The Renin-Angiotensin System and the Exocrine Pancreas

Mark C Chappell*, Debra I Diz, Patricia E Gallagher
Hypertension and Vascular Disease Center, Wake Forest University School of Medicine. Winston- Salem, North Carolina, USA
Corresponding Author
Mark C Chappell
Gray 67A, Hypertension and Vascular Disease Center
Wake Forest University School of Medicine
Medical Center Blvd
Winston-Salem, NC 27157-1032
Phone +1-336-716.9236
Fax +1-336-716.0269
E-mail [email protected]
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An accumulating body of evidence strongly indicates a local tissue renin-angiotensin system in the pancreas of a various species. In contrast to the majority of tissues that primarily express the angiotensin type 1 (AT1) receptor, the pancreas is one of the few tissues that contain a significant proportion of the AT2 subtype. Moreover, our findings indicate a greater distribution angiotensin II binding sites in the exocrine pancreas. Although the physiological aspects of a local pancreatic renin-angiotensin system remain largely unexplored, recent studies in our laboratory utilizing an acinar cell model demonstrate both functional AT1 and AT2 receptors. Indeed, we show that the AR42J cell line expresses all components of an angiotensin system including the mRNA for renin, angiotensinogen, angiotensin converting enzyme (ACE), AT1a, AT1b and AT2 receptors. Thus, these cells may be of particular value to study the interplay of the AT1 and AT2 receptors to regulate cell growth and potentially



Angiotensin II; Angiotensinogen; Glucocorticoids; Pancreas; Peptidyl- Dipeptidase A; Protein-Tyrosine-Phosphatase; Receptors, Angiotensin


ARB: AT1 receptor blocker; Sarthran: [Sar1,Thre8]-angiotensin II


In 1991, we published the first study demonstrating key components that comprise an intrinsic renin-angiotensin system (RAS) within the canine pancreas [1, 2]. These studies documented the expression of the bioactive peptides angiotensin II, angiotensin III and angiotensin-(1-7), both protein and mRNA levels of the precursor angiotensinogen, as well as the distribution of the AT2 and AT1 receptor subtypes. Subsequent studies by other investigators reported comparable findings in the rat, mouse and human pancreas [3, 4, 5]. Indeed, in one of the few reports to study the in vivo regulation of pancreatic angiotensin II receptors, Ghiani and Massini [6] demonstrated an increase in angiotensin II binding sites in the pancreas of normotensive rats maintained on a high-salt diet. Although angiotensin II receptors were distributed throughout the pancreas, the highest density of sites, at least in the dog and monkey (see below), comprised the AT2 receptor subtype and localized to acinar cells and the ductal epithelium [2, 7]. Indeed, the pancreas is one of the few tissues that primarily express the AT2 receptor subtype. At the time of our initial report, the AT2 receptor had not been cloned and no functional data had been attributed to this receptor subtype. Only in the last several years has a more complete understanding of the functional role of the AT2 receptor emerged. In this brief review, we assess the recent data on the AT2 receptor and the potential influence on the functional aspects of the exocrine pancreas. We also present several novel aspects on the regulation and function of the AT2 receptor utilizing the AR42J acinar cell line.

Pancreatic Angiotensin II Receptor Characterization

As shown in Figure 1 (top left panel), in vitro receptor autoradiography of angiotensin II receptors in the primate pancreas using the nonselective angiotensin ligand 125I-[Sar1,Thre8]- angiotensin II (Sarthran) revealed the distribution of sites throughout the tissue, but with the highest density on acinar cells [7, 8]. The majority of Sarthran binding (>80%) was attenuated by the AT2 selective antagonist PD123319 (Figure 1, bottom left panel). High resolution emulsion autoradiography of this tissue revealed a very high expression of Sarthran binding surrounding the islet cells and a lower density of sites within the islet field (Figure 1, top right panel); addition of the PD123319 compound essentially abolished binding (bottom right panel). These findings in the monkey and those in the dog, demonstrating the predominant expression of the AT2 subtype in exocrine components of the pancreas, prompted further investigation of angiotensin II receptors and other components of the RAS in an acinar cell model. We characterized angiotensin II receptor binding in the AR42J acinar cell line and reported a high density of binding sites (>300 fmol/mg protein) [9]. Similar to monkey and dog tissues, the majority of these receptors were the AT2 subtype with a minority of sites (<15%) competed for by the AT1 antagonist losartan. Although the proportion of AT1 receptors expressed in the AR42J cell line was small, application of angiotensin II to cells loaded with the fluorescent calcium dye Fura-2 resulted in an immediate and significant increase in intracellular calcium. The angiotensin IIdependent rise in calcium was abolished by the AT1 antagonist, but was not modified by AT2 antagonists [9]. Consistent with our data, subsequent studies by others also reported AT1- dependent changes in intracellular calcium by angiotensin II [10]. The biochemical characterization of the AT2 binding sites utilized cross-linking of radiolabeled Sarthran and SDS/PAGE fractionation. These studies revealed an AT2 site with a molecular mass of approximately 110 kilodaltons (kDa) that was substantially greater than the predicted mass of 40 kDa based on the protein sequence of the AT2 receptor. Analysis of the AT2 sequence indicated a high number of glycosylation sites which likely influences the larger molecular mass observed in these cells, as well that reported in other tissues [11]. Similar to other reports, we could not demonstrate internalization of the AT2 receptor, another characteristic quite distinct from the rapid down-regulation of the AT1 receptor subtype following agonist binding [12].
Regarding the functional aspects of the AT2 receptor, several groups demonstrated a link to activation of tyrosine phosphatase activity [13, 14]. In the AR42J cells, activation of somatotostatin receptors increased tyrosine phosphatase activity and inhibited cell proliferation [15]. We find that in the presence of AT1 blockade, angiotensin II increased vanadate-inhibitable tyrosine phosphatase activity as measured with para-nitrophenol phosphate (Figure 2). In the presence of both AT2 and AT1 antagonists, angiotensin II did not change phosphatase activity. Interestingly, these data are consistent with a recent report by Elbaz et al. [16] who demonstrated an AT2- dependent reduction in the phosphorylation of the activated insulin receptor in intact AR42J cells. These authors found that both the AT2 antagonist PD123319 and a tyrosine phosphatase inhibitor abolished this response [16]. Moreover, others reported that AT2 activation may contribute to the dephosphorylation of the MAP kinases ERK1 and ERK2 [17, 18] . The activation of tyrosine phosphatase activity may underlie the antiproliferative actions generally associated with stimulation of the AT2 receptor [17, 19, 20]. Currently, we do not know whether angiotensin II exerts proliferative or anti-proliferative actions in these cells; the growth effects may likely depend on the relative balance of both receptor subtypes and the overall potency of their cellular signals.
In the AR42J cells, we are investigating the regulation of the AT2 receptor subtype. As shown in Figure 3, treatment with the glucocorticoid agonist dexamethasone resulted in a significant decline in PD123319-sensitive binding within six hours and a maximal decrease in binding by 24 hours [21]. The addition of cortisol also substantially reduced binding, but other steroid agents including estrogen, and aldosterone had little or no effect (Figure 3). Although not shown, saturation analysis of the dexamethasone-induced inhibition of the AT2 binding reflected a decrease in the number of receptor sites (Bmax) and no change in the relative affinity (KD) of the receptor to the Sarthran ligand. Consistent with the decrease in receptor number, the assessment of AT2 mRNA levels by RT-PCR revealed an almost complete inhibition of mRNA expression by dexamethasone in these cells (Figure 4). In contrast, estrogen treatment had no effect on angiotensin II binding or AT2 mRNA expression. Further studies are necessary to determine whether this reduction in AT2 mRNA results from an attenuation in transcriptional activity or decreased mRNA stability. However, our results are quite consistent with those of Kijima et al. [22] who reported that dexamethasone treatment reduced AT2 mRNA levels in the adrenomedullary PC12 cell line. In their study, dexamethasone treatment primarily reduced the message stability to attenuate AT2 mRNA half-life [22]. In view of the contrasting actions of AT1 and AT2 receptors, glucocorticoids are known to increase AT1 binding and AT1 mRNA, as well as ACE activity [23, 24]. Thus, glucocorticoidinduced hypertension may comprise a shift in the balance of effects between the AT1 and AT2 receptors in the presence of elevated levels of angiotensin II. Glucocorticoid down-regulation of the AT2 receptors may also be relevant to the recent findings that endogenous glucocorticoids suppress apoptosis in an induced- pancreatitis model [25]. Leung and colleagues [26, 27] demonstrated up-regulation of the pancreatic RAS including increased expression of AT2 mRNA in a chronic model of hypoxia, as well as augmented angiotensinogen in induced pancreatitis. In this regard, perhaps the activation of a pancreatic RAS, particularly the AT2 receptor, may promote cellular apoptosis and influence pancreatitis. Transient upregulation of the AT2 receptor has been reported in other tissues such as brain and kidney [12].
Finally, in view of our incomplete understanding of the generation of angiotensin II in the pancreas and other tissues such as the kidney, heart and brain, we have begun to investigate the expression of additional components of the pancreatic RAS in the AR42J cells. As shown in Figure 5, molecular analysis using RT-PCR revealed that the AR42J cells express mRNA for both AT1a and AT1b isotypes, as well as that for renin, angiotensinogen and ACE. Indeed, to our knowledge this is the first demonstration that this pancreatic cell line exhibits all components of the RAS. Although the expression of these components may result from the transformed phenotype, the AR42J cells constitute a unique cell model to explore the processing of angiotensin II and angiotensin I. Indeed, these cells may more closely model an autocrine system in which the local production of angiotensin II or other active metabolites acting through different receptor subtypes may feedback to influence its tissue of origin. This may be of particular relevance in hypertensive patients as AT1 receptor blockers (ARBs) may supplant ACE inhibitors and other antihypertensive treatments. ARB treatment not only blocks AT1 receptors, but significantly increases angiotensin II levels (due to the disinhibition of renin release) that may result in greater activation of the AT2 and other receptor subtypes. Furthermore, the acinar cell model may be of relevance to study more novel components of the RAS such as the AT4 receptor and the biologically active ligands, angiotensin-(3-8) and angiotensin-(3-7); these endogenous peptides exhibit high affinity for the AT4 binding site [12, 28]. Although a high density of AT4 sites are found in a number of tissues including the heart, adrenal gland, and the vascular endothelium, whether this site is expressed on the exocrine or endocrine elements of the pancreas is not known at this time. In addition, numerous studies demonstrate a functional role for angiotensin-(1-7) in the vasculature, brain and kidney that is mediated by a non-AT1,-AT2 receptor [29, 30]. Indeed, elevated levels of angiotensin-(1-7) contribute to the anti-hypertensive actions of ACE inhibitors and AT1 receptor antagonists [31,32]. Although we originally measured significant angiotensin-(1-7) levels in the dog pancreas, whether this peptide influences pancreatic function is also unknown.

Future Perspectives

The RAS cascade has historically been viewed as a key factor in the role of cardiovascular regulation primarily to maintain arterial blood pressure and water and sodium balance, with its regulation and expression predominantly controlled through the kidney. Although relatively few studies addressed the role of angiotensin II or other active fragments in the regulation of endocrine or exocrine aspects of the pancreas, substantial evidence indicates local systems in other tissues that may exhibit paracrine or autocrine actions. Indeed, the data from other tissues provide a new understanding that this important hormonal system is actually a pleiotropic system encompassing both vasopressor/depressor and proliferative/antiproliferative actions. Moreover, the regulation of individual components of the system is tissue specific and may be under the control of local factors. Thus, the mechanisms of the angiotensin system now include multiple receptors, different ligands and a diverse number of target tissues including the pancreas.

Figures at a glance

Figure 1 Figure 2 Figure 3 Figure 4 Figure 5
Figure 1 Figure 2 Figure 3 Figure 4 Figure 5


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