Samenvatting
Introduction: The nonapeptide oxytocin (OT) is synthesized in hypothalamic nuclei and widely distributed over the central nervous system. OT can act as a hormone or a neuromodulator to regulate a variety of brain functions such as learning and memory [1]. Moreover, central OT is essential for social recognition [2], modulates stress and anxiety responses [3] and exerts antidepressant-like effects [4]. Lew et al. showed that OT is rapidly cleaved by insulin-regulated aminopeptidase (IRAP) in vitro [5]. In the present study, we aimed to investigate whether OT is also an ex vivo and in vivo substrate of IRAP and whether modulation of IRAP would interfere with the antidepressant-like effects of OT.
Methods: To unequivocally verify that OT is a substrate of IRAP ex vivo, OT radio-immunassay (RIA) and electro-spray ionisation mass spectrometry (ESI-MS) were used to study the degradation profile of OT in cortical membranes of IRAP wild-type (WT) and KO mice. In addition, cortical aminopeptidase activity was determined by incubating these membranes with the substrate l-leucine-p-nitroanilide. Rats were implanted with microdialysis probes in the hippocampus and in vivo OT levels were determined in dialysates before and after the administration of the IRAP inhibitor angiotensin IV (Ang IV). An open field (OF) and forced swim test (FST) was used to study locomotor behavior and to screen OT for antidepressant-like activity, respectively.
Results and discussion: In cortical membranes of IRAP WT and KO mice, no degradation of OT could be detected by the RIA. However, preliminary microdialysis experiments show that IRAP is responsible for the degradation of OT, since the perfusion of Ang IV seem to increase OT levels in rat hippocampal dialysates. In addition, preliminary ESI-MS results point to the presence of OT metabolites in WT mice.
Burbach et al. showed that the complete nonapeptide structure of OT is required for full endocrine activity, whereas OT metabolites have strong central activities which are different of the parent molecule [6]. We found an antidepressant-like effect of 0.15 mg/kg OT in middle-aged female WT mice, compared to saline treated controls. OF results excluded that these differences are due to locomotor problems. In addition, middle-aged female IRAP KO mice treated with saline or OT did not show significant differences in immobility. In line with the findings of Burbach, these data suggest that OT is a substrate of IRAP in vivo and that OT metabolites rather than the intact neuropeptide are responsible for antidepressant-like activity.
Older female WT and KO mice treated with saline, on the contrary, continued to struggle, trying to escape from the cylinder in the FST. Enzymatic studies showed that IRAP levels are declined in these mice, however, we suggest that the markedly reduced immobility in old mice is indicative of a problem in understanding the context due to aging rather than the presence of OT metabolites.
References
1. Burbach JPH, Bohus B, Kovacs GL, Van Nispen JW, Greven HM, De Wied D (1983) Oxytocin is a precursor of potent behaviourally active neuropeptides. European Journal of Pharmacology 94:125-131.
2. Ferguson JN, Aldag JM, Insel TR, Young LJ (2001) Oxytocin in the medial amygdala in essential for social recognition in the mouse. The Journal of Neuroscience 21:8278-8285.
3. Windle RJ, Shanks N, Lightman SL, Ingram CD (1997) Central oxytocin administration reduces stress-induced corticosterone release and anxiety behavior in rats. Endocrinology 138:2829-2834.
4. Nowakowska E, Kus K, Bobkiewicz-Kozkowska T, Hertmanowska H (2002) Role of neuropeptides in antidepressant and memory improving effects of venlafaxine. Polish Journal of Pharmacology 54:605-613.
5. Lew RA, Mustafa T, Ye S, McDowall SG, Chai YS, Albiston AL (2003) Angiotensins AT4 ligands are potent, competitive inhibitors of insulin regulated aminopeptidase (IRAP). Journal of Neurochemistry 86:344-350.
6. Burbach JPH, Lebouille JLM (1983) Proteolytic conversion of arginine-vasopressin and oxytocin by brain synaptic membranes. The Journal of Biological Chemistry 258:1487-1494.
Methods: To unequivocally verify that OT is a substrate of IRAP ex vivo, OT radio-immunassay (RIA) and electro-spray ionisation mass spectrometry (ESI-MS) were used to study the degradation profile of OT in cortical membranes of IRAP wild-type (WT) and KO mice. In addition, cortical aminopeptidase activity was determined by incubating these membranes with the substrate l-leucine-p-nitroanilide. Rats were implanted with microdialysis probes in the hippocampus and in vivo OT levels were determined in dialysates before and after the administration of the IRAP inhibitor angiotensin IV (Ang IV). An open field (OF) and forced swim test (FST) was used to study locomotor behavior and to screen OT for antidepressant-like activity, respectively.
Results and discussion: In cortical membranes of IRAP WT and KO mice, no degradation of OT could be detected by the RIA. However, preliminary microdialysis experiments show that IRAP is responsible for the degradation of OT, since the perfusion of Ang IV seem to increase OT levels in rat hippocampal dialysates. In addition, preliminary ESI-MS results point to the presence of OT metabolites in WT mice.
Burbach et al. showed that the complete nonapeptide structure of OT is required for full endocrine activity, whereas OT metabolites have strong central activities which are different of the parent molecule [6]. We found an antidepressant-like effect of 0.15 mg/kg OT in middle-aged female WT mice, compared to saline treated controls. OF results excluded that these differences are due to locomotor problems. In addition, middle-aged female IRAP KO mice treated with saline or OT did not show significant differences in immobility. In line with the findings of Burbach, these data suggest that OT is a substrate of IRAP in vivo and that OT metabolites rather than the intact neuropeptide are responsible for antidepressant-like activity.
Older female WT and KO mice treated with saline, on the contrary, continued to struggle, trying to escape from the cylinder in the FST. Enzymatic studies showed that IRAP levels are declined in these mice, however, we suggest that the markedly reduced immobility in old mice is indicative of a problem in understanding the context due to aging rather than the presence of OT metabolites.
References
1. Burbach JPH, Bohus B, Kovacs GL, Van Nispen JW, Greven HM, De Wied D (1983) Oxytocin is a precursor of potent behaviourally active neuropeptides. European Journal of Pharmacology 94:125-131.
2. Ferguson JN, Aldag JM, Insel TR, Young LJ (2001) Oxytocin in the medial amygdala in essential for social recognition in the mouse. The Journal of Neuroscience 21:8278-8285.
3. Windle RJ, Shanks N, Lightman SL, Ingram CD (1997) Central oxytocin administration reduces stress-induced corticosterone release and anxiety behavior in rats. Endocrinology 138:2829-2834.
4. Nowakowska E, Kus K, Bobkiewicz-Kozkowska T, Hertmanowska H (2002) Role of neuropeptides in antidepressant and memory improving effects of venlafaxine. Polish Journal of Pharmacology 54:605-613.
5. Lew RA, Mustafa T, Ye S, McDowall SG, Chai YS, Albiston AL (2003) Angiotensins AT4 ligands are potent, competitive inhibitors of insulin regulated aminopeptidase (IRAP). Journal of Neurochemistry 86:344-350.
6. Burbach JPH, Lebouille JLM (1983) Proteolytic conversion of arginine-vasopressin and oxytocin by brain synaptic membranes. The Journal of Biological Chemistry 258:1487-1494.
| Originele taal-2 | English |
|---|---|
| Titel | 13th International conference on in vivo methods |
| Status | Published - 2010 |