Reciprocal paracrine pathways link atrial natriuretic peptide and somatostatin secretion in the antrum of the stomach

Regulatory Peptides 110 (2003) 101 – 106 www.elsevier.com/locate/regpep

Reciprocal paracrine pathways link atrial natriuretic peptide and somatostatin secretion in the antrum of the stomach W.R. Gower Jr. a, R.W. McCuen b, A. Arimura c, D.A. Coy d, J.R. Dietz a, C.S. Landon a, M.L. Schubert b,* b

a Department of Medicine, University of South Florida and James A. Haley Veterans Hospital, Tampa, FL, USA Department of Medicine, Medical College of Virginia/Virginia Commonwealth University and McGuire Veterans Affairs Medical Center, Richmond, VA, USA c Department of Medicine, Tulane University, Belle Chase, LA, USA d Department of Medicine, Tulane University Medical Center, New Orleans, LA, USA

Received 27 February 2002; received in revised form 26 August 2002; accepted 26 August 2002

Abstract Atrial natriuretic peptide (ANP) as well as its receptor, NPR-A, have been identified in gastric antral mucosa, suggesting that ANP may act in a paracrine fashion to regulate gastric secretion. In the present study, we have superfused antral mucosal segments obtained from rat stomach to examine the paracrine pathways linking ANP and somatostatin secretion in this region. ANP (0.1 pM to 0.1 AM) caused a concentration-dependent increase in somatostatin secretion (EC50, 0.3 nM). The somatostatin response to ANP was unaffected by the axonal blocker tetrodotoxin but abolished by addition of the selective NPR-A antagonist, anantin. Anantin alone inhibited somatostatin secretion by 18 F 3% ( P < 0.005), implying that endogenous ANP, acting via the NPR-A receptor, stimulates somatostatin secretion. Somatostatin (1 pM to 1 AM) caused a concentration-dependent decrease in ANP secretion (EC50, 0.7 nM) that was abolished by addition of the somatostatin subtype 2 receptor (sst2) antagonist, PRL2903. Neutralization of ambient somatostatin with somatostatin antibody (final dilution 1:200) increased basal ANP secretion by 70 F 8% ( P < 001), implying that endogenous somatostatin inhibits ANP secretion. We conclude that antral ANP and somatostatin secretion are linked by paracrine feedback pathways: endogenous ANP, acting via the NPR-A receptor, stimulates somatostatin secretion, and endogenous somatostatin, acting via the sst2 receptor, inhibits ANP secretion. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Reciprocal paracrine pathways; Atrial natriuretic peptide; Somatostatin; Antrum

1. Introduction The family of atrial natriuretic peptides contains at least six peptides [1]. Three of these peptides, i.e., atrial natriuretic peptide (ANP, also known as atrial natriuretic factor), brain natriuretic peptide, and C-type natriuretic peptide, originating from the C-terminus of their prohormones, have similar structure and biological activities. ANP, first identified in cardiac atrial myocytes, has subsequently been identified in a variety of extracardiac tissues including the stomach [2,5,6]. The biologic functions of ANP are mediated primarily via * Corresponding author. Division of Gastroenterology, McGuire Veterans Affairs Medical Center, code 111N, 1201 Broad Rock Boulevard, Richmond, VA 23249, USA. Tel.: +1-804-675-5021; fax: +1-804-6755816. E-mail address: [email protected] (M.L. Schubert).

the natriuretic peptide receptor subtype A (NPR-A, also known as guanylyl cyclase-A (GC-A) or NPR1) through the generation of cGMP [4,7,8]. ANP also binds to NPR-C (also known as NPR3), a transmembrane cell surface receptor lacking guanylyl cyclase activity, which was originally thought to act primarily as a natriuretic peptide clearance receptor [4,9,10]. In the stomach, ANP has been reported to relax smooth muscle cells [11] and either inhibit or stimulate acid secretion [12]. In anesthetized rats, low doses of ANP augmented vagally induced acid secretion whereas higher doses reduced vagally induced acid secretion as well as acid secretion induced by carbachol [12]. It should be noted that studies in conscious animals, in which agonists are administered systemically, cannot distinguish between central and peripheral actions of ANP and do not necessarily reflect the action of ANP released in the stomach.

0167-0115/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 0 11 5 ( 0 2 ) 0 0 2 0 6 - 9

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Using ribonuclease protection analysis and RT-PCR, we have detected the expression of mRNA for both ANP and its receptor, NPR-A, in antral mucosa of rat stomach [3,13]. In antrum, the ANP-expressing cells were identified as enterochromaffin (EC) cells by dual immunohistochemistry and colorimetric in situ hybridization [3]. Although ANP secreted from atrial myocytes into the circulation is thought to act systemically to elicit natriuresis and diuresis in the kidney [14], the fact that ANP and functional ANP receptors are coexpressed in antral mucosa suggests that ANP may function in a paracrine fashion. In the present study, we have used antral mucosal segments obtained from rat stomach, a preparation that retains intact paracrine pathways, to examine the effect of endogenous ANP on somatostatin secretion and the effect of endogenous somatostatin on ANP secretion. The results indicate that ANP and somatostatin secretion, in antral mucosa, are linked by a paracrine feedback pathway: ANP, acting via the NPR-A receptor, stimulates somatostatin secretion and somatostatin, in turn, acting via the sst2 receptor, inhibits ANP secretion.

2. Materials and methods 2.1. Materials ANP, the selective NPR-A receptor antagonist, anantin [15 –17], the specific NPR-C agonist, cANP4-23 [15,18,19], and somatostatin-14 were obtained from Bachem (King of Prussia, PA). The axonal blocker, tetrodotoxin, was obtained from Sigma (St. Louis, MO). Somatostatin antiserum S775 was raised by A. Arimura [20] and the selective somatostatin subtype 2 receptor (sst2) antagonist, PRL2903, was synthesized by D. Coy [21]. 2.2. Superfusion of antral segments The studies were performed on superfused mucosal segments of rat antrum as previously described [22]. Sprague – Dawley rats, weighing 250 –400 g, were deprived of food overnight but allowed free access to water containing 10% glucose. The animals were anesthetized with 20% urethane (0.25 ml/50 g body weight), injected intraperitoneally. The muscle layer was stripped off the antrum and a segment, about 1 cm2, was obtained. Each piece of tissue (average weight: 178 F 8 mg) was cut into six to eight segments, washed with saline, and placed on a porous grid separating the two halves of a minichamber (Swinnex 25, 1.4 ml volume, Millipore Bedford, MA). Krebs bicarbonate solution containing 0.2% bovine serum albumin, 4% dextran, and 4.5 mM glucose was perfused into the bottom of the chamber at the rate of 1 ml/min and the effluent collected via a catheter leading from a small aperture at the top of the chamber. The perfusate was gassed with 95% O2 and 5% CO2. Drugs were delivered at the rate of 0.1 ml/min via a

side arm close to the inlet. The entire preparation was contained within a chamber maintained at 37 jC. The protocols were approved by the Virginia Commonwealth University Institutional Animal Care and Use Committee and the McGuire Research and Development Committee. 2.3. Experimental design A 30-min equilibration period was followed by an 80min sampling period. The sampling period consisted of a 30-min control basal period, a 20-min period during which ANP was superfused at various concentrations (0.1 pM to 0.1 AM) either alone or in combination with the competitive NPR-A receptor antagonist, anantin, or the axonal blocker, tetrodotoxin, and a final 30-min control period. In other experiments, antral mucosal segments were superfused for 20 min with somatostatin (1 pM to 1 AM), somatostatin antibody (final dilution 1:200), anantin (0.1 AM), or the NPR-C agonist, cANP4-23 (10 pM to 0.1 AM). In some experiments, the sst2 antagonist, PRL2903 (100 nM), was superfused for 20 min together with somatostatin (10 nM). One-milliliter samples of the superfusate were obtained at 5-min intervals and stored in 0.5-ml aliquots at 20 jC for subsequent measurement of somatostatin and ANP by radioimmunoassay. 2.4. Radioimmunoassay Somatostatin concentration was measured in duplicate by radioimmunoassay as described in detail previously [23]. Somatostatin antibody 1001 (final dilution 1:66,000) was a gift from Dr. Tadataka Yamada and Dr. John DelValle, University of Michigan. [125I]-somatostatin was purchased from New England Nuclear (Boston, MA). The limit of detection was 5 pg/ml of sample and the IC50 was 47 F 9 pg/ml of sample (mean F SD, n = 7 assays). Interassay and intraassay coefficients of variability were 12% and 8%, respectively. ANP was extracted from pooled aliquots of effluent collected for 5 min and its concentration measured in duplicate by radioimmunoassay as described in detail previously [3]. ANP-containing superfusates were applied to Amprep-mini C18 columns equilibrated with 0.1 N acetic acid and eluted with a mixture of acetonitrile and 0.1 M trifluoroacetic acid (60:40). The eluate was dried under nitrogen, resuspended in a minimal volume of radioimmunoassay buffer, and then assayed for immunoreactive ANP. The limit of detection was 0.64 pg/tube and the IC50 was 12 F 2 pg/tube (mean F SD, n = 5 assays). Interassay and intraassay coefficients of variability were 9% and 7%, respectively. 2.5. Data analysis Somatostatin and ANP secretion were expressed as the mean increase or decrease, in picograms per minute or as

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percent change, from the preceding basal level during the 5 min immediately preceding the experimental period. Changes in secretion were tested for significance using Student’s t test for unpaired values. All values are given as means F SE of n experiments on different animals. Concentrations eliciting 50% of maximal response (EC50) were calculated using sigmoid plot logistics (Fig.P program, Biosoft, Cambridge, UK).

3. Results 3.1. Basal somatostatin and ANP secretion In rat antral mucosal segments, mean basal somatostatin and ANP secretion were reproducible between experiments and reverted to initial control levels at the end of the experimental period (somatostatin secretion: 33 F 5 pg/min (start) and 33 F 6 pg/min (end); ANP secretion: 13 F 1 pg/ min (start) and 11 F 2 pg/min (end)).

Fig. 2. Time course for the effect of atrial natriuretic peptide (ANP; 10 nM) alone (closed circles) and in combination with the axonal blocker tetrodotoxin (TTX, 5 AM, open squares) or the NPR-A antagonist anantin (100 nM, open circles) on basal somatostatin secretion in rat antral mucosal segments. Dotted line indicates level of basal secretion. Data are means F SE of six experiments each.

3.2. Effect of ANP on somatostatin secretion Superfusion of rat antral mucosal segments for 20 min with ANP, ranging from 0.1 pM to 0.1 AM, caused a concentration-dependent increase in somatostatin secretion (Fig. 1). The EC50 value for stimulation of somatostatin secretion was 3.2  10 10 and maximal stimulation of somatostatin secretion (60 F 9% above basal level, P < 0.005, n = 6), expressed as the integrated 20-min response, was obtained at a concentration of 10 nM. The responses were not significantly affected by the axonal blocker tetrodotoxin (5 AM, n = 6) (Fig. 2). The specific NPR-C receptor agonist, cANP4-23, ranging 0.1 pM to 0.1 AM, had no significant effect on somatostatin secretion (n = 4 – 6 each).

Fig. 1. Effect of ANP (0.1 pM to 0.1 AM) on basal somatostatin secretion in rat antral mucosal segments. Data are means F SE of six to eight experiments each. *Significant difference from basal levels at P < 0.01.

3.3. Effect of somatostatin on ANP secretion Superfusion of rat antral mucosal segments for 20 min with somatostatin, ranging from 1 pM to 1 AM, caused a concentration-dependent decrease in ANP secretion (Fig. 3). The EC50 value for inhibition of somatostatin secretion was 7  10 10 and maximal inhibition of somatostatin secretion (51 F 7% below basal level, P < 0.001, n = 6), expressed as the integrated 20-min response, was obtained at a concentration of 10 nM. The responses were abolished by the sst2 antagonist, PRL2903 (Fig. 4).

Fig. 3. Effect of somatostatin (1 pM to 1 AM) on basal ANP secretion in rat antral mucosal segments. Data are means F SE of six to eight experiments each. *Significant difference from basal levels at P < 0.01.

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Fig. 4. Time course for the effect of somatostatin alone (10 nM, closed circles) and in combination with the sst2 antagonist PRL2903 (100 nM, open circles) on basal atrial natriuretic peptide (ANP) secretion in rat antral mucosal segments. Dotted line indicates level of basal secretion. Data are means F SE of six experiments each.

Fig. 6. Time course for the effect of somatostatin antibody (1:200) on basal atrial natriuretic peptide (ANP) secretion in rat antral mucosal segments. Dotted line indicates level of basal secretion. Data are means F SE of six experiments.

3.4. Effect of NPR-A receptor antagonist on somatostatin secretion

3.5. Effect of somatostatin antibody on ANP secretion

The involvement of NPR-A in ANP-stimulated somatostatin secretion in the antrum was tested by using the selective NPR-A receptor antagonist, anantin [15 – 17]. Addition of anantin (0.1 AM) to the superfusate abolished ANP-stimulated somatostatin secretion (n = 6) (Fig. 2). Of particular note, superfusion of rat antral mucosal segments with anantin (0.1 AM) alone for 20 min caused a decrease in somatostatin secretion (integrated response: 18 F 3% below basal level; P < 0.005, n = 6) (Fig. 5), implying that endogenous ANP, acting via the NPR-A receptor, exerts a stimulatory influence on somatostatin secretion.

Fig. 5. Time course for the effect of the NPR-A antagonist anantin (0.1 AM) on basal somatostatin secretion in rat antral mucosal segments. Dotted line indicates level of basal secretion. Data are means F SE of six experiments.

To determine whether endogenous somatostatin regulates ANP secretion, experiments were performed under conditions in which somatostatin was neutralized. Superfusion of rat antral mucosal segments with somatostatin antibody alone (final dilution 1:200) for 20 min caused an increase in ANP secretion (integrated response: 70 F 8% above basal level, P < 0.001, n = 6) (Fig. 6), indicating that endogenous somatostatin exerts a tonic inhibitory influence on ANP secretion.

4. Discussion In the stomach, somatostatin-containing D cells are located in proximity to their target cells, i.e., parietal and enterochromaffin-like (ECL) cells in the fundus and gastrin cells in the antrum [24]. The functional correlate of this anatomic coupling is a tonic restraint exerted by somatostatin on the secretion of histamine, gastrin, and acid [25 –31]. The actions of somatostatin are mediated via interaction with a family of seven transmembrane domain receptors designated sst1 to sst5 [32]. Molecular and pharmacological techniques indicate that sst2 is the predominant receptor subtype on parietal, ECL, and gastrin cells [21,33 – 38]. Recently, atrial natriuretic peptide (ANP) was identified in enterochromaffin (EC) cells of the gastric antrum [3]. ANP exerts many of its actions through interaction with NPR-A, a particulate guanylyl cyclase-linked receptor [4]. In some tissues, the effects of ANP may be mediated via NPR-C, a transmembrane receptor lacking guanylyl cyclase activity [9,10]. The presence of ANP as well as its receptor, NPR-A, in antral mucosa [3,39] led us to

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postulate that ANP may function in a paracrine fashion to regulate gastric endocrine secretion, specifically somatostatin secretion. In the present study, we have demonstrated, for the first time, the existence of a feedback paracrine pathway coupling locally synthesized ANP and somatostatin in the antrum of the stomach. Endogenous ANP, acting via the NPR-A receptor, stimulates somatostatin secretion and endogenous somatostatin, in turn, acting via the sst2 receptor, inhibits ANP secretion. The evidence on which this conclusion is based can be summarized as follows. First, the NPR-A receptor antagonist, anantin, decreased somatostatin secretion, implying that endogenous ANP, acting via the NPR-A receptor, stimulates somatostatin secretion. In support of this notion, ANP caused a concentration-dependent stimulation of somatostatin secretion in rat antral mucosal segments. Second, somatostatin secretion induced by ANP in antral mucosal segments was abolished by addition of the NPR-A receptor antagonist, anantin. Although it has been suggested that ANP can have biological actions mediated also by signaling activity via NPR-C [9,10,40, 41], this was not evident in the present study. The specific NPR-C receptor agonist, cANP4-23, superfused in the same concentrations as ANP, had no significant effect on somatostatin secretion. Third, the somatostatin response to ANP was unaffected by addition of the axonal blocker, tetrodotoxin, suggesting a direct paracrine effect of ANP on somatostatin secretion. This concentration of tetrodotoxin (5 AM) was previously shown to block neurally mediated peptide secretion in this preparation [42,43]. Fourth, somatostatin antibody significantly increased ANP secretion, implying that endogenous somatostatin exerts a paracrine restraint on ANP secretion. In support of this notion, somatostatin caused a concentration-dependent inhibition of ANP secretion. The effect of somatostatin was abolished by addition of the sst2 antagonist, PRL2903, suggesting that ANP-containing EC cells possess this receptor subtype. A model depicting the reciprocal paracrine pathways linking ANP-containing EC cells and somatostatin-containing D cells of the antrum is illustrated in Fig. 7. The precise physiologic role of ANP in regulating gastric endocrine and exocrine secretion and the precise physiologic mechanisms responsible for releasing antral ANP are not known. Stimulation of somatostatin secretion, however, may represent one mechanism by which ANP inhibits gastrin [13] and acid secretion [12]. Furthermore, it has been reported that prolonged fasting decreases steady-state antral ANP mRNA levels in rats [3] and, in preliminary studies, we have shown that release of ANP from rat antrum can be regulated by activation of cholinergic and noncholinergic intramural neurons [44]. The effect of fasting and intramural neural activation on ANP synthesis and secretion lead us to postulate that ANP may play a role in the regulation of

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Fig. 7. Model illustrating the dual paracrine pathways linking atrial natriuretic peptide (ANP) and somatostatin (SST) secretion in the antrum of the stomach. ANP, released from enterochromaffin (EC) cells, interacts with NPR-A receptors on D cells to stimulate somatostatin secretion. Somatostatin, in turn, interacts with sst2 receptors on EC cells to inhibit ANP secretion.

protein- and distension-induced gastrin and acid secretion [42,45,46].

Acknowledgements This work was supported by grants from the Veterans Administration Medical Research Fund.

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