All animal procedures were conducted in agreement with the guiding principles for the care and use of animals approved by the Society for Neuroscience. Intracerebroventricular Injections in Anesthetized Rats All compounds injected ICV or i.v. of EC33 alone caused a dose-dependent decrease in BP by blocking the formation of brain but not systemic AngIII. This is corroborated by the fact that the selective APN inhibitor, PC18 (2-amino-4-methylsulfonyl butane thiol), administered alone via the ICV route, increases BP. This pressor response was blocked by prior treatment with the angiotensin type 1 (AT1) receptor antagonist, losartan, showing that blocking the action of APN on AngIII metabolism leads to an increase in endogenous AngIII levels, resulting in BP increase, through interaction with AT1 receptors. These data demonstrate that AngIII is a major effector peptide of the brain RAS, exerting tonic stimulatory control over BP. Thus, APA, the enzyme responsible for the formation of brain AngIII, represents a potential central therapeutic target that justifies the development of APA inhibitors as central antihypertensive agents. the N-terminal aspartate of AngII to generate AngIII. In contrast, aminopeptidase N (APN, EC 184.108.40.206), another zinc-metallopeptidase (18, 19) from the same family, the gluzincins (20), hydrolyzes the N-terminal arginine of AngIII to generate angiotensin IV (21, 22) (Fig. ?(Fig.1).1). In an attempt to define the respective roles of brain AngII and AngIII in the central control of cardiovascular functions, we recently have developed highly selective APA and APN inhibitors: the compound EC33 [(S)-3-amino-4-mercaptobutyl sulfonic acid] specifically inhibits APA whereas the compounds EC27 [(S)-2-amino-pentan-1,5-dithiol] and PC18 (2-amino-4-methylsulfonyl butane thiol) specifically inhibit APN (22C24). Using these new tools, we demonstrated previously that AngIII and not AngII, as shown at the periphery, is one of the main effector peptides of the brain RAS in the central control of vasopressin release and supraoptic vasopressinergic neuron activity (21, 22, 25). Open in a separate window Figure 1 Metabolic pathways of AngII and AngIII in the brain involving zinc-metallopeptidases. EC33, APA inhibitor; PC18, APN inhibitor. This prompted us to delineate the respective roles of AngII and AngIII in the central control of arterial blood pressure (BP) by blocking each of Rabbit Polyclonal to TCF7 their metabolic pathways with selective APA and APN inhibitors, respectively. If brain AngIII proves to be the active peptide of the brain RAS in the control of BP, this study will allow, in addition, to demonstrate the efficacy of APA inhibitors as central antihypertensive agents in an experimental hypertension model, the SHR rat. Materials and Methods Drugs. EC33 and PC18 were synthesized by the laboratory of B. P. Roques (Institut National de la Sant et de la Recherche Mdicale, Unit 266; and Centre National de la Recherche Scientifique, UMR 8600) as described previously (23, 24). Human AngII and human AngIII were purchased from Sigma. The AT1 receptor antagonist losartan was obtained from DuPont, and the AT2 receptor antagonist PD123319 was purchased from Research Biochemicals (Natick, MA). Animals. Experiments were performed on mature normotensive Wistar Kyoto (WKY) rats and SHR weighing 300C350 g. These animals were obtained from Iffa Credo (LArbresle, France) and kept under artificial light (12-h light/12-h dark cycle) with a normal standard diet (Usine alimentation Rationnelle; Epinay-sur-Orge, France) and water given ad libitum. All animal procedures were conducted in agreement with the guiding principles for the care and use of animals approved by the Xanomeline oxalate Society for Neuroscience. Intracerebroventricular Injections in Anesthetized Rats All compounds injected ICV or i.v. were dissolved in sterile 0.9% saline, and the pH was adjusted to 7.0 with 0.1 M NaOH. Mature male SHR and WKY rats were anesthetized with 100 mg/kg i.p. Inactin [5-ethyl-2-(1methylpropyl)-2-thiobarbiturate Research Biochemicals] and placed in a stereotaxic apparatus (Kopf Instruments, Tujunga, CA). A 26-gauge stainless steel guide cannula was implanted just above Xanomeline oxalate the roof of the right lateral ventricle (stereotaxic coordinates with respect to bregma: 1-mm caudal and 1.5-mm lateral; ref. 26) and was lowered 4 mm below the surface of the skull. The guide cannula was anchored to the skull by using acrylic dental cement. Peptides and inhibitors were injected by inserting a 33-gauge stainless steel internal cannula into the guide cannula so that it extended 1 mm beyond the tip of the guide into the lateral ventricle. Xanomeline oxalate This injector was connected to a 10-l.