Quantitative PCR was used to analyse the expression of GluR1, GluR2, GluR2 flip, GluR2 flop and GluR3 mRNA in animals after ischemia and tolerance-inducing ischemia. The ischemic animals showed a decrease in the GluRs to approximately 30%, except for GluR2-flip, which decreased to 75%. The tolerance animals displayed regulation of GluR1 to 75%, GluR2 and GluR2-flop to 283% and 265% respectively. We did not find a correlation between GluR2 regulation and cell loss in the ischemic group. The selective upregulation of GluR2/GluR2 flop in tolerant animals indicates a possible mechanism for enhanced AMPA receptor desensitisation leading to tolerance to ischemia.

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The potentiating effects of cyanide on the inhibition of rat liver mitochondrial monoamine oxidase-A & B and of ox liver mitochondrial MAO-B by pheniprazine [(1-methyl-2-phenylethyl)hydrazine] has been studied. Pheniprazine was shown to behave as a mechanism-based MAO inhibitor. For rat liver MAO-B, the initial non-covalent step was characterized by dissociation constant (K (i)) of 2450 nM and the first-order rate constant (k (+2)) for the covalent adduct formation was 0.16 min(-1). As a reversible inhibitor it was selective towards rat liver MAO-A (K (i) = 420 nM) but the rate of irreversible inhibition of that enzyme was considerably slower (k (+2) = 0.06 min(-1)). MAO-B from ox liver more closely resembled MAO-A from the rat in sensitivity to reversible inhibition by pheniprazine (K (i) = 450 nm) but it was closer to rat liver MAO-B in rate of irreversible inhibition (k (+2) = 0.29 min(-1)). The K (i) values were significantly decreased in the presence of KCN but there was little effect on the k (+2) values. However, sensitivities of the different enzymes to KCN varied widely and considerably higher concentrations of KCN were required for this effect to be apparent with the rat liver mitochondrial MAO-A than with MAO-B from rat and ox liver. The kinetic behaviour of cyanide activation was consistent with partial (non-essential) competitive activation in all cases.

To analyse GluR2 regulations in the rat hippocampal CA1 region following global and tolerance-inducing ischemia in situ hybridization (ISH) and quantitative PCR (Q-PCR) was applied. In addition, cDNA was synthesised from two different primer combinations in order to elucidate possible differences in the adenylation state of GluR2 mRNA. Following global ischemia, ISH and Q-PCR both showed reductions to half of control levels of GluR2 mRNA in consent with previously published results. Following tolerance induction, ISH showed no changes, whereas PCR analysis showed up-regulation to 228% of control value for the general cDNA synthesis, and no change for the specific cDNA synthesis. This indicates that tolerance-inducing ischemia does not increase the amount of GluR2 mRNA; instead polyadenylation of the existing GluR2 mRNA pool takes place.

Tolerance against ischemia can be induced in the CA1 region of the hippocampus of the brain. In gerbils tolerance evolvement is blocked by the NMDA-antagonist MK-801. To examine this mechanism in rats, MK-801 was administered i.p. 1 h prior to tolerance inducing ischemia. Body temperature and activity were monitored before and after ischemia, and show that MK-801 results in hyperthermia immediately after the injection, the post-ischemic body temperature remain elevated until 5 h post-ischemia in spite of the animals being less active than control animals. Histology shows that pre-treatment with MK-801 does not affect the CA1 neuronal density, and we thus conclude that for the used rat model, MK-801 does not affect development of ischemic tolerance.