Angiotensin II mediates catecholamine and neuropeptide Y secretion in human adrenal chromaffin cells through the AT 1 receptor Claudia Cavadas a , Daniela Grand a , Franc ßois Mosimann b , Maria Dulce Cotrim c , Carlos Alberto Fontes Ribeiro d , Hans R. Brunner a , Eric Grouzmann a, * a Division of Hypertension and Vascular Medicine, Centre Hospitalier Universitaire Vaudois, 1011 Lausanne, Switzerland b Department of Surgery, Centre Hospitalier Universitaire Vaudois, 1011 Lausanne, Switzerland c Laboratory of Pharmacology, Faculty of Pharmacy, University of Coimbra, 3000 Coimbra, Portugal d Department of Pharmacology, IBILI, Faculty of Medicine, University of Coimbra, 3000 Coimbra, Portugal Received 24 May 2002; received in revised form 30 September 2002; accepted 2 October 2002 Abstract The aim of the present work was to study the effect of angiotensin II (Ang II) on catecholamines and neuropeptide Y (NPY) release in primary cultures of human adrenal chromaffin cells. Ang II stimulates norepinephrine (NE), epinephrine (EP) and NPY release from perifused chromaffin cells by 3-, 2- and 12-fold, respectively. The NPY release is more sustained than that of catecholamines. We found that the receptor-AT 2 agonist, T 2 -(Ang II 4 – 8) 2 has no effect on NE, EP and NPY release from chromaffin cells. We further showed that Ang II increases intracellular Ca 2+ concentration ([Ca 2+ ] i ). The selective AT 1 -receptor antagonist Candesartan blocked [Ca 2+ ] i increase by Ang II, while T 2 -(Ang II 4–8) 2 was ineffective. These findings demonstrate that AT 1 stimulation induces catecholamine secretion from human adrenal chromaffin cells probably by raising cytosolic calcium. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Human adrenal chromaffin cells; Catecholamines; Neuropeptide Y; Angiotensin II; AT 1 receptor; AT 2 receptor 1. Introduction It is well established that angiotensin II (Ang II) is able to stimulate catecholamine release from bovine, rat and porcine adrenal chromaffin cells [1–7]. However, controversy exists about whether AT 1 or AT 2 receptors are involved in this effect in several species [6–12]. No study is available to clearly demonstrate, in vitro, the effect of Ang II on human adrenal medulla on catecholamine release since this subject has been debated almost 40 years ago [13]. Neuropeptide Y (NPY) is a 36-amino-acid peptide present in the adrenal medulla of many species, including humans [14–16] and is co-localized with catecholamines in the same granules [17]. Functionally, NPY potentiates the effect of various agonists such as Ang II and norepinephrine (NE) in addition to exerting a direct contractile effect on the vasculature [18]. We have previously found that NPY, like nicotine, increases catecholamine release from human chromaffin cells through the NPY y3 receptor [19]. The aim of this work was to study the effects of Ang II on NE, epinephrine (EP) and NPY secretions in human adrenal chromaffin cells in culture. 2. Materials and methods 2.1. Peptides and antagonists Angiotensin II was purchased from Novabiochem (Lau- felfingen, Switzerland). T 2 -(Ang II 4–8) 2 is a template- assembled peptide agonist for AT 2 receptors made of two angiotensin II 4–8 pentapeptide fragments (Ang II 4–8) 2 , attached to a carrier molecule (T 2 ) which alone did not bind to either AT 1 or AT 2 receptors. Binding assays showed that in the presence of an AT 1 antagonist, T 2 -(Ang II 4–8) 2 com- pletely inhibited the specific binding of 125 I-AII to the AT 2 receptors of a rat adrenal membrane preparation and that half-maximal inhibition (IC 50 ) occurred at the concentration of 2 Â 10 À 7 M. In contrast, T 2 -(Ang II 4 – 8) 2 at the concen- tration of 10 À 5 M did not bind to AT 1 receptors of rat aortic 0167-0115/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII:S0167-0115(02)00253-7 * Corresponding author. Present address: Centre Hospitalier Universi- taire Vaudois, Division of Clinical Pharmacology and Toxicology, 1011 Lausanne, Switzerland. Tel.: +41-21-314-0741; fax: +41-21-314-4266. E-mail address: eric.grouzmann@chuv.hospvd.ch (E. Grouzmann). www.elsevier.com/locate/regpep Regulatory Peptides 111 (2003) 61 – 65