Oxidative Stress Poster Session
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Introduction
Intracellular Ca overload, excitotoxic activity of glutamate and concomitant generation of free radicals are thought to play the prominent role as triggering factors in the pathogenesis of brain ischemic-reperfusion injury (IRI).(1) Intracellular dysregulation of Ca homeostasis is proposed as critical step responsible for delayed neuronal death of selective regions of hippocampus, but it is not yet clear which sources of Ca and which pathways are involved (2).
The plasma membrane Ca2+ pump (PMCA) is ubiquitously expressed protein with the sole high affinity Ca extrusion mechanism in the plasma membrane(3). It is believed to play a major role in maintaining a basal level of [Ca ]i.Evidence also suggests a more active role for the PMCAs in returning [Ca]i to basal levels after cellular depolarization. Both PMCA and organellar Ca pump (SERCA) are the main determinants for lowering cytosolic Ca at physiological conditions in hippocampal neurones, whereas the Na, Ca-exchanger and mitochondria seem to play a minor role (4).
The Na pump is the enzyme responsible for the major portion of brain energy expenditure and the Na gradient. A quantitative Western blot technique was used to assess the level of proteins in the cells of gerbil CNS involved in ion homeostasis after ischemia-reperfusion injury.
Materials and Methods
Antibodies against isoforms of PMCA were provided by Dr. Guerini, Zürich (5), against alpha 3 isoform of Na+ pump by Dr. Presley, Dallas. Membrane proteins prepared by differential centrifugation were separated by Laemmli gels and Western blotted. Bands stained by diaminobenzidine were digitized by means of Eagle Eye and quantified. Immediately after isotermic (37 degrees C) ischemic insult for 10 min (ischemic group) in halothane anesthesia and/or after 10 days after the operation (reperfusion group), the male gerbils were decapitated. Controls were sham-operated animals (6).
Results
The gene products of PMCA were detected in the hippocampus, cerebral cortex and cerebellum. However, they showed a distict distribution pattern. The PMCA1 isoform is the most abundant in the cortex and hippocampus. The PMCA2 was detected in a lesser amount comparing to PMCA1 and was highest in the cerebellum and in a moderate amount in the cortex. Weakly stained PMCA3 was localized in the cerebellum and to the hippocampus. InsP3 receptor and SERCA are the most abundant in cerebellum and hippocampus. Ischemia and reperfusion leads to a significant decrease of PMCA immuno-signal. This decrease could be ascribed to the loss of PMCA1 signal, especially in hippocampus. The IRI selectively down-regulates also the levels of InsP3 receptor I.
No statistically significant changes have been detected in the levels of SERCA 2b and reticular calreticulin as well as alpha 3 isoform of Na pump.
Calcium acts in neurones as an important second messenger, it also acts as an essential mediator of neuronal cell damage. It is widely accepted that ischemic injury results first from continuous depolarization of the neuronal cells following activation of glutamate excitotoxicity (1). Recent studies suggest that cell death may occur subsequent to loss of Ca homeostasis. Cerebral ischemia is known to alter proteosynthetic and proteolytic machinery. Selective decrease of inositol 1,4,5 trisphosphate mRNA - and NMDA- receptors has been shown after different types of cerebral ischemia(7,8,9). Loss of immuno-signal for PMCA is also in concordance with histochemical study, where a massive disappearance of PMCA activity after IRI was revealed. Alteration of PMCA1 and PMCA2 at the mRNA and protein levels has been revealed in seizure- induced delayed neuronal death by kainic acid of hippocampal neurons. Moreover, calcium alone mediates changes in the expression of alternative spliced PMCA variants which suggests possible regulatory mechanism by which cell may coordinate its response to environmental clues(10). PMCA protein level could reflect its decrease translation due to decreased mRNA level (or its lower stability), could be due to decreased number of total cells or due to combination of both. Since yield of protein in control and ischemic brain equals (beta- tubulin level does not differ between control and ischemic tissue) and no data are available on the regulation of PMCA isoforms on gene level or controlled isoform proteolysis, we have no answer yet at which level the changes occur.
Table 1.
Semi/quantitation of PMCA isoforms distribution in gerbil brain regions
PMCA isoforms Cerebral Cortex Hippoacpmus Cerebellum
----------------------------------------------------------
PMCA 1 ++++ +++ ++
PMCA 2 + very low ++
PMCA 3 n.d. ++ +
Table 2.
Levels of ion transporting proteins in gerbil forebrain after IRI as
determined by quantitative Western blotting.
PMCA1 InsP3R SERCA2b CR alpha3 Na pump
Control 100 100 100 100 100
Ischem. 103 95 103 101 98
Reperf. 58 70 97 102 96
Discussion and Conclusion
Evidence has been presented by Western blot analysis for detection of PMCA three gene products in the cerebral cortex, hippocampus and cerebellum, however isoform variants showed a distinct distribution pattern. Global forebrain ischemia and prolonged reperfusion triggered significant decrease of PMCA immumo-signal, which could be ascribed to the loss of PMCA1 isoform, especially in the hippocampus. The IRI selectively down-regulates also the levels of InsP3 receptor I. No statistically significant changes have been detected in the levels of SERCA 2b as well as alpha 3 isoform of Na pump. Studies on distribution of the PMCA, SERCA and alpha3- Na pump on the brain from various animals including our study revealed distinct localization of splice variants to specific brain cells. Isoform expression and alternate splicing seems a viable tool for PMCA regulation in vivo.
Ischemia is known to alter proteo-synthetic and proteolytic machinery. We suppose that alteration of number of ion transport proteins during recirculation period can contribute to the changes which lead to derangement of ion homeostasis and may participate and/or follow the delayed death of hippocampal neurons.
References
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