Enzyme activity of insulin-regulated aminopeptidase: role in pentylenetetrazol-induced seizures in mice

BACKGROUND: The peptide angiotensin IV (Ang IV) is known to facilitate acquisition in spatial memory tasks and to influence seizure susceptibility. Ang IV has been shown to be antiepileptogenic in a mouse model of pentylenetetrazol (PTZ) kindling.1, 2 It is suggested that one of the mechanisms by which Ang IV exerts these effects is by inhibiting insulin-regulated aminopeptidase (IRAP) and consequently prolonging the half-life of neuropeptides in the brain. Indeed, Stragier et al. found that the Ang IV-mediated inhibition of pilocarpine induced seizures in rats was prevented by a somatostatin-2 receptor antagonist, suggesting the involvement of somatostatin-14 (sst-14).3
AIMS: To unequivocally unravel the involvement of IRAP in seizure generation, IRAP knock-out (KO) mice and their wild-type (WT) littermates were subjected to an intravenous tail infusion of PTZ, which is an established acute model of generalised seizures. With ex vivo enzymatic and radio immunoassays, we also aimed to compare respectively the aminopeptidase activity and the degradation profile of labelled sst-14 in cortical homogenates of IRAP WT and KO mice.
RESULTS: Adult male and female WT and KO mice showed no differences in locomotor activity, as showed by an open field test. Basal EEG monitoring specified that WT and KO mice did not display spontaneous convulsions. Challenging these mice intravenously with PTZ resulted in an increased threshold in male KO mice in comparison with male WT mice. The PTZ thresholds were significantly higher for the myoclonic twitch, clonus without loss of reflexes and clonus with loss of reflexes. These behavioural data were confirmed by video-EEG monitoring. Female mice did not show any differences in PTZ thresholds. Enzymatic studies using the synthetic substrate L-leucine-p-nitroanilide in brain cortex membranes of WT mice showed that 70% of the aminopeptidase activity was represented by IRAP and 30% by aminopeptidase-N (AP-N). In agreement, AP-N was the only active aminopeptidase in KO mice. Subsequently, these brain cortex membranes were incubated for increasing time periods with exogenously added sst-14. Preliminary data indicate that there was a time-dependent degradation of sst-14 which was similar in membranes from WT as compared to KO mice.
CONCLUSIONS: This study shows that male IRAP KO mice are less sensitive to the development of generalized seizures following PTZ administration and that IRAP is involved in seizure generation. Unfortunately, the mechanism by which IRAP inhibition leads to anticonvulsive effects remains elusive since we showed it is not due to cumulation of sst-14.