44,45 Likewise, Davies Figure 2.

mRNA export from NEVs.

12'

Poly(A) stimulation of NTPase activity.

10-

Group (n=3 each) NIC NIC + poly(A} ISCH ISCH + poly(A) R2 R2 + poly(A) R6 R6 + poly(A)

NTPase Activity (nM ADP/mg protein/rain 15+2 23_+2 13+2 31_+4 18+1 25_+3 16_+4 29+2

P

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6-

• NIC • ISCH • R2 * R6 []

Control

<.02 <.001 <.05 <.02

Brain NEVswere prepared from each group, and the rate of hydrolysis of 2 mM ATP by the NEV NTPase was measured as described in the text. Polyadenosinewas incorporated into the NEVs during preparation. ATP hydrolysiswith and without poly(A)was compared within each group by use of Student's ttest of the regression slopes. In all groups, NEV NTPasewas stimulated by pely(A) (NIC, 53%; ISCH, 131%; R2, 41%; R6 81%). There was no stimulation of the NEV NTPase activity when the pely(A)was external to the NEV (15+2 versus 17_+1nM ADP/mg protein/minute, P~.50). Data expressed as rnean+SEM.

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ng RNA/mgprotein

Table 2.

,

,

.

1

2

3

,

,

4 5 Time(rain)

.

.

,

.

6

7

8

9

NEVs were prepared from each group and poly(A)+ mRNA was incorporated into the NEV as described in the text. Each point is the mean of three animals, and lines are fit by means of linear regression. The control group uses NEVs from normal brain with poly(A)+ incorporated into the NEVa,but ATP was omitted from the reaction. Neither ischemia nor ischemia and reperfusien altered the paly(A)+ mRNA export rate (F-1.04, G.60; ANOVA of the regression slopes).

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et al showed that nanomolar radical concentrations were sufficient to cause damage in the primary, secondary, and tertiary structure and a 50-fold increased degradation rate in some proteins. 46-49 However, we did not find any evidence of free radical-induced damage to genomic or mitochondrial DNA22 using the same animal model used in this study Similarly, golbergrova et a152 and Krause et a153 did not find any evidence of protein peroxidation in the reperfused brain, although Oliver et al reported some evidence of protein peroxidation. 54 This general lack of radical damage to the internal macromolecules of neurons may provide important insight into the location of the radical species. Whatever the free radical species that initiates lipid peroxidation 55-5r, it must be formed at the site of catalytic quantities of a °'free" transition metal, probably iron in the case of biological systems. 5s,59 There does not appear to be any storage of iron in neurons. Immunohistochemical localization of ferritin and transferrin showed that they are found in the perineuronal and perifascicular oligodendrocytes and are absent from adult neurons. 6°,61 Moreover, the absolute ratio of ferric to ferrous iron is the main factor in initiation of lipid peroxidation.34 Whereas ferrous iron is highly soluble at physiologic pH, formation of iron hydroxides limits the ferric iron concentration in aqueous solutions to about 10-is mol/L62, effectively prohibiting lipid peroxidation initiation. These findings and the paucity of damage to intracellular macromolecules we have observed lead us to suggest that the iron-mediated free radical attack on neurons during brain reperfusion originates outside of the neuron. This theory is supported by the detection of free radicals in the interstitial space by means of spin-trapping and microdialysis during brain ischemia and reperfusion. 63 There is, however, little correspondence between the distribution of iron-containing glia and the selectively vulnerable neurons. 64 This may suggest that preferential lipid peroxidation of neurons in the selectively vulnerable zones is related to mechanisms involving membrane lipid composition (ie, a higher content of polyunsaturated fatty acids represents a better target), a failure of cellular repair mechanisms to keep up with the damage rate 65, a failure of cellular defenses against free radical damage, or increased local radical precursor generation. Gutteridge and Halliwell have proposed that extracellular antioxidant defenses depend mainly on inactivation of iron complexes before they form reactive oxidants. 66 For example, in human serum, ceruloplasmin and transferrin account for only 4% of the total proteins present but account for almost all of the antioxidant activity This ira-

8 14

plies that removal of iron is the main defense mechanism. In this regard, the absence of ferritin and transfemn in the vulnerable neurons may be most unfortunate. In addition, levels of the oxygen radical-scavenging enzymes catalase and glutathione peroxidase are decreased 6r or absent 68, respectively, from the selectively vulnerable areas. Moreover, there is evidence that superoxide dismutase (SOD) and catalase are ineffective in the presence of catalytic amounts of iron. 69 The suppression of protein synthesis in the reperfused brain may prevent the manufacture of necessary defense or repair proteins. Matsuyama et al showed increased production of SOD mRNA in the CA 1 layer of the hippocampus following 5 minutes of transient forebrain ischemia but a decrease in SOD protein. 7° This example may be a generalized phenomenon. Proteins in a family of inducible antioxidant enzymes, recently characterized as the "dectrophile counterattack" group, which includes glutathione transferases, NAD(P)H:quinone reductase, UDP-glucuronsyltransferases, and epoxide hydrolase rl,r2, share AP-1 consensus sequences in the promoter regions of their genes and are induced by barbiturates, which have long been known to exert pretreatment neuroprotective effects. 73 The hea W c-fos (a component of AP-174) transcription response seen in the selectively vulnerable neurons 75,76 during reperfusion may represent a response to radical damage, but failure to subsequently make c-fos protein 7r,rs may represent an important block of the neurons' attempt to defend themselves from radical-mediated damage. Several alternate sites of disruption in the transcription-translation pathway would produce nuclear retention of mRNA and suppression of protein synthesis during reperfusion. The mRNA transport system can be thought of as an "assembly line" that carries newly minted heterogenous nuclear RNA from the site of transcription through the posttranscriptional processing system to specific sites in the cytoplasm, where it is translated. Studies have shown strikingly localized, curvilinear tracks of specific transcripts in the nucleus rg, an asymmetric distribution of the components of the splicing system in the nucleus 8°, the apparent transport of mRNA along defined intranuclear "tracks" that extend from specific genes toward a single or small subset of nuclear pores sl, and specific subcellular localization of mRNA messages. 82-84 Disturbance of this assembly line would be expected to cause a backup in mRNA transport and contribute to decreased protein synthesis after an ischemic insult. Several examples of disruption of the assembly line--including posttranscriptional processing, collapse of the cytoskele-

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ton, and failure of translation initiation--are found in the hyperthermia-induced heat-shock model. When Drosophila melanogastercells are subjected to heat shock, a transient, generalized block of the splicing system persists for hours after the insult sS, and unspliced messages accumulate in the nucleus, s6 Once blocked, unspliced transcripts do not appear to reenter the splicing pathway and are not transported to the cytoplasm. Non-introncontaining transcripts are not affected because they do not require splicing. After translocation across the nuclear membrane, most cytoplasmic mRNA remains attached to the cytoskeleton. 87,88 Evidence suggests that translation may depend on this association because nearly all of the mRNA actively engaged in protein synthesis is associated with the cytoskeleton.89-91 Hyperthermia-induced heat shock causes collapse of the cytoskeleton92,93 and heat-shock protein synthesis is necessary for reformation of the cytoskeleton. 94 Disruption and restitution of polyribosomes and, presumably, translation, was coincident with the changes in the cytoskeletal organization. 9~ Finally, there may be a failure to form the ribosomal initiation complex required for translation of the mRNA. Two potential mechanisms for inhibition of the formation of the initiation complex are changes in the activities of eukaryotic initiation factor 4E (eIF-4E), which binds to the mRNA 5" cap and escorts the mRNA to the ribosome, and eIF-2, which regulates the overall rate of protein synthesis. 1 Hyperthermia-induced heat shock decreases the ability of eIF-4E to bind to the 5" cap on the mRNA and decreases protein synthesis. 96 Addition of eIF4E derived from normal ceils restores the affinity of elF4E for the 5" mRNA cap and rescues protein synthesis. 9r The activity of elF-4E has not been studied during ischemia and reperfusion. Similarly, heat shock alters the phosphorylation of eIF-20~98,99, producing severe inhibition of protein synthesis. Hu and Wieloch, using a carotid occlusion/hypotension rat model, found decreased elF-2 activity in the brain which was attributed to a decrease in elF-2B activity; however, they did not examine protein synthesis. 100 It remains to be determined to what extent the heat-shock-like response that follows ischemia and reperfusion parallels the hyperthermia-induced heat shock case. In summary, we found no evidence for reperfusioninduced inhibition of the NEV NTPase or of poly(A) + mRNA transport, although these systems are very sensitive to in vitro radical attack. These findings, taken together with other evidence, suggest that the main subcellular target of radical damage during reperfusion is

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the plasmalemma. Direct inhibition of nuclear cytoplasmic transport of mRNA cannot account for the observed inhibition of protein synthesis and nuclear retention of newly synthesized mRNA, and further study is needed of other potential mechanisms--including cytoskeletal degeneration and failure of formation of the initiation complex-involved in these phenomena. REFERENCES 1. Krause GS, Tiffany BR: Suppression of protein synthesis in the reperfused brain. Stroke 1993;24:747-756. 2, Dienel GA, Pulsinelli WA, Duffy TE: Regional protein synthesis in rat brain following acute hemispheric ischernia. J Neurochem1980;35:1216-1226. 3. Bodsch W, Barbier A, Oehmichen M, et ah Recovery of monkey brain after prelenged ischernia, II. Protein synthesis and rnerpholegic alterations. J CerebBloodFlow Metab 1986;6:22-33. 4. Thilrnann R, Xie Y, Kleihues P, et al: Persistent inhibition of protein synthesis precedes delayed neuronal death in post-ischernic gerbil hippocarnpus. Acta Neuropatho11986;71:88-93. 5. Kiessling M, Xie Y, KleJhues P: Regionally selective inhibition of cerebral protein synthesis in the rat during hypoglycemia and recovery. J Neurochem1984;43:1507-1514. 6. Pulsinelli WA, Brierley JB, Plum F: Temporal profile of neuronal damage in a model of transient ferebrain ischemia. Ann Neural1982;11:491-498. 7. Widmann R, Kumiwa T, Bonnekoh P, et aL [14C]leucine incorporation into brain proteins in gerbils after transient ischernia: Relationship to selective vulnerability of hippecarnpus. J Neurochem1991 ;56:789-796. 8. Matsurnoto K, Yamada K, Hayakawa T, et ah RNA synthesis and processing in the gerbil brain after transient hindbrain ischernia. NeuralRes 1990;12:45-48. 9. Sakaguchi T, Yamada K, Hayakawa T, et ah Malfunction of gone expression as a possible cause of delayed neuronal death. No To Shinkei1988;40:629-635. 10. Marune M, Yanagihara T: Progressive loss ef messenger RNA and delayed neuronal death following transient cerebral ischernia in gerbils. NeurosdLett1990;115:155-160. 11. Dworetzky SI, Fe[dherr CM: Trenslocatien of RNA-coated gold particles through the nuclear pores of eocytes. J CellBiol 1988;106:575-584. 12. Forbes DJ: Structure and function of the nuclear pore complex. AnnuRevCeil Biol 1992;8:495-527. 13. Agutter PS, McArdle 14J, McOaldin B: Evidence for involvement of nuclear envelope nucleoside triphosphatase in nucleocytoplasrnic translocation of ribonucleoprotein. Nature 1976;263:165-167, 14. Agutter PS, McCaldin B, McArdle M J: Importance of rnarnrnalian nuclear-envelope nucleoside triphesphatase in nucleo-cytoplasrnie transpert of ribenucleoproteins. BiochemJ 1979;182:811-819. 15. Kender-Koch C, Riedel N, Valentin R, et ah Characterization of an ATPase on the inside af ratliver nuclear envelopes by affinity labeling. EurJSiochem1982;127:285-289. 16. White BC, Daya A, DeGracia DJ, et al: Fluorescent histochernical localization of lipid peraxidation during brain reperfusion following cardiac arrest. Acta Neuropathol(Berlin)1993;86:1-9. 17. Enseleit WH, Darner FR, Jarrott DM, et al: Cerebral phasphelipid cantent and NA+K+-ATPase activity during ischemie and post-ischernic reperfusion in the rnongorian gerbil JNeurochem 1984;43:320-327. 18. MacMillan V: Cerebral NA+K+-ATPase activity during exposure to and recovery from acute ischemia. J CerebBloodFlowMetab 1982;2:457-465. 19. Galdberg W J, Watson BD, Busto R, et al: Concurrent measurement of (NA+K+)-ATPase activity and lipid peroxides in rat brain following reversible global ischemia. NeurochemRes 1984;9:1737-1747. 20. Chen JW, Zhang I_, Lian X, et al: Effect of hydroxyl radical on Na(+)-K(+)-ATPase activity of the brain microsoma[ membranes. CellBiol Int Rep1992;16:927-936.

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43. Petit CK, Babiak T: Early proliferative changes in astrocytes in post-ischemic noninfarcted rat brain. Ann Neurol1982;11:510-518. 44. Rhaese HJ, Freese E: Chemical analysis ef DNA alterations. 1. Base liberation and backbone breakage of DNA and oligedeoxyadenylic acid induced by hydrogen peroxide and hydroxylamine. BiachimBiophysActa 1968;155:476-490. 45. Rhaese HJ, Freese E, Melzer MS: Chemical analysis of DNA alterations. II. Alteration and liberation of bases of deoxyneucleotides and deoxyneucJosides induced by hydrogen peroxide and hydrexylamine. BiechimBiophysActa 1968;I 55:491-504.

70. Matsuyama T, Mishishita H, Nakamura H, et el: Induction of copper-zinc superexide dismutase in gerbil hippocampus after ischemia. J CerabBloodFlew Metab 1993;13:135-144. 71. Prestera T, Zhang Y, Spencer SR, et al: The electrophile counterattack response: Protection against eeoplasia and toxicity. Adv EnzRegu11993;33:281-296. 72. Prestera T, Haltzclaw WD, Zhang Y, et al: Chemical and molecular regulation of enzymes that detoxify carcinogens. ProcNatlAcedSci USA 1993;90:2965-2969. 73. Safar P: Resuscitation from clinical death: Pathephysielog[o limits and therapeutic potentials. Crit CareMed 1988;16:923-941.

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74. Angel P, Kafin M: The role of Jun Fos and the AP-1 complex in cell-proliferation and transformation. BiechimBiephysActa 1991 ;1072:129-157.

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Reprint no. 47/1/63453 Address for reprints:

76. Nowak TS Jr, Ikeda J, Nakajima T: 70 kDa heat shock protein and c-los gone expressionafter transient ischemia. Stroke 1990;21(supp1110:167-111. 77. Uemura Y, Kowall NW, Beal MF: Globat ischemia induces NMDA receptor-mediated c-fos expression in neurons resistant to injury in gerbil hippocampus. Brain Roe 1991;542:343-347.

"

78. Jorgensen M, Deckert J, Wright 13,eta[: Delayed c-fos prote-oncogeneexpression in the rat hippocampus induced by transient grobal ischemia: an in situ hybridization study. Brain Res 1989;484:393-398. 79. LawrenceJB, Singer RH, Marselle LM: Highly localized tracks of specific transcripts within interphase nuclei visualized by in situ hybridization. Cefl1989;57:493-502. 80. Specter DL: Higher order nuclear organization: Three-dimensionaldistribution of small ribonucleopretein particles. ProcNeff Acad Sci USA 1990;87:147-151.

Gary Krause, MD, MS Department of Emergency Medicine Wayne State University 4201 St Antoine Detroit, Michigan 48201 313-577-3187 Fax 313-745-3653

81. Nobel G: Gone gating: A hypothesis. PrecNat/Acad Sci USA 1985;82:8527-8529. 82. Capco DG, Jeffery WR: Regional accumulation of vegetal pole poly (A)+ RNA injected into fertilized Xenopuseggs. Nature 1981;294:255-257. 83. Agutter PS: RNA transport, in Between Nucleus and Cytoplasm. New York: Chapman& Hall, 1991, pp 97-110. 84. Russell B, Dix DJ: Mechanisms for the intracellurar distribution of mRNA: In situ hybridization studies in muscle. Am J Physio11992;262:C1-C8. 85. Yost HJ, Lindquiet S: RNA splicing is interrupted by heat shock and is rescued by heat shock protein synthesis. Cell 1986;45:185-193. 86. Yost HJ, Lindquist S: Translation of unspliced transcripts after heat shock. Science 1988;242:1544-1548. 87. Jeffery WRL: Messenger RNA in the cytoskeletal framework: Analysis by in situ hybridization. J Cell Die/1982;95:1-7. 88. Lenk R, Ransom L, KaufmannY, et ah A cytoskeletal structure with associated polyribosemes obtained from HeLacells. Cell 1977;10:67-78. 89. CerevraM, Dreyfuss G, Penman S: Messenger RNA is translated when associated with the cytoskeletal framework in normat end VSWinfected HeLacells. Cell1981;23:113-120. 90. Ornetles DA, Fey EG, PenmanS: Cytochalasinreleases mRNA from the cytoskeletal framework and inhibits protein synthesis. Me/Cell Bio11986;6:1650-1662. 91. Van Venrooij WJ, Sillikens PTG.Van EekelenCAG, et el: On the association of mRNA with the cytoskeleton in uninfected and adenovirus-infected human KB cells. Exp Cell Res 1981;135:79-91. 92. Walter MF, Petersen NS, Biessmann H: Heat shock causes the collapse of the intermediate filament cytoskeleton in Drosophila embryos. Dev Genet 1990;11:279-279. 93. van Bergen en Henegouwen PM, LinnemansAM: Heat shock gene expression and cytoskeJetal alterations in mouse neurob/astomacells. Exp Cell Res 1987;171:367-375. 94. Jordi WJ, van Dongen G, RamaekersFC, et al: Studies on a possible relationship between alteration in the cytoske]eton and induction of heat shock protein synthesis in mammalian cells. Int J Hyperthermia 1985;1:69-83. 95. Shyy TT, Asch BB, Asch HL: Concurrentcollapse of keratin filaments aggregation of organerlasand inhibition of protein synthesis during the heat shock response in mammary epithelial cells. J Cell Bio11989;108:997-1008. 96. LamphearBJ. Panniers R: Heat shock impairs the interaction of cap-binding protein complex with 5" mRNA cap. J Biel Chem1991;266:2789-2799. 97. LamphearBJ, PanniersR: Cap binding protein complex that restores protein synthesis in heat-shocked Erlich cell lysates contains highly phosphorylatedelF-4E. J Biol Chem 1990;265:5333-5336. 98. ScorseneKA, PanniersR, Rowlands AG, et el: Phosphorylationof eukaryotic initiation factor 2 during physiological stresses which affect protein synthesis. JBiol Chem1987;262:14538-14543. 99. Duncan R, HersheyJWB: Heat shock-induced translational alterations in HeLa ceils. JBiol Chem 1964;259:11882-11889. 100. Hu DR, Wieloch T: Stress-inducedinhibition of protein synthesis initiation: Modulation of initiation factor 2 and guanine nuclootide exchangefactor activities following transient ischemia in the rat. J Neurosci 1993;13:1830-1838.

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