Rationale: 

The peptide angiotensin IV (Ang IV) has been shown to be antiepileptogenic in a mouse model of pentylenetetrazol (PTZ) kindling. It was 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 anticonvulsive substrate 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).

Methods: 

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. Via ex vivo enzymatic studies using the synthetic substrate L-leucine-p-nitroanilide, the aminopeptidase activity in cortical homogenates of IRAP WT and KO mice was measured. The degradation profile of labelled sst-14 in these homogenates was determined at different time intervals (0, 30, 60, 120 and 180 minutes) by using radio immuno assays (RIA).

Results: 

Compared to male WT mice, male KO mice showed significantly increased PTZ thresholds for myoclonic twitch, clonus without loss of reflexes and clonus with loss of reflexes. Female KO mice did not show any differences in PTZ thresholds compared to female WT littermates. However, the PTZ doses needed to induce the different behavioural manifestations were similar in male KO en female WT and KO mice. Enzymatic studies in brain cortex membranes of WT mice, using the synthetic IRAP inhibitor AL-11 and the synthetic AP-N inhibitor 7B, showed that 70% of the aminopeptidase activity was represented by IRAP. The remaining 30% was aminopeptidase-N (AP-N), non-IRAP and non-AP-N. In agreement, AP-N, non-IRAP and non-AP-N were the only active aminopeptidases in KO mice. Subsequently, these brain cortex membranes were incubated with exogenously added sst-14. RIA data indicate a time-dependent degradation of sst-14 which was similar in male WT and KO mice. Female KO mice, however, showed 25% less degradation of sst-14 in comparison with female WT.

Conclusions: 

This study shows that IRAP is involved in seizure generation, since male IRAP KO mice are less sensitive to the development of generalized seizures following PTZ administration. Unfortunately, the mechanism by which IRAP inhibition leads to anticonvulsive effects remains elusive as our comparable study between males and females showed that it is not due to the accumulation of sst-14.