role of the ryanodine receptor in ischemic brain damage—localized reduction of ryanodine receptor...

Cellular and Molecular Neurobiology, Vol. 19, No. 1, 1999

Role of the Ryanodine Receptor in Ischemic BrainDamage—Localized Reduction of Ryanodine ReceptorBinding During Ischemia in Hippocampus CA1

Hiroyuki Nozaki,1,3 Kortaro Tanaka,1 Shintaro Gomi,1 Ban Mihara,2

Shigeru Nogawa,1 Eiichiro Nagata,1 Taro Kondo,1 and Yasuo Fukuuchi,1

Received December 28, 1995; accepted February 16, 1996

SUMMARY

1. The ryanodine receptor has recently been shown to play a pivotal role in theregulation of intracellular Ca21 concentration via Ca21-induced Ca21 release (CICR). Effectsof ischemia on CICR in the brain tissue, however, remain largely unknown since only afew reports have been published on this subject. In this paper we report on work in thisarea by our group and review related progress in this field.

2. We examined alterations of ryanodine receptor binding and local cerebral bloodflow (LCBF) at 15 min, 30 min, and 2 hr after occlusion of the right common carotidartery in the gerbil brain. A quantitative autoradiographic method permitted simultaneousmeasurement of these parameters in the same brain. The LCBF was significantly reducedin most of the cerebral regions on the occluded side during each time period of ischemia.In contrast, only in the hippocampus CA1 on the occluded side was a significant reductionin ryanodine binding found at 15 min, 30 min and 2 hr after the occlusion.

3. These findings suggest that suppression of ryanodine binding in the hippocampusCA1 may be attributable to a regionally specific perturbation of CICR and that thisperturbation may be closely associated with the pathophysiological mechanism that leadsto the selective ischemic vulnerability of this region.

4. Other recent studies have also reported an important role for ryanodine receptorsin neuronal injury such as the delayed neuronal death in the hippocampus CA1. Thesedata suggest that derangement of CICR is likely to be involved in acute neuronal necrosisas well as in delayed neuronal death in ischemia.

5. Further studies on clarifying the role of CICR in ischemic brain damage are neededin order to develop new therapeutic strategies for stroke patients.

KEY WORDS: ryanodine receptor; cerebral ischemia; second messenger; Ca21.

INTRODUCTION

The calcium ion (Ca21) has become a major focus of attention in the last decadedue to its possible role in mediating ischemic neuronal death (Miller, 1988). The

1 Department of Neurology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo160-8582, Japan.

2 Mihara Memorial Hospital, Gunma 372, Japan.3 To whom correspondence should be addressed.

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0272-4340/99/0200–0119$16.00/0 1999 Plenum Publishing Corporation

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mechanisms of intracellular Ca21 overload involve two main routes: influx from theextracellular Ca21 and release from the internal Ca21 pools. Influx of extracellularCa21 in ischemia could be mediated through channels linked to transmitter-specificreceptors such as N-methyl-D-aspartate (NMDA) preferring receptors (Benvenisteet al., 1984; Rothman and Olney, 1986) or voltage-sensitive channels (Choi, 1988).Intracellularly, the endoplasmic reticulum contains at least two types of releasableCa21 stores, of which one is released by inositol 1,4,5-trisphosphate (IP3) via IP3

receptors (IP3-induced Ca21 release; IICR) (Berridge and Irvine, 1989), and theother is released by Ca21 itself in a process referred to as Ca21-induced Ca21 release(CICR) via the ryanodine receptor (Henzi and MacDermott, 1992).

Ryanodine is a plant alkaloid, which selectively binds to the ryanodine receptorlocated either on the sarcoplasmic or on the endoplasmic reticulum, and inducesCICR. The ryanodine receptor contains an amino acid sequence similar to the IP3

receptor (Furuichi et al., 1989; Sudhof et al., 1991; Yoshikawa et al., 1992). An invitro study on brain slices indicated that approximately one-third of the elevatedintracellular [Ca21] during ischemia may be derived from extracellular Ca21 influx,indicating that the remaining two-thirds is provided by intracellular Ca21 stores(Mitani et al., 1993). Intracellular Ca21 pools thus appear to play a critical role inthe regulation of free cytosolic Ca21 concentration and related cellular activity underischemic conditions.

In a previous study, we observed a significant reduction in IP3 binding localizedto the hippocampus CA1 after 2- and 6-hr hemispheric ischemia in the gerbil brain(Nagata et al., 1994; Nagata, 1995). These findings suggest that changes in IP3

receptor binding may be related to selective vulnerability of the hippocampus CA1.A genetically engineered IP3 receptor type 1-deficient mouse has recently beendeveloped (Matsumoto et al., 1996), which may help to clarify the role of thisreceptor in ischemic brain damage.

The ryanodine receptor, however, has been given less attention, and its rolein various physiological as well as pathological conditions is still largely unknown.In this review, we briefly describe the fundamental features of the ryanodine recep-tor, then describe our studies on the alteration of ryanodine receptor binding duringthe acute ischemic phase in the gerbil brain, and finally, review other related studieson CICR in ischemic brain damage.

RYANODINE RECEPTOR PHYSIOLOGY

The ryanodine receptor is present in many tissues, where it mediates depolariza-tion-mediated activation or, more commonly, Ca21-mediated release of intracellularCa21-store release (Fig. 1) (Simpson et al., 1995). The ryanodine receptor is ahomotetramer composed of 550- to 565-kDa monomers, one of the largest intracellu-lar proteins (Henzi and MacDermott, 1992). Ryanodine receptors are usually di-vided into three main subtypes as shown in Table I (Meissner, 1994): the skeletalmuscle type (RYR-1) (Takeshima et al., 1989), cardiac muscle type (RYR-2) (Nakaiet al., 1990; Otsu et al., 1990), and brain type (RYR-3) (Hakamata et al., 1992). Thethree types of ryanodine receptor subunits are encoded by different genes having

Role of Ryanodine Receptor in Ischemic Damage 121

Fig. 1. Schema of Ca21 induced Ca21 release. The ryanodine re-ceptor is located on the endoplasmic reticulum, where it plays animportant role in regulation of the intracellular Ca21 level viacalcium-induced calcium release (CICR). Re, receptor; G, GTP-binding protein; PLC, phospholipase C; DAG, diacylglycerol;PKC, protein kinase C; IP3 , inositol 1,4,5-trisphosphate; ER, endo-plasmic reticulum; PAF, platelet activating factor; CICR, Ca21-induced Ca21 release; IICR, IP3-induced Ca21 release; FKBP, FK-506 binding protein.

Table I. Ryanodine Receptor Subtypes

Subtype Location Caffeine sensitivity

RYR-1 Skeletal muscle, cerebellar Purkinje cells 1RYR-2 Heart, stomach, endothelial cells, brain (widespread) 11RYR-3 Epithelial cells, smooth muscle, brain (restricted regions) 2

60–70% homology (McPherson and Campbell, 1993; Meissner 1994). It has recentlybeen demonstrated that several forms of ryanodine receptor are expressed in thebrain.

At least three distinct genes encoding for the ryanodine receptor are expressedin the mammalian brain: RYR-1 appears to be expressed exclusively in cerebellarPurkinje neurons (Kuwajima et al., 1992). RYR-2 is expressed uniformly throughoutthe brain (Nakai et al., 1990; Kuwajima et al., 1992) and appears to be the majorform of the ryanodine receptor in the brain (McPherson and Campbell, 1993).RYR-3 is abundantly expressed in the corpus striatum, thalamus, and hippocampus,(Hakamata et al., 1992; Furuichi et al., 1994).

Endogenous modulators of ryanodine receptors appear to include Mg21, ade-nine nucleotides such as ATP, calmodulin, and various protein kinases. Cyclicadenosine 59-diphosphoribose (cyclic ADP ribose; cADPR) has also been proposedas an important endogenous activator of ryanodine receptors (Furuichi et al., 1994;Hua et al., 1994), due to its preponderance in a wide variety of tissues. FKBP-12(FK-506 binding protein; cis–trans-peptidylprolylisomerase) is reported to be closelyassociated with the ryanodine receptor/calcium channel (Jayaraman et al., 1992;Brillantes et al., 1994), and its function is to modulate the activity of the calciumchannel (Timerman et al., 1993).

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Exogenous modulators such as caffeine and a low concentration of ryanodinealso activate or enhance Ca21 release (Kuba, 1994; Simpson et al., 1995), althoughRYR-3 appears to release Ca21 in response to ryanodine but not to caffeine (TableI) (Meissner, 1994).

ALTERATION OF RYANODINE RECEPTOR BINDING DURINGISCHEMIA IN THE HIPPOCAMPUS CA1

Six-Hour Ischemia

In order to evaluate the influence of cerebral ischemia on CICR, alterationsin ryanodine receptor binding and local cerebral blood flow (LCBF) were examinedat 6 hr after occlusion of the right common carotid artery in the gerbil brain (Nozakiet al., 1995b). An autoradiographic method developed in our laboratory enabledus to measure both parameters within the same brain (Takashima et al., 1990;Tanaka et al., 1991). Animals attaining ischemic scores of more than 5 (McGraw,1977) at 1 hr postocclusion were used. LCBF was measured using [14C]iodoantipyrineat 6 hr postocclusion or sham operation (Sakurada et al., 1978; Tanaka et al., 1991).Ryanodine receptor binding was evaluated in vitro using [3H]ryanodine (15 nM)as a specific ligand (Padua et al., 1991; Nozaki et al., 1995b; 1996; 1997). Nonspecificryanodine binding was evaluated using incubation with [3H]ryanodine and unlabeledryanodine(15 eM), while specific ryanodine binding was calculated by subtractingthe nonspecific binding from the total binding.

Figure 2 shows the results of Scatchard analysis of specific ryanodine binding

Fig. 2. Scatchard plot of specific ryanodine binding. The dataillustrate that each brain region contains a single class of bindingsites (Nozaki et al., 1995b).


in the normal gerbil brain as reported previously (Nozaki et al., 1995b). These dataindicate that each brain region contains a single class of ryanodine binding sites.

Representative color-coded autoradiograms of LCBF and ryanodine bindingat the level of the hippocampus are shown in Fig. 3. LCBF was significantly reducedin most of the cerebral regions on the occluded side. In contrast, a significantreduction in ryanodine binding sites was noted only in the hippocampus CA1 onthe occluded side (Nozaki et al., 1995b). Ryanodine receptor immunoreactivityexamined with a specific antibody against ryanodine receptor protein, however, didnot reveal any differences between the ischemic and the sham groups on eitherside, suggesting that ryanodine receptors may not undergo significant morphologicaldegradation despite significant reduction in binding (Nozaki et al., 1995b).

No significant correlation was noted between LCBF values and ryanodinebinding in any region except for the hippocampus CA1. Figure 4 illustrates therelationship between LCBF values and ryanodine binding in the hippocampus CA1.Although a range of [3H]ryanodine binding was obtained, these values tended to

Fig. 3. Representative color-coded autoradiograms of LCBF and ryanodine binding at the level ofthe hippocampus. The viewer’s right is the right side of the brain. The upper autoradiograms wereobtained from animals who underwent sham operation, whereas the lower images were obtainedfrom those who sustained 6-hr occlusion. Left: LCBF; right, ryanodine binding. LCBF was significantand homogeneously reduced in each of the cerebral regions on the occluded side compared withthe sham group. In contrast, a significant reduction in ryanodine binding was localized to the hippocam-pus CA1 on the occluded side compared to that seen in the sham group (Nozaki et al., 1995b).

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Fig. 4. Correlation between LCBF and specific ryanodinebinding in the hippocampus CA1 6 hr postischemia. Filledcircles represent data from the occluded side, and filled squaresfrom the nonoccluded side in the ischemia group. Open circlesrepresent data from the occluded side, and open squares fromthe nonoccluded side in the sham-operated group. Note thereduction in ryanodine binding when LCBF fell below 20 ml/100 g/min (Nozaki et al., 1995b).

be low when LCBF was less than 20 ml/100 g/min. In contrast, [3H]ryanodinebinding was relatively constant when LCBF was above 20 ml/100 g/min.

Fifteen-Minute to Two-Hour Ischemia

Figs. 5 and 6 summarize LCBF and ryanodine binding examined at 15 min, 30min, and 2 hr postocclusion of the right common carotid artery, respectively. Thedata for sham-operated animals are also shown. Although LCBF was decreasedsignificantly, to less than 10 ml/100 g/min in most of the cerebral regions on theoccluded side (Fig. 5), significant reductions in ryanodine binding sites were localizedto the hippocampus CA1 on the occluded side during each period of ischemia (Fig.6) (Nozaki et al., 1995a; 1996; 1997).

Suppression of ryanodine binding in the hippocampus CA1 could be explainedby a regionally specific perturbation of CICR in these regions. Such perturbationmay become evident as early as after 15 min of ischemia and may be related tothe pathophysiological mechanism of selective ischemic vulnerability of this region.

Ryanodine Receptor Binding Study in Ischemia: Discussion

The above data demonstrated that significant decreases in ryanodine bindinglocalized to the hippocampus CA1 from 15 min to 6 hr of ischemia. Since a homoge-neous reduction of LCBF was observed in all regions except the cerebellum on theoccluded side, this indicates that localized decreases in ryanodine binding in thehippocampus CA1 may represent a regionally specific reaction to ischemia and thatCICR may become impaired during the acute ischemic phase in this region.


Fig. 5. Local cerebral blood flow. LCBF was significantly reduced, to less than 10 ml/100g/min, in most of the cerebral regions on the occluded side at each ischemic time point. Theleft and right panels show the nonoccluded and occluded sides of the brain, respectively.Significant differences from the sham group: *P , 0.05; **P , 0.01.

Fig. 6. Ryanodine binding. A significant reduction in ryanodine binding sites was noted onlyin the hippocampus CA1 on the occluded side at each ischemic time point. The left and rightpanels show the nonoccluded and occluded sides of brain, respectively. Significant differencesfrom the sham group: *P , 0.05; **P , 0.01.

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The localized reduction of ryanodine binding in the hippocampus CA1 demon-strated in the present study may be related to any of the following mechanisms:(1) dephosphorylation of the ryanodine receptor, (2) a high accumulation of intracel-lular Ca21, (3) morphological changes in the ryanodine receptor, (4) ischemic vulner-ability of a specific subtype of ryanodine receptor, (5) alteration of the FKBP, or(6) a reduction of cADPR.

The ryanodine receptor is a substrate for phosphorylation by cyclic AMP-dependent protein kinase (cAMP-DPK) and Ca21/calmodulin-dependent kinase II.The phosphorylated ryanodine receptor exhibits an increase in ryanodine bindingas well as an enhanced channel activity (McPherson and Campbell, 1993), and theryanodine receptor in the brain appears to be a better substrate for phosphorylationthan the skeletal ryanodine receptor (Takasago et al., 1989, 1991; Yoshida et al.,1992).

In a separate study, we found that cyclic AMP binding sites, an indicatorof the total amount of particulate cAMP-DPK, were significantly reduced in thehippocampus CA1 on the ischemic side at 30 min and 2 hr of ischemia in the gerbilbrain (Tanaka et al., 1996). The reduction of ryanodine receptor binding in thehippocampus CA1 was relatively stable during 15 min to 2 hr of ischemia as shownin Fig. 6. These observations suggest that specific reduction of ryanodine bindingin the hippocampus CA1 may be associated with inhibition of cAMP-DPK.

On the other hand, the reduction of cAMP-DPK in ischemic hippocampusCA1 was found to become gradually more pronounced over the time course ofischemia (Tanaka et al., 1995; 1996). Moreover, cAMP binding in the pyramidalcell bodies was not decreased after 15 min of ischemia (Tanaka et al., 1997), whileryanodine binding was already suppressed at the same time point. Taken together,the reduction in ryanodine binding in the present study may not be directly relatedto the inhibition of cAMP-DPK.

Ryanodine binding is inhibited by a high concentration of intracellular Ca21

(Michalak et al., 1988), and it is well known that a profound accumulation ofintracellular Ca21 occurs, especially in the hippocampus CA1, after ischemic insult(Miller, 1988). Significant elevation of intracellular Ca21 is a likely cause of theryanodine binding inhibition seen in the present study.

Although light microscopic investigation revealed no apparent loss of neuronsin any of the regions examined, severe ischemia may have induced ultrastructuralalterations. We found that ryanodine receptor immunoreactivity, determined withspecific antiryanodine receptor antibody, did not reveal any noticeable differencesbetween the ischemic and control groups after 6-hr hemispheric ischemia in thegerbil brain (Nozaki et al., 1995b). This indicates that the protein constituents ofthe ryanodine receptor may not have been totally destroyed in the present study.Similarly, because IP3 receptor binding was not reduced in the hippocampus CA1after 30 min of ischemia (Nagata, 1995), this suggests that nonspecific structuraldestruction of the endoplasmic reticulum cannot explain the present findings. Verysubtle morphological or conformational changes of ryanodine receptor protein thatcould cause decreased binding cannot be ruled out in the present study. Furtherstudies including electron microscopic examinations are needed to analyze in detailstructural alterations of the ryanodine receptor that could occur during ischemia.


There are three main types of ryanodine receptor as described in the initialpart of this review: the skeletal muscle type (RYR-1), cardiac type (RYR-2), andbrain type (RYR-3). The RYR-3 is abundantly expressed in localized regions suchas the corpus striatum, thalamus, and hippocampus (Hakamata et al., 1992). Thelocalized distribution may indicate that Ca21 regulation in these regions is differentfrom that in other areas of the brain, and these regions roughly correspond toareas where delayed neuronal death occurs after cerebral hypoxia (Kirino, 1982;Hakamata et al., 1992). In the preset study, however, only hippocampus CA1 re-vealed a significant reduction in ryanodine binding. This suggests that decreasedbinding cannot be fully explained on the basis of receptor subtype alone.

FKBP has recently been found to be tightly associated with the ryanodinereceptor under normal conditions (Timerman et al., 1994). Snyder and co-workers(Steiner et al., 1992; Lyons et al., 1995) recently found FKBP to be present inneuronal tissue (Steiner et al., 1992). Since FKBP is regarded as a gating proteinfor the ryanodine receptor (Timerman et al., 1993), alterations of FKBP inducedby ischemia could elicit a reduction in ryanodine binding. In this context, FK-506has recently been reported to be cytoprotective against the excitatory amino acid(NMDA) (Liu et al., 1992; Dawson et al., 1993) and focal ischemic insult (Sharkeyand Butcher, 1994). FK-506, an immunosuppresant, binds to FKBP, and the resul-tant FK-506-FKBP complex leads to detachment of FKBP from the ryanodinereceptor. FKBP is especially abundant in the hippocampus CA1 (Steiner et al., 1992),and alterations in FKBP induced by ischemia are currently under investigation inour laboratory.

cADPR synthesized from NAD1 is an endogenous activator of the nonskeletaltype of ryanodine receptor, and this substance may play a role similar to IP3 inCa21 signaling (Meszaros et al., 1993; White et al., 1993). Extracts from rabbit brainhave been found to contain cADPR-synthesizing enzymes (Rushinko and Lee,1989). cADPR has no effect on skeletal muscle RYR-1 so its messenger functionis restricted to RYR-2 and perhaps also to RYR-3 (Berridge, 1993). Since alterationsof cADPR have as yet to be reported in cerebral ischemia, we cannot directlyspeculate on the relationship of cADPR to the present findings. If the level ofcADPR is reduced by ischemic insult, however, this could cause a reduction inryanodine binding.

CALCIUM MOBILIZATION AND CEREBRAL ISCHEMIA

Intracellular calcium overload has been proposed as being one of the mainfactors triggering ischemic brain injury (Miller, 1988; Kennedy, 1989). As describedbefore, an in vitro study on brain slices has indicated that approximately one-thirdof the elevated intracellular [Ca21] after ischemic stimulation was derived fromextracellular Ca21 influx, so that the remaining two-thirds would be expected tocome from intracellular Ca21 stores (Mitani et al., 1993).

Previous studies (Hirashima et al., 1985; Ikeda et al., 1986; Abe et al., 1987)have consistently reported enhanced activity of phospholipase C in brain tissue inresponse to ischemia, and a transient increase in IP3 concentration in the brain has

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been described in a decapitation ischemia model (Lin et al., 1993). In transientischemia, IP3 binding sites in the hippocampus CA1 have been reported to undergoa gradual decrease after the onset of ischemia (Onodera and Kogure, 1989). In aprevious study, we observed a significant reduction in IP3 binding localized to thehippocampus CA1 after 2- and 6-hr hemispheric ischemia in the gerbil brain (Nagataet al., 1994; Nagata, 1995). These findings suggest that IICR plays an importantrole in the selective vulnerability of the hippocampus CA1.

Studies using primary cultures of cerebral cortical neurons have revealed thatdantrolene, a specific inhibitor of CICR (Ellis and Carpenter, 1972; Van Winkle,1976), provided complete protection against glutamate-induced neurotoxicity(Frandsen and Schousboe, 1991). Furthermore, an in vivo investigation demon-strated that dantrolene protected against delayed neuronal death in a transientischemia model in the gerbil (Zhang et al., 1993). Mitani et al. (1993) found thatmobilization of Ca21 from intracellular stores was reduced to roughly half by dantro-lene administration. These findings also support the role of CICR in ischemicbrain damage.

CONCLUSIONS

The results of our ryanodine binding study as well as those of other reportssuggest that CICR plays an important role in ischemia-induced acute neuronalinjury, as well as delayed neuronal death. CICR is, however, controlled by variousintracellular mechanisms as described above, and the major factors leading to CICRalterations during ischemia are largely unknown at present. Further studies areclearly warranted in order to elucidate the precise mechanisms of CICR alterationsin cerebral ischemia.

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role of the ryanodine receptor in ischemic brain damage—localized reduction of ryanodine receptor...

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