Chapter 6 Importance of the Local Renin-Angiotensin System in Pancreatic Disease Po Sing Leung Department of Physiology, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong 1. INTRODUCTION The pancreas is structurally made up of two organs in one: the exocrine gland, consisting of acinar cells and duct cells that produce digestive enzymes and sodium bicarbonate, respectively; the endocrine gland, consisting of four islet cells, namely α-, β-, δ- and PP- cells that produce glucagon, insulin, somatostatin and pancreatic polypeptide, respectively. The exocrine pancreas’ major function is to secrete digestive enzymes, including amylase, lipase and proteases that are responsible for the normal digestion of our daily foodstuff; while sodium bicarbonate is critical for the neutralization of gastric chyme entering the duodenum. The endocrine pancreas’ major function is to secrete the four islet hormones that maintain glucose homeostasis in our body. The exocrine and endocrine functions are finely regulated by neurocrine, endocrine, paracrine and/or intracrine mechanisms (Solomon 1994; Cluck et al 2005; Toskes 1998). Dysregulation of these pathways thus leads to such pancreatic diseases as pancreatitis, cystic fibrosis, pancreatic cancer and diabetes mellitus. The local mechanisms that regulate pancreatic exocrine and endocrine physiology and pathophysiology remain poorly understood. However, a recently-identified local pancreatic renin-angiotensin system (RAS) is of considerable interest due to its involvement in major pancreatic functions. Components of this pancreatic RAS are subject to upregulation by various 131 U. Lendeckel and Nigel M. Hooper (eds.), Proteases in Gastrointestinal Tissue, 131-152. © 2006 Springer. Printed in the Netherlands 132 PO SING LEUNG Chapter 6 physiological and pathological conditions such as hypoxia, pancreatitis, type 2 diabetes mellitus (T2DM), and islet transplantation (Leung and Carlsson 2001; Leung and Chappell 2003). Emerging data from our laboratory and others indicate that activation of the pancreatic RAS could influence cell inflammatory responses, driving apoptosis, fibrosis, and generation of reactive oxygen species observed in pancreatitis, islet transplantation and T2DM (Leung 2005; Leung and Carlsson 2005). The elucidation of the regulatory pathways of pancreatic RAS activation and the consequent oxidative stress-induced pancreatic cell dysfunction has the potential to significantly improve our understanding of pancreatic physiology and pathophysiology. Ultimately, understanding the local pancreatic RAS should lead to new insights into the development of novel therapeutic strategies in the prevention and treatment of patients with pancreatitis, pancreatic cancer, islet transplantation and T2DM. 2. THE RENIN-ANGIOTENSIN SYSTEM 2.1 Circulating RAS The circulating RAS is an endocrine system best known for its regulation of blood pressure and fluid homeostasis (Peach 1977; Reid et al 1978). These regulatory functions are mediated largely by potent actions on the vascular smooth muscle and on renal reabsorption of electrolyte and water via direct tubule actions and via the stimulation of aldosterone and vasopressin (Lumber 1999; Matsusaka and Ichikawa 1997). This classic RAS consists of several components: the liver-derived precursor angio- tensinogen, two critical enzymes for the system, namely kidney renin and membrane-bound pulmonary angiotensin-converting enzyme (ACE). The sequential actions of these two enzyme generate plasma angiotensin I (1-10) and angiotensin II (1-8), respectively, the latter being the physiologically active element of the RAS. In addition, alternate enzymes to renin and ACE produce a number of bioactive peptides including angiotensin III (2-8), angiotensin IV (3-8) and angiotensin (1-7). Angiotensin II and these bioactive peptides mediate their specific functions via respective cellular transmembrane receptors of target tissues and organs (Leung 2004). Figure 1 summarizes the biosynthetic cascade for the RAS using renin and ACE and other alternate enzymes, which are linked by the bioactive peptide products along with their respective receptors. 6. Importance of the Local RAS in Pancreatic Disease 133 Angiotensinogen Renin Angiotensin I Kallikrein ACE AT1 & AT2 receptor Angiotensin II Aminopeptidase A AT1/AT2 receptor Angiotensin III AT3 receptor Aminopeptidase B/N Angiotensin IV AT4 receptor Propylendopeptidase Angiotensin (1-7) ACE-2 AT7 receptor Figure 1: An outline of the RAS depicting its biologically active peptides generated by various angiotensin-processing peptidases, along with their respective receptors. 2.2 Renin and angiotensin-converting enzyme Renin (EC 3.4.23.15) is an aspartyl protease, one of the key enzymes of the RAS. It is synthesized as a zymogen prorenin and subsequently activated by proteolytic cleavage. The gene coding for renal renin has 10 exons in human and 9 in rodents. A high degree of sequence homology is found among these renin isoforms (Hardman et al 1984; Hobart et al 1984). Active renin cleaves its substrate angiotensinogen to angiotensin I; however, the inactive renin, i.e. preprorenin and prorenin are the precursors of active renin and they are found in circulating blood plasma, amniotic fluid and kidney (Lumbers 1971; Day and Luetscher 1975; Nielsen and Poulsen 1988). The afferent arteriolar juxtaglomerular cells of kidney act as the site of renin production for the RAS (Hackenthal 1990). The preprorenin synthesized is rapidly hydrolyzed by signal protease to give prorenin. The prorenin is then converted to active renin and is secreted via a regulated pathway (Pratt et al 1983). The renin gene is expressed in many tissues besides the kidneys, including the vascular endothelium and islet beta cells of the pancreas (Leung et al 1999; Tahmasebi et al 1999) and may show species selectivity, as evidenced by its expression in the submandibular glands of the mouse but not the rat (Morris et al 1980). 134 PO SING LEUNG Chapter 6 ACE (EC 3.4.15.1) is a membrane-bound zinc ectoenzyme that functions as dipeptidyl carboxypeptidase (also called peptidyl-dipeptidase A, kininase II, peptidase P, and carboxycathepsin). Its major function is to process angiotensin I to angiotensin II and degrade bradykinin by removal of a dipeptide from the C-terminus. Other bioactive peptides such as metenkephalin, substance P, tachykinins, and prohormone convertase are also substrates for ACE (Coates 2003). Two isoforms of ACE are expressed in mammals: a germinal isoform (gACE) required for male fertility, and a somatic isoform (sACE) which plays a critical for the RAS (Corvol et al 1995). Until now, the clinical application of ACE inhibitors (e.g. captopril and ramipril) has been for the treatment of hypertension, diabetic nephropathy and heart failure (Dell’Italia et at 2002). In the pancreas, ACE has been identified in islet cells and in the vascular endothelium of pancreatic islets (Reddy et al 1995; Carlsson et al 1998). ACE activity and ACE mRNA have also been detected in the rat pancreas (Ip et al 2003a). 2.3 Other angiotensin-processing peptidases Apart from renin and ACE, a raft of angiotensin-processing peptidases is involved in the generation and metabolism of active angiotensin peptides. These enzymes include, to name but a few, the chymase, cathepsin G, chymotrypsin, trypsin, tonin, kallikrein, ACE-2 and other exopeptidases as well as endopeptidases. The existence of these enzymes has expanded the classic view of RAS to a more contemporary model of “angiotensin- generating systems” that recognizes the contribution of alternate pathways (Sernia 2001). These peptidases act directly on angiotensin I and/or angiotensin II as well as the precursor angiotensinogen to generate a number of bioactive peptides with varying physiological activities, such as angiotensin (1-7), angiotensin III and angiotensin IV (Campbell 2003). Of particular interest in this context is the discovery of a novel peptidase termed ACE-2, which is the first human homologue of ACE. Like ACE, ACE-2 acts as a carboxypeptidase; however, ACE-2 hydrolyzes a single residue either from angiotensin II (Pro7-Phe8) or angiotensin I (His9-Leu10) to generate angiotensin (1-7) and angiotensin (1-9), respectively (Rice et al 2004). ACE- 2 also cleaves other peptides, such as dynorphin, apelin and bradykinin. A physiological role for ACE-2 has been implicated in hypertension, heart function and diabetes and, perhaps more importantly, as a receptor of the severe acute respiratory syndrome coronarvirus (Warner et al 2004). Figure 2 depicts the peptide linkages that are cleaved by the angiotensin-processing peptidases. In the pancreas, kallikrein has been isolated in the dog and rat (Hojima et al 1977). It is a peptidase capable of generating angiotensin II directly from its precursor angiotensinogen (Arakawa and Maruta 1980; 6. Importance of the Local RAS in Pancreatic Disease 135 Arakawa 1996). In addition, a number of serine proteases capable of forming angiotensin II from angiotensin I and/or angiotensinogen have been identified in the pancreas (Sasaguri et al 1999). Chymotrypsin Chymase Aminopeptidase A Tonin ACE ACE-2 ACE-2 Asp1 – Arg2 – Val3 – Tyr4 – Ile5 – His6 – Pro7 – Phe8 – His9 – Leu10 Carboxypeptidase Propylendopeptidase Trypsin Endopeptidase *Aminopeptidase B/N Figure 2 : Different angiotensin-processing peptidases including endopeptidase, aminopepti- dase and carboxypeptidase that cleave peptide linkages from the interior, aminoterminal and