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Amylin-induced suppression of ANP secretion through receptors for CGRP1 and salmon calcitonin
Regulatory Peptides 117 (2004) 159 – 166 www.elsevier.com/locate/regpep
Amylin-induced suppression of ANP secretion through receptors for CGRP1 and salmon calcitonin Feng Lian Piao, Chunhua Cao, Jeong Hee Han, Sung Zoo Kim, Kyung Woo Cho, Suhn Hee Kim * Department of Physiology, Institute for Medical Sciences, Chonbuk National University Medical School, 2 – 20 Keum-Am-Dong-San, Jeonju 561-180, South Korea Received 13 June 2003; received in revised form 25 September 2003; accepted 1 October 2003
Abstract Amylin cosecretes with insulin from pancreatic h-cells and shows high sequence homology with CGRP, adrenomedullin, and salmon calcitonin. This study aimed to investigate the effect of amylin on the atrial hemodynamics and ANP release from rat atria and to identify its receptor subtypes. Isolated perfused left atria from either control or streptozotocin-treated rats were paced at 1.3 Hz. The concentration of ANP was measured by radioimmunoassay and the translocation of ECF was measured by [3H]-inulin clearance. Rat amylin increased atrial contractility and suppressed the release of ANP. Rat CGRP showed similar effects but was approximately 300-fold more potent than amylin. Pretreatment with receptor antagonist for CGRP1 [rat a-CGRP (8-37)] or salmon calcitonin [acetyl-(Asn30, Tyr32)-calcitonin(8-32), (AC 187)] blocked the suppressive effect of ANP release and the positive inotropic effect by rat amylin. However, receptor antagonists for amylin [amylin (8-37), acetyl-amylin] did not block those effects. Amylin (8-37), acetyl-amylin, or rat a-CGRP (8-37) alone accentuated the release of ANP with no changes in atrial contractility. The effect of rat amylin and rat amylin (8-37) on the ANP release was attenuated in streptozotocin-treated rats. We suggest that amylin suppressed ANP release with increased atrial contractility through receptors for CGRP1 and salmon calcitonin and the attenuation of amylin and its antagonist on ANP release from streptozotocin-treated rat atria may be due to the downregulation of amylin receptor. D 2003 Elsevier B.V. All rights reserved. Keywords: Atrial contractility; Amylin; CGRP; Calcitonin; Diabetes; Natriuretic peptide; Receptors
1. Introduction Amylin or islet amyloid polypeptide [1,2] is a 37-amino acid that is colocalized with insulin in the central granule core of pancreatic h-cell secretory granules and cosecreted with insulin in response to hyperglycemia and arginine in a fixed ratio [3,4]. Amylin shares homology with a group of peptides including calcitonin gene-related peptide (CGRP, 45%), salmon calcitonin (33%), adrenomedullin, and rat calcitonin (18%) [5]. Amylin crossacts to the receptors for CGRP and salmon calcitonin [6,7], as well as amylin-preferring receptors [8]. A number of effects of amylin have been reported including counteraction of insulin on the regulation of carbohydrate metabolism, inhibition of osteoclastic activity,
* Corresponding author. Tel.: +82-63-274-9788; fax: +82-63-274-9892. E-mail address: [email protected] (S.H. Kim). 0167-0115/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2003.10.005
vasodilation, and anorectic effects [9]. Amylin is also involved in the development of hypertension through enhancement of renin release, stimulation of sodium/water reabsorption, and acute elevation of blood pressure [10,11]. Bell and McDermott [12] have demonstrated that amylin stimulates a positive contractile response in rat ventricular myocytes by interaction with CGRP1 receptor. CGRP also has positive inotropic effect with accentuation of atrial natriuretic peptide (ANP) secretion in rat atrial strips [13]. However, the receptor subtypes involved in the cardiac effects of CGRP are still controversial. In addition, little is known about the effect of amylin on ANP secretion and its receptor subtypes. Hyperinsulinemic states are associated with hyperamylinaemia [5,9]. Plasma concentration of amylin is elevated in non-insulin dependent diabetes mellitus (DM) [14] and essential hypertension [15]. However, a loss of amylin secretion is reported after treatment with streptozotocin because of destruction of pancreatic h-cells [16]. The aim
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of the present study was to investigate the effects of amylin on atrial contractility and ANP release using isolated rat atria and to identify its receptor subtypes. The possibility of modification of amylin effects was also studied in streptozotocin-induced DM rats. To assess the functional role of receptor for amylin or CGRP1, we used the selective amylin receptor antagonists [amylin (8-37) and acetyl-amylin] [8] or CGRP1 receptor antagonist [CGRP (8-37)] [17]. To study effects mediated by the receptor for salmon calcitonin, we used the selective salmon calcitonin receptor antagonist [acetyl-(Asn30, Tyr32)-calcitonin(8-32), AC 187] [18].
n = 6), rat a-CGRP (8-37) (1 AM, n = 6), or (AC187) (1 AM, n = 6) in HEPES buffer was introduced into the atrial lumen after a 10-min control collection period. Group 4 was atria perfused with amylin in the presence of receptor antagonist. Receptor antagonist for amylin [amylin (8-37) or acetyl-amylin], CGRP1 [rat a-CGRP (837)], or salmon calcitonin (AC187) (0.3 AM, all n = 6) in HEPES buffer was administered as a pretreatment at 40 min after start of the perfusion. Then, rat amylin (0.3 AM) was simultaneously infused after a 10-min control collection
2. Methods 2.1. Animals Male Sprague – Dawley rats weighing 300 to 350 g were used. In order to induce insulin-dependent DM, rats were received streptozotocin (100 mg/kg, ip) and sacrificed on the seventh day. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). 2.2. Preparation of perfused beating rat atria Isolated perfused beating atria were prepared by using a previously described method [19,20], with minor modifications. In brief, left atrium was dissected from the heart after sacrifice and fixed into a Tygon cannula. The cannulated atrium was transferred into an organ chamber, immediately perfused with oxygenated HEPES buffer solution at 36.5 jC, and paced at 1.3 Hz (duration, 0.3 ms; voltage, 40 V), as described previously [20]. The composition of the HEPES buffer solution was as follows: NaCl 118 mM, KCl 4.7 mM, CaCl2 2.5 mM, MgSO4 1.2 mM, NaHCO3 25 mM, HEPES 10 mM, glucose 10 mM, and bovine serum albumin 0.1%. The pericardial buffer solution contained [3H]-inulin to measure the translocation of extracellular fluid (ECF). Intra-atrial pressure was measured throughout the experiments. After stabilization for 100 min, the perfusate was collected at 2-min intervals at 4 jC. 2.3. Experimental protocols Experiments were performed with five groups. Group 1 was atria perfused with HEPES buffer as timecontrol (n = 6). Group 2 was atria perfused with amylin or CGRP. Rat amylin (Bachem Bubendorf, Switzerland, 0.1, 0.3, 1 AM, n = 6 –8) or rat CGRP (Bachem 1 nM, n = 5) was introduced into the atrial lumen after a 10-min control collection period, and perfusate was collected for 50 min. Group 3 was atria perfused with receptor antagonists. Rat amylin (8-37) (0.3, 1 AM, all n = 6), rat acetyl-amylin (1 AM,
Fig. 1. Effect of rat amylin on pulse pressure (A), ECF translocation (B), ANP secretion (C), and ANP concentration (D) in isolated perfused beating rat atria. After a 100-min control period, atrial perfusate was collected for 10 min at 2-min intervals as a control and then rat amylin (0.1 or 0.3 AM, n = 6 or 8) was perfused into atrial lumen. Values were expressed as ratio of last five experimental values exposed to amylin, as compared to mean of five control values. Amylin increased pulse pressure and decreased ANP secretion. The secretion of ANP in terms of ECF translocation (interstitial ANP concentration) was markedly decreased by amylin. ECF translo, ECF translocation; ANP conc, ANP concentration; CONT, control atria; , , amylin-infused atria. *, significantly different from atria perfused with 0.1 AM amylin, P < 0.05.
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period. All antagonists used in this study were purchased from Bachem. Group 5 was amylin or amylin (8-37)-perfused atria from diabetes mellitus rats. Rat amylin (0.3 AM, n = 7) or rat amylin (8-37) (1 AM, n = 9) was introduced into the atrial lumen after a 10-min control collection period. 2.4. RIA of ANP The concentration of immunoreactive ANP in the perfusate was measured by using specific RIA, as described previously [19,21]. RIA was performed in Tris-acetate buffer (0.1 M, pH 7.4) containing neomycin (0.2%), EDTA (1 mM), soybean trypsin inhibitor (50 benzoyl arginine ethyl ester units/ml), aprotinin (200 Kallikrein inhibiting unit/ml), phenylmethylsulfonyl fluoride (0.4 mg%), sodium azide (0.02%) and bovine serum albumin (1%). Standard and samples were incubated with anti-ANP antibody and [125I]-ANP for 24 h at 4 C. Bound forms were separated from free form using charcoal suspension or second antibody. RIA for ANP was done on the day of experiments and all samples in an experiment were analyzed in a single assay. The molar concentration of ANP release was calculated by the ANP secretion divided by ECF translocation and 3060 [molecular mass for ANP(1-28)] and was expressed in AM [22]. 2.5. Measurement of ECF translocation We previously reported a two-step sequential mechanism of ANP secretion from the atria [22]. First, atrial release of ANP into the interstitial space occurs by means of atrial
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stretching, and second, the released ANP is translocated into the atrial lumen, concomitantly with ECF translocation due to contraction. The radioactivity of [3H]-inulin in the atrial perfusate was measured by using a liquid scintillation counter (Tris-Carb 23-TR; A Packard Bioscience Downers Grove, IL). The amount of ECF translocated through the atrial wall was calculated by total radioactivity in perfusate divided by radioactivity in pericardial reservoir and atrial wet weight and was expressed in Al/min/g. 2.6. Statistical analysis The results are given as the mean F SEM. The statistical significance of the differences was assessed using ANOVA followed by Bonferroni test. Student’s t-test was also used. The critical level of significance was set at P < 0.05.
3. Results 3.1. Effects of amylin on atrial pulse pressure and ANP release After stabilization, perfusate was collected five times every 2 min to serve as a control period and then, amylin was infused at a concentration of 0.1, 0.3 or 1 AM. Fig. 1 shows the relative changes in pulse pressure, ECF translocation, ANP secretion, and ANP concentration by amylin (0.1, 0.3 AM) with time, as compared to control group. In time-control group, the basal pulse pressure, ECF translocation, and ANP secretion were 4.20 F 0.29 mm Hg, 56.81 F 17.96 Al/min/g, and 11.39 F 1.22 ng/min/g, respec-
Fig. 2. Percent changes in pulse pressure (A), ECF translocation (B), ANP secretion (C), and ANP concentration (D) by rat amylin (0.1, 0.3, 1 AM, n = 6 – 8) and rat a-calcitonin gene-related peptide (CGRP, 1 nM, n = 5). Values were expressed as percent changes of last five-experimental values exposed to amylin or CGRP, as compared to mean of five-control values. Amylin caused an increase in pulse pressure in a dose-dependent manner. The decrease in ANP secretion in terms of ECF translocation by amylin was not dose-dependent. CGRP showed the similar effect to amylin but the potency was approximately 300-fold greater than amylin. Legends are the same as in Fig. 1. #P < 0.05 vs. corresponding value.
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tively, which were not significantly different from basal values in atria perfused with amylin. Amylin at a dose of 0.1 and 0.3 AM caused increases in pulse pressure in a dosedependent manner (Fig. 1A). Amylin caused a decrease in ANP secretion (Fig. 1C) without changes in ECF translocation (Fig. 1B). The released ANP from atrial myocytes into the interstitial space is translocated into the atrial lumen, concomitantly with ECF translocation [19 – 22]. Therefore, the ANP secretion in terms of ECF translocation, which means the interstitial ANP concentration (Fig. 1D), was markedly suppressed. Fig. 2 shows the percent changes in pulse pressure, ECF translocation, and ANP secretion obtained from mean of five control values and last five experimental values exposed to amylin or CGRP. Amylin caused increases in pulse pressure in a dose-dependent manner (Fig. 2A). Decrease in ANP secretion by amylin was not dose-dependent (Fig. 2C). The ANP secretion in terms of ECF translocation was also decreased (Fig. 2D). Rat CGRP caused a decrease in ANP secretion with increase in pulse pressure. Rat CGRP was approximately 300-fold more potent than amylin. 3.2. Effects of receptor antagonists on atrial pulse pressure and ANP release Fig. 3 shows the relative changes in pulse pressure, ECF translocation, ANP secretion, and ANP concentration by amylin antagonist [amylin (8-37), 0.3 and 1 AM] with time, as compared to control group. No significant change in pulse pressure was found by amylin (8-37) (Fig. 3A). Amylin (8-37) at a dose of 0.3 AM did not cause any significant changes in ECF translocation and ANP secretion (Fig. 3B and C). Amylin (8-37) at a dose of 1 AM caused an increase in ANP secretion without changes in ECF translocation. Therefore, the ANP secretion in terms of ECF translocation (interstitial ANP concentration) was markedly accentuated by 1 AM amylin (8-37) (Fig. 3D). Fig. 4 shows the relative percent changes in pulse pressure, ECF translocation, and ANP secretion by receptor antagonists for amylin, CGRP and salmon calcitonin. No significant changes in pulse pressure and ECF translocation were found by all antagonists (1 AM) used in this study (Fig. 4A). Receptor antagonists for amylin, amylin (8-37) and acetyl-amylin, caused an accentuation of ANP secretion in terms of ECF translocation (Figs. 4C and D). Receptor antagonist for CGRP1, rat a-CGRP (8-37), also caused an accentuation of ANP secretion but receptor antagonist for salmon calcitonin, AC187, did not. 3.3. Modification of amylin effects on pulse pressure and ANP release with receptor antagonists In order to modify amylin-induced suppression of ANP release and positive inotropic effect, receptor antagonist (0.3 AM) was added as a pretreatment at 40 min after start of the perfusion. Fig. 5 shows the percent changes in pulse
Fig. 3. Effect of amylin receptor antagonist, rat amylin (8-37), on pulse pressure (A), ECF translocation (B), ANP secretion (C), and ANP concentration (D) in isolated perfused beating rat atria. After a 100-min control period, atrial perfusate was collected for 10 min at 2-min intervals as a control and then amylin (8-37) (0.3 or 1 AM, all n = 6) was perfused into atrial lumen. Values were expressed as ratio of last five experimental values exposed to amylin (8-37), as compared to mean of five-control values. Rat amylin (8-37) did not show any significant changes in pulse pressure. High dose of amylin (8-37) gradually increased ANP secretion in terms of ECF translocation. CONT, control atria; , , rat amylin (8-37)infused atria. *, **, significantly different from atria perfused with 0.3 AM amylin, P < 0.05, P < 0.01, respectively. Other legends are the same as in Fig. 1.
pressure, ECF translocation, and ANP secretion by amylin in the presence of receptor antagonist. The pretreatment of rat a-CGRP (8-37) completely blocked positive inotropic effect of amylin and also attenuated the suppression of ANP release (Fig. 5A and C). AC 187 also attenuated positive inotropic effect of amylin and completely blocked the suppression of ANP release. However, amylin (8-37) or acetyl-amylin did not modify the effects of amylin on the pulse pressure, ECF translocation, and ANP secretion (Fig. 5).
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Fig. 4. Percent changes in pulse pressure (A), ECF translocation (B), ANP secretion (C), and ANP concentration (D) by various receptor antagonists. Receptor antagonists for amylin [rat amylin (8-37), n = 6; acetyl-amylin, n = 6] and CGRP1 [rat a-CGRP (8-37), n = 6] caused an increase in ANP secretion with no significant changes in pulse pressure and ECF translocation. However, antagonist for salmon calcitonin, AC187 (n = 6) did not cause any significant changes. CONT, time control; AC-amylin, acetyl-amylin; CGRP (8-37), rat a-calcitonin gene-related peptide (8-37); AC187, acetyl-(Asn30, Tyr32)-calcitonin (8-32). *P < 0.05, **P < 0.01 vs. the time control group. Other legends are the same as in Fig. 1.
Fig. 5. Modification of amylin-induced suppression of ANP secretion and positive inotropism by the pretreatment of various antagonists. Amylin (8-37) and acetyl-amylin did not show any significant changes in amylin effects. However, rat a-CGRP (8-37) and AC187 blocked the suppression of ANP release and the increased pulse pressure by rat amylin. CONT, rat amylin-perfused atria; Amylin (8-37), rat amylin (8-37)-pretreated atria; AC-amylin, acetyl-amylinpretreated atria; CGRP (8-37), rat a-calcitonin gene-related peptide (8-37)-pretreated atria; AC187, acetyl-(Asn30, Tyr32)-calcitonin (8-32)-pretreated atria. **P < 0.01 vs. the time control atria. Other legends are the same as in Fig. 1.
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Fig. 6. Relative percent changes in pulse pressure (A), ECF translocation (B), ANP secretion (C), and ANP concentration (D) by rat amylin (0.3 AM, n = 7) and rat amylin (8-37) (1 AM, n = 9) in streptozotocin-treated diabetes mellitus rats. Positive inotropic effect by amylin did not change in diabetes mellitus rats. Changes in ECF translocation and ANP secretion by amylin and amylin (8-37) were significantly attenuated in diabetes mellitus rats. CONT, control rat; DM, diabetes mellitus rats. *P < 0.05, **P < 0.01 vs. control rat. Other legends are the same as in Fig. 1.
3.4. Modification of amylin and amylin (8-37) effects in DM rats The fasting blood sugar level in DM rats was 466.0 F 12.3 mg/100 ml (n = 9, P < 0.001), which was higher than control rats (126.0 F 3.5 mg/100 ml, n = 12). The basal levels of ECF translocation and ANP secretion were not different from control rats. Fig. 6 shows the percent changes in pulse pressure, ECF translocation, ANP secretion, and ANP concentration by rat amylin or rat amylin (8-37) in streptozotocin-treated DM rats. The increased pulse pressure by rat amylin in DM rats was not different from control rats (Fig. 6A). A decrease in ANP release by rat amylin and an increase in ANP release by rat amylin (8-37) were attenuated in DM rats, as compared to control rats (Fig. 6C).
4. Discussion We have shown that rat amylin caused a suppression of ANP release with increased atrial contractility, whereas its receptor antagonists caused an accentuation of ANP release in isolated perfused beating rat atria. The potency of amylin was approximately 300-fold less than CGRP. The antagonistic actions of rat a-CGRP (8-37) and AC187 on amylin effects suggest that both receptors for CGRP1 and salmon calcitonin mediate positive inotropic response and suppression of ANP release by amylin in rat atria. Additionally, the effects of amylin and amylin (8-37) on ANP release were attenuated in streptozotocin-treated rats. The structural similarity of amylin with CGRP, adrenomedullin and salmon calcitonin suggest that they may act via common receptors [5]. Two classes of CGRP receptors have been suggested that one with high affinity mediates a
positive contractile response and a second one with low affinity mediates a relaxant response [26]. They showed biphasic contractile response to CGRP but monophasic response to amylin. The proportion of two classes of CGRP receptors in the atrium and different affinity to receptors in terms of concentrations may be one of the possible factors to influence the effects of amylin. Amylin binds with high affinity to receptors selective for CGRP and salmon calcitonin in a number of tissues [6,7]. A positive contractile response of amylin in rat ventricular myocytes [12] is reported by interaction with CGRP1 receptor. In porcine myocardium [23] and human myocardial trabeculae [24], functional CGRP1 and CGRP2 receptors may mediate a positive inotropic effect by a-CGRP and amylin also has a potential positive inotropic effect. Therefore, the receptor subtypes involved in the cardiac effects of CGRP and amylin are still controversial. In the present study, we demonstrated that amylin caused dose-dependent increases in atrial contractility. The direct positive inotropic effect of amylin was blocked by receptor antagonist for CGRP1 [CGRP (8-37)] and receptor antagonist for salmon calcitonin (AC 187) but not by receptor for amylin [amylin (8-37), acetyl-amylin]. These results are consistent with other report showing amylin-induced positive contractile response blocked by rat a-CGRP (8-37) but not by amylin (8-37) [12,23]. The positive inotropic effect by amylin showed approximately 300-fold less potent than rat a-CGRP. The interaction of amylin with weak affinity at CGRP1 receptors coupled to a positive inotropic response is in agreement with studies in which the peptide was infused and found to be approximately 100-fold less potent as a peripheral vasodilator than CGRP [25]. The lower potency of amylin may be due to low affinity to CGRP1 receptor or differences in receptor density in species and tissues. These results suggest that the positive inotropic effect of amylin in isolated rat
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atria may be mediated by receptors for both CGRP1 and salmom calcitonin. The secretion of ANP is closely related to atrial hemodynamics, such as increased contractility and heart rate [21]. Our results show that amylin as well as CGRP had a suppressive effect of ANP release with increased contractility and its antagonists except AC187 had an accentuated effect of ANP release without changes in contractility. Positive inotropism is one of important factors, which influences ANP release. Our data are not consistent with the report showing the stimulation of ANP secretion by CGRP in rat beating atrial strips [13]. They show biphasic ANP secretory response to CGRP with increased atrial tension mediated by cAMP. The discrepancy may be partly due to differences in atrial model used for the study. Ca2 + and cAMP act as negative regulators of ANP secretion in isolated perfused atria [27,28] but as positive regulators in atrial strips [13,29] even though the exact reason is not clear. The relationship between Ca2 + and ANP secretion is still debatable. To identify the receptor subtypes responsible for the suppression of ANP release by amylin, we used 0.3 Amol/ l of antagonists, which did not show their own antagonistic effects. Amylin-induced suppression of ANP release was blocked by rat a-CGRP1 and AC187 but not by amylin (837) and acetyl-amylin. Our findings are in a good agreement with other report demonstrating the antagonistic effects of rat a-CGRP (8-37) and AC187 on the inhibitory effect of amylin in rat soleus muscle [18]. It has been reported that despite of little sequence homology, rat amylin and salmon calcitonin compete with high affinity for a single class of binding sites in the region of the rat brain [30]. As shown in Fig. 5, our results show that the inhibitory effect of increased pulse pressure by rat a-CGRP (8-37) seems to be more potent than by AC187. On the other hand, the blocking effect of suppression of ANP release by AC187 seems to be more potent than by rat a-CGRP (8-37). No appreciable inhibitory effects by amylin (8-37) and acetylamylin were found on those effects. These results may be due to not only differences in binding affinities of amylin to multiple receptors but also differences in receptor densities and distributions. These observations indicate that the effect of amylin on the ANP release is mediated by specific receptors for CGRP1 and salmon calcitonin. We demonstrated antagonism of endogenous ligand versus exogenous amylin in Figs. 4 and 5. None of the antagonists added to beating atria modulated contractility, however, the amylin and CGRP antagonists accentuate ANP release. On the other hand, if amylin is used as agonist, both CGRP and calcitonin receptor antagonist inhibit amylinmediated inotropic effects as well as ANP release effects. These results suggest that other ligand rather than amylin may be an endogenous ligand released from beating rat atria. It is highly probable that adrenomedullin released by the heart may be responsible for the antagonistic effects demonstrated in Fig. 4 because of a similarity of adreno-
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medullin to amylin. It has been shown that endogenous adrenomedullin inhibits ANP expression in cultured neonatal cardiomyocytes via a CGRP1 receptor [31]. The difference may be partly due to experimental materials or parameters studied. It has previously been shown that amylin is co-secreted with insulin [3] and that hyperinsulinemic states are associated with hyperamylinaemia [5]. In various animal models of insulin resistance with obesity, glucose tolerance and hypertension, amylin concentrations are markedly elevated [14]. Ogawa et al. [16] showed a loss of amylin secretion after treatment with streptozotocin because of destruction of pancreatic h-cells. It is possible that the response of ANP secretion to amylin may be different depending on the secretory function of pancreatic h-cells. The present study shows an attenuation of responsiveness of ANP secretion to amylin and amylin (8-37) without differences in pulse pressure in streptozotocin-treated DM. It may be due to changes in receptor density, or sensitivity. More studies are needed to define the pathophysiological involvement of amylin in the development of hypertension with different types of DM.
Acknowledgements The authors thank Dr. Mie-Jae Lim for her valuable critics and comments. The authors also thank Kyong-Sook Kim for her secretarial support. This work was supported by Korea Science and Engineering Foundation (98-0403-1001-5) and the Korea Research Foundation (2000-015FP0023), Republic of Korea.
References [1] Cooper GJS, Willis AC, Clark A, Turner RC, Sim RB, Teid KBM. Purification and characterization of a peptide from amyloid-rich pancreases of type-2 diabetic patients. Proc Natl Acad Sci U S A 1987; 84:8628 – 32. [2] Westermark P, Wernstedt A, Wilander E, Hayden DW, O’Brien TD, Johnson KH. Amyloid fibrils in human insulinoma and islets of Langerhans of the diabetic cat are derived from neuropeptide-like protein also present in normal islet cell. Proc Natl Acad Sci U S A 1987;84: 3881 – 5. [3] Fehmann HC, Weber V, Goke R, Goke B, Arnold R. Cosecretion of amylin and insulin from isolated rat pancreas. FEBS Lett 1990;262: 279 – 81. [4] Inoue K, Hisatomi A, Umeda F, Nawata H. Release of amylin from perfused rat pancreas in response to glucose, arginine, beta-hydroxybutyrate, and gliclazide. Diabetes 1991;40:1005 – 9. [5] Cooper GJ. Amylin compared with calcitonin gene-related peptide: structure, biology, and relevance to metabolic disease. Endocrine Rev 1994;15:163 – 201. [6] Chantry A, Leighton B, Day AJ. Cross-reactivity of amylin with calcitonin-gene-related peptide binding sites in rat liver and skeletal muscle membranes. Biochem J 1991;277:139 – 43. [7] Rink TJ, Beaumont K, Koda J, Young A. Structure and biology of amylin. TIPS 1993;14:113 – 8. [8] Deems RO, Cardinaux F, Deacon RW, Young DA. Amylin or CGRP
166
[9] [10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
F.L. Piao et al. / Regulatory Peptides 117 (2004) 159–166 (8-37) fragments reverse amylin-induced inhibition of 14C-glycogen accumulation. Biochem Biophys Res Commun 1991;181:116 – 20. Edwards BJ, Morley JE. Amylin. Life Sci 1992;51:1899 – 912. Wookey PJ, Tikellis C, Du HC, Qin HF, Sexton PM, Cooper ME. Amylin binding in rat renal cortex, stimulation of adenylyl cyclase, and activation of plasma renin. Am J Physiol 1996;270:F289 – 94. Wookey PJ, Cooper ME. Amylin: physiological roles in the kidney and a hypothesis for its role in hypertension. Clin Exp Phamacol Physiol 1998;25:653 – 60. Bell D, McDermott BJ. Activity of amylin at CGRP1-preferring receptors coupled to positive contractile response in rat ventricular cardiomyocytes. Regul Pept 1995;60:125 – 33. Schiebinger RJ, Santora AC. Stimulation by calcitonin gene-related peptide of atrial natriuretic peptide secretion in vitro and its mechanism of action. Endocrinology 1989;124:2473 – 9. Gill AM, Yen TT. Effects of ciglitazone on endogenous plasma islet amyloid polypeptide and insulin sensitivity in obese-diabetic viable yellow mice. Life Sci 1991;48:703 – 10. Kautzky-Willer A, Thomaseth K, Pacini G, Clodi M, Ludvik B, Streli C, et al. Role of islet amyloid polypeptide secretion in insulin-resistant humans. Diabetologia 1994;37:188 – 94. Ogawa A, Harris V, McCorkle SK, Unger RH, Luskey KL. Amylin secretion from the rat pancreas and its selective loss after streptozotocin treatment. J Clin Invest 1990;85:973 – 6. Chiba T, Yamaguchi A, Yamatani T, Nakamura A, Morishita T, Inui T, et al. Calcitonin gene-related peptide receptor antagonist human CGRP-(8-37). Am J Physiol 1989;256:E331 – 5. Beaumont K, Pittner RA, Moore CX, Wolfe-Lopez D, Prickett KS, Young AA, et al. Regulation of muscle glycogen metabolism by CGRP and amylin: CGRP receptors not involved. Br J Pharmacol 1995;115:713 – 5. Cho KW, Seul KH, Kim SH, Seul KM, Ryu H, Koh GY. Reduction volume dependence of immunoreactive atrial natriuretic peptide secretion in isolated perfused rabbit atria. J Hypertens 1989;7: 371 – 5. Han JH, Cao C, Kim SZ, Cho KW, Kim SH. Decreases in ANP
[21]
[22]
[23]
[24]
[25]
[26] [27]
[28]
[29]
[30] [31]
secretion by lysophosphatidylcholine through PKC. Hypertension 2003;41:1380 – 5. Cho KW, Seul KH, Kim SH, Seul KM, Koh GY. Atrial pressure, distension, and pacing frequency in ANP secretion in isolated perfused rabbit atria. Am J Physiol 1991;260:R39 – 46. Cho KW, Seul KH, Kim SH, Koh GY, Seul KM, Hwang YH. Sequential mechanism of atrial natriuretic peptide secretion in isolated perfused rabbit atria. Biochem Biophys Res Commun 1990;172: 423 – 31. Ole Saetrum O, de Vries R, Tom B, Edvinsson L, Saxena PR. Positive inotropy of calcitonin gene-related peptide and amylin on porcine isolated myocardium. Eur J Pharmacol 1999;385:147 – 54. Ole Saetrum O, Kasbak P, de Vries R, Saxena PR, Edvinsson L. Positive inotropy mediated via CGRP receptors in human myocardial trabeculae. Eur J Pharmacol 2000;397:373 – 82. van Rossum D, Menard DP, Fournier A, St-Pierre S, Quirion R. Autoradiographic distribution and receptor binding profile of [125I]Bolton Hunter-rat amylin binding sites in the rat brain. J Pharmacol Exp Ther 1994;270:779 – 87. Kim D. CGRP activates the muscarinic-gated potassium current in atrial cells. Eur J Physiol 1991;418:338 – 45. Cho KW, Kim SH, Seul KH, Hwang YH, Kook YJ. Effect of extracellular calcium depletion on the two-step ANP secretion in perfused rabbit atria. Regul Pept 1994;52:129 – 37. Cui X, Wen JF, Jin JY, Xu WX, Kim SZ, Kim SH, et al. Protein kinase-dependent and Ca2 +-independent cAMP inhibition of ANP release in beating rabbit atria. Am J Physiol Regul Integr Comp Physiol 2002;282:R1477 – 89. Schiebinger RJ, Li Y, Cragoe Jr EJ. Calcium dependency of frequency-stimulated atrial natriuretic peptide secretion. Hypertension 1994;23:710 – 6. Beaumont K, Kenney MA, Young AA, Rink TJ. High affinity amylin binding sites in rat brain. Mol Pharmacol 1993;44:493 – 7. Autelitano DJ, Ridings R. Adrenomedullin signalling in cardiomyocytes is dependent upon CRLR and RAMP2 expression. Peptides 2001;22:1851 – 7.
Amylin-induced suppression of ANP secretion through receptors for CGRP1 and salmon calcitonin Feng Lian Piao, Chunhua Cao, Jeong Hee Han, Sung Zoo Kim, Kyung Woo Cho, Suhn Hee Kim * Department of Physiology, Institute for Medical Sciences, Chonbuk National University Medical School, 2 – 20 Keum-Am-Dong-San, Jeonju 561-180, South Korea Received 13 June 2003; received in revised form 25 September 2003; accepted 1 October 2003
Abstract Amylin cosecretes with insulin from pancreatic h-cells and shows high sequence homology with CGRP, adrenomedullin, and salmon calcitonin. This study aimed to investigate the effect of amylin on the atrial hemodynamics and ANP release from rat atria and to identify its receptor subtypes. Isolated perfused left atria from either control or streptozotocin-treated rats were paced at 1.3 Hz. The concentration of ANP was measured by radioimmunoassay and the translocation of ECF was measured by [3H]-inulin clearance. Rat amylin increased atrial contractility and suppressed the release of ANP. Rat CGRP showed similar effects but was approximately 300-fold more potent than amylin. Pretreatment with receptor antagonist for CGRP1 [rat a-CGRP (8-37)] or salmon calcitonin [acetyl-(Asn30, Tyr32)-calcitonin(8-32), (AC 187)] blocked the suppressive effect of ANP release and the positive inotropic effect by rat amylin. However, receptor antagonists for amylin [amylin (8-37), acetyl-amylin] did not block those effects. Amylin (8-37), acetyl-amylin, or rat a-CGRP (8-37) alone accentuated the release of ANP with no changes in atrial contractility. The effect of rat amylin and rat amylin (8-37) on the ANP release was attenuated in streptozotocin-treated rats. We suggest that amylin suppressed ANP release with increased atrial contractility through receptors for CGRP1 and salmon calcitonin and the attenuation of amylin and its antagonist on ANP release from streptozotocin-treated rat atria may be due to the downregulation of amylin receptor. D 2003 Elsevier B.V. All rights reserved. Keywords: Atrial contractility; Amylin; CGRP; Calcitonin; Diabetes; Natriuretic peptide; Receptors
1. Introduction Amylin or islet amyloid polypeptide [1,2] is a 37-amino acid that is colocalized with insulin in the central granule core of pancreatic h-cell secretory granules and cosecreted with insulin in response to hyperglycemia and arginine in a fixed ratio [3,4]. Amylin shares homology with a group of peptides including calcitonin gene-related peptide (CGRP, 45%), salmon calcitonin (33%), adrenomedullin, and rat calcitonin (18%) [5]. Amylin crossacts to the receptors for CGRP and salmon calcitonin [6,7], as well as amylin-preferring receptors [8]. A number of effects of amylin have been reported including counteraction of insulin on the regulation of carbohydrate metabolism, inhibition of osteoclastic activity,
* Corresponding author. Tel.: +82-63-274-9788; fax: +82-63-274-9892. E-mail address: [email protected] (S.H. Kim). 0167-0115/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2003.10.005
vasodilation, and anorectic effects [9]. Amylin is also involved in the development of hypertension through enhancement of renin release, stimulation of sodium/water reabsorption, and acute elevation of blood pressure [10,11]. Bell and McDermott [12] have demonstrated that amylin stimulates a positive contractile response in rat ventricular myocytes by interaction with CGRP1 receptor. CGRP also has positive inotropic effect with accentuation of atrial natriuretic peptide (ANP) secretion in rat atrial strips [13]. However, the receptor subtypes involved in the cardiac effects of CGRP are still controversial. In addition, little is known about the effect of amylin on ANP secretion and its receptor subtypes. Hyperinsulinemic states are associated with hyperamylinaemia [5,9]. Plasma concentration of amylin is elevated in non-insulin dependent diabetes mellitus (DM) [14] and essential hypertension [15]. However, a loss of amylin secretion is reported after treatment with streptozotocin because of destruction of pancreatic h-cells [16]. The aim
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of the present study was to investigate the effects of amylin on atrial contractility and ANP release using isolated rat atria and to identify its receptor subtypes. The possibility of modification of amylin effects was also studied in streptozotocin-induced DM rats. To assess the functional role of receptor for amylin or CGRP1, we used the selective amylin receptor antagonists [amylin (8-37) and acetyl-amylin] [8] or CGRP1 receptor antagonist [CGRP (8-37)] [17]. To study effects mediated by the receptor for salmon calcitonin, we used the selective salmon calcitonin receptor antagonist [acetyl-(Asn30, Tyr32)-calcitonin(8-32), AC 187] [18].
n = 6), rat a-CGRP (8-37) (1 AM, n = 6), or (AC187) (1 AM, n = 6) in HEPES buffer was introduced into the atrial lumen after a 10-min control collection period. Group 4 was atria perfused with amylin in the presence of receptor antagonist. Receptor antagonist for amylin [amylin (8-37) or acetyl-amylin], CGRP1 [rat a-CGRP (837)], or salmon calcitonin (AC187) (0.3 AM, all n = 6) in HEPES buffer was administered as a pretreatment at 40 min after start of the perfusion. Then, rat amylin (0.3 AM) was simultaneously infused after a 10-min control collection
2. Methods 2.1. Animals Male Sprague – Dawley rats weighing 300 to 350 g were used. In order to induce insulin-dependent DM, rats were received streptozotocin (100 mg/kg, ip) and sacrificed on the seventh day. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). 2.2. Preparation of perfused beating rat atria Isolated perfused beating atria were prepared by using a previously described method [19,20], with minor modifications. In brief, left atrium was dissected from the heart after sacrifice and fixed into a Tygon cannula. The cannulated atrium was transferred into an organ chamber, immediately perfused with oxygenated HEPES buffer solution at 36.5 jC, and paced at 1.3 Hz (duration, 0.3 ms; voltage, 40 V), as described previously [20]. The composition of the HEPES buffer solution was as follows: NaCl 118 mM, KCl 4.7 mM, CaCl2 2.5 mM, MgSO4 1.2 mM, NaHCO3 25 mM, HEPES 10 mM, glucose 10 mM, and bovine serum albumin 0.1%. The pericardial buffer solution contained [3H]-inulin to measure the translocation of extracellular fluid (ECF). Intra-atrial pressure was measured throughout the experiments. After stabilization for 100 min, the perfusate was collected at 2-min intervals at 4 jC. 2.3. Experimental protocols Experiments were performed with five groups. Group 1 was atria perfused with HEPES buffer as timecontrol (n = 6). Group 2 was atria perfused with amylin or CGRP. Rat amylin (Bachem Bubendorf, Switzerland, 0.1, 0.3, 1 AM, n = 6 –8) or rat CGRP (Bachem 1 nM, n = 5) was introduced into the atrial lumen after a 10-min control collection period, and perfusate was collected for 50 min. Group 3 was atria perfused with receptor antagonists. Rat amylin (8-37) (0.3, 1 AM, all n = 6), rat acetyl-amylin (1 AM,
Fig. 1. Effect of rat amylin on pulse pressure (A), ECF translocation (B), ANP secretion (C), and ANP concentration (D) in isolated perfused beating rat atria. After a 100-min control period, atrial perfusate was collected for 10 min at 2-min intervals as a control and then rat amylin (0.1 or 0.3 AM, n = 6 or 8) was perfused into atrial lumen. Values were expressed as ratio of last five experimental values exposed to amylin, as compared to mean of five control values. Amylin increased pulse pressure and decreased ANP secretion. The secretion of ANP in terms of ECF translocation (interstitial ANP concentration) was markedly decreased by amylin. ECF translo, ECF translocation; ANP conc, ANP concentration; CONT, control atria; , , amylin-infused atria. *, significantly different from atria perfused with 0.1 AM amylin, P < 0.05.
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period. All antagonists used in this study were purchased from Bachem. Group 5 was amylin or amylin (8-37)-perfused atria from diabetes mellitus rats. Rat amylin (0.3 AM, n = 7) or rat amylin (8-37) (1 AM, n = 9) was introduced into the atrial lumen after a 10-min control collection period. 2.4. RIA of ANP The concentration of immunoreactive ANP in the perfusate was measured by using specific RIA, as described previously [19,21]. RIA was performed in Tris-acetate buffer (0.1 M, pH 7.4) containing neomycin (0.2%), EDTA (1 mM), soybean trypsin inhibitor (50 benzoyl arginine ethyl ester units/ml), aprotinin (200 Kallikrein inhibiting unit/ml), phenylmethylsulfonyl fluoride (0.4 mg%), sodium azide (0.02%) and bovine serum albumin (1%). Standard and samples were incubated with anti-ANP antibody and [125I]-ANP for 24 h at 4 C. Bound forms were separated from free form using charcoal suspension or second antibody. RIA for ANP was done on the day of experiments and all samples in an experiment were analyzed in a single assay. The molar concentration of ANP release was calculated by the ANP secretion divided by ECF translocation and 3060 [molecular mass for ANP(1-28)] and was expressed in AM [22]. 2.5. Measurement of ECF translocation We previously reported a two-step sequential mechanism of ANP secretion from the atria [22]. First, atrial release of ANP into the interstitial space occurs by means of atrial
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stretching, and second, the released ANP is translocated into the atrial lumen, concomitantly with ECF translocation due to contraction. The radioactivity of [3H]-inulin in the atrial perfusate was measured by using a liquid scintillation counter (Tris-Carb 23-TR; A Packard Bioscience Downers Grove, IL). The amount of ECF translocated through the atrial wall was calculated by total radioactivity in perfusate divided by radioactivity in pericardial reservoir and atrial wet weight and was expressed in Al/min/g. 2.6. Statistical analysis The results are given as the mean F SEM. The statistical significance of the differences was assessed using ANOVA followed by Bonferroni test. Student’s t-test was also used. The critical level of significance was set at P < 0.05.
3. Results 3.1. Effects of amylin on atrial pulse pressure and ANP release After stabilization, perfusate was collected five times every 2 min to serve as a control period and then, amylin was infused at a concentration of 0.1, 0.3 or 1 AM. Fig. 1 shows the relative changes in pulse pressure, ECF translocation, ANP secretion, and ANP concentration by amylin (0.1, 0.3 AM) with time, as compared to control group. In time-control group, the basal pulse pressure, ECF translocation, and ANP secretion were 4.20 F 0.29 mm Hg, 56.81 F 17.96 Al/min/g, and 11.39 F 1.22 ng/min/g, respec-
Fig. 2. Percent changes in pulse pressure (A), ECF translocation (B), ANP secretion (C), and ANP concentration (D) by rat amylin (0.1, 0.3, 1 AM, n = 6 – 8) and rat a-calcitonin gene-related peptide (CGRP, 1 nM, n = 5). Values were expressed as percent changes of last five-experimental values exposed to amylin or CGRP, as compared to mean of five-control values. Amylin caused an increase in pulse pressure in a dose-dependent manner. The decrease in ANP secretion in terms of ECF translocation by amylin was not dose-dependent. CGRP showed the similar effect to amylin but the potency was approximately 300-fold greater than amylin. Legends are the same as in Fig. 1. #P < 0.05 vs. corresponding value.
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tively, which were not significantly different from basal values in atria perfused with amylin. Amylin at a dose of 0.1 and 0.3 AM caused increases in pulse pressure in a dosedependent manner (Fig. 1A). Amylin caused a decrease in ANP secretion (Fig. 1C) without changes in ECF translocation (Fig. 1B). The released ANP from atrial myocytes into the interstitial space is translocated into the atrial lumen, concomitantly with ECF translocation [19 – 22]. Therefore, the ANP secretion in terms of ECF translocation, which means the interstitial ANP concentration (Fig. 1D), was markedly suppressed. Fig. 2 shows the percent changes in pulse pressure, ECF translocation, and ANP secretion obtained from mean of five control values and last five experimental values exposed to amylin or CGRP. Amylin caused increases in pulse pressure in a dose-dependent manner (Fig. 2A). Decrease in ANP secretion by amylin was not dose-dependent (Fig. 2C). The ANP secretion in terms of ECF translocation was also decreased (Fig. 2D). Rat CGRP caused a decrease in ANP secretion with increase in pulse pressure. Rat CGRP was approximately 300-fold more potent than amylin. 3.2. Effects of receptor antagonists on atrial pulse pressure and ANP release Fig. 3 shows the relative changes in pulse pressure, ECF translocation, ANP secretion, and ANP concentration by amylin antagonist [amylin (8-37), 0.3 and 1 AM] with time, as compared to control group. No significant change in pulse pressure was found by amylin (8-37) (Fig. 3A). Amylin (8-37) at a dose of 0.3 AM did not cause any significant changes in ECF translocation and ANP secretion (Fig. 3B and C). Amylin (8-37) at a dose of 1 AM caused an increase in ANP secretion without changes in ECF translocation. Therefore, the ANP secretion in terms of ECF translocation (interstitial ANP concentration) was markedly accentuated by 1 AM amylin (8-37) (Fig. 3D). Fig. 4 shows the relative percent changes in pulse pressure, ECF translocation, and ANP secretion by receptor antagonists for amylin, CGRP and salmon calcitonin. No significant changes in pulse pressure and ECF translocation were found by all antagonists (1 AM) used in this study (Fig. 4A). Receptor antagonists for amylin, amylin (8-37) and acetyl-amylin, caused an accentuation of ANP secretion in terms of ECF translocation (Figs. 4C and D). Receptor antagonist for CGRP1, rat a-CGRP (8-37), also caused an accentuation of ANP secretion but receptor antagonist for salmon calcitonin, AC187, did not. 3.3. Modification of amylin effects on pulse pressure and ANP release with receptor antagonists In order to modify amylin-induced suppression of ANP release and positive inotropic effect, receptor antagonist (0.3 AM) was added as a pretreatment at 40 min after start of the perfusion. Fig. 5 shows the percent changes in pulse
Fig. 3. Effect of amylin receptor antagonist, rat amylin (8-37), on pulse pressure (A), ECF translocation (B), ANP secretion (C), and ANP concentration (D) in isolated perfused beating rat atria. After a 100-min control period, atrial perfusate was collected for 10 min at 2-min intervals as a control and then amylin (8-37) (0.3 or 1 AM, all n = 6) was perfused into atrial lumen. Values were expressed as ratio of last five experimental values exposed to amylin (8-37), as compared to mean of five-control values. Rat amylin (8-37) did not show any significant changes in pulse pressure. High dose of amylin (8-37) gradually increased ANP secretion in terms of ECF translocation. CONT, control atria; , , rat amylin (8-37)infused atria. *, **, significantly different from atria perfused with 0.3 AM amylin, P < 0.05, P < 0.01, respectively. Other legends are the same as in Fig. 1.
pressure, ECF translocation, and ANP secretion by amylin in the presence of receptor antagonist. The pretreatment of rat a-CGRP (8-37) completely blocked positive inotropic effect of amylin and also attenuated the suppression of ANP release (Fig. 5A and C). AC 187 also attenuated positive inotropic effect of amylin and completely blocked the suppression of ANP release. However, amylin (8-37) or acetyl-amylin did not modify the effects of amylin on the pulse pressure, ECF translocation, and ANP secretion (Fig. 5).
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Fig. 4. Percent changes in pulse pressure (A), ECF translocation (B), ANP secretion (C), and ANP concentration (D) by various receptor antagonists. Receptor antagonists for amylin [rat amylin (8-37), n = 6; acetyl-amylin, n = 6] and CGRP1 [rat a-CGRP (8-37), n = 6] caused an increase in ANP secretion with no significant changes in pulse pressure and ECF translocation. However, antagonist for salmon calcitonin, AC187 (n = 6) did not cause any significant changes. CONT, time control; AC-amylin, acetyl-amylin; CGRP (8-37), rat a-calcitonin gene-related peptide (8-37); AC187, acetyl-(Asn30, Tyr32)-calcitonin (8-32). *P < 0.05, **P < 0.01 vs. the time control group. Other legends are the same as in Fig. 1.
Fig. 5. Modification of amylin-induced suppression of ANP secretion and positive inotropism by the pretreatment of various antagonists. Amylin (8-37) and acetyl-amylin did not show any significant changes in amylin effects. However, rat a-CGRP (8-37) and AC187 blocked the suppression of ANP release and the increased pulse pressure by rat amylin. CONT, rat amylin-perfused atria; Amylin (8-37), rat amylin (8-37)-pretreated atria; AC-amylin, acetyl-amylinpretreated atria; CGRP (8-37), rat a-calcitonin gene-related peptide (8-37)-pretreated atria; AC187, acetyl-(Asn30, Tyr32)-calcitonin (8-32)-pretreated atria. **P < 0.01 vs. the time control atria. Other legends are the same as in Fig. 1.
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Fig. 6. Relative percent changes in pulse pressure (A), ECF translocation (B), ANP secretion (C), and ANP concentration (D) by rat amylin (0.3 AM, n = 7) and rat amylin (8-37) (1 AM, n = 9) in streptozotocin-treated diabetes mellitus rats. Positive inotropic effect by amylin did not change in diabetes mellitus rats. Changes in ECF translocation and ANP secretion by amylin and amylin (8-37) were significantly attenuated in diabetes mellitus rats. CONT, control rat; DM, diabetes mellitus rats. *P < 0.05, **P < 0.01 vs. control rat. Other legends are the same as in Fig. 1.
3.4. Modification of amylin and amylin (8-37) effects in DM rats The fasting blood sugar level in DM rats was 466.0 F 12.3 mg/100 ml (n = 9, P < 0.001), which was higher than control rats (126.0 F 3.5 mg/100 ml, n = 12). The basal levels of ECF translocation and ANP secretion were not different from control rats. Fig. 6 shows the percent changes in pulse pressure, ECF translocation, ANP secretion, and ANP concentration by rat amylin or rat amylin (8-37) in streptozotocin-treated DM rats. The increased pulse pressure by rat amylin in DM rats was not different from control rats (Fig. 6A). A decrease in ANP release by rat amylin and an increase in ANP release by rat amylin (8-37) were attenuated in DM rats, as compared to control rats (Fig. 6C).
4. Discussion We have shown that rat amylin caused a suppression of ANP release with increased atrial contractility, whereas its receptor antagonists caused an accentuation of ANP release in isolated perfused beating rat atria. The potency of amylin was approximately 300-fold less than CGRP. The antagonistic actions of rat a-CGRP (8-37) and AC187 on amylin effects suggest that both receptors for CGRP1 and salmon calcitonin mediate positive inotropic response and suppression of ANP release by amylin in rat atria. Additionally, the effects of amylin and amylin (8-37) on ANP release were attenuated in streptozotocin-treated rats. The structural similarity of amylin with CGRP, adrenomedullin and salmon calcitonin suggest that they may act via common receptors [5]. Two classes of CGRP receptors have been suggested that one with high affinity mediates a
positive contractile response and a second one with low affinity mediates a relaxant response [26]. They showed biphasic contractile response to CGRP but monophasic response to amylin. The proportion of two classes of CGRP receptors in the atrium and different affinity to receptors in terms of concentrations may be one of the possible factors to influence the effects of amylin. Amylin binds with high affinity to receptors selective for CGRP and salmon calcitonin in a number of tissues [6,7]. A positive contractile response of amylin in rat ventricular myocytes [12] is reported by interaction with CGRP1 receptor. In porcine myocardium [23] and human myocardial trabeculae [24], functional CGRP1 and CGRP2 receptors may mediate a positive inotropic effect by a-CGRP and amylin also has a potential positive inotropic effect. Therefore, the receptor subtypes involved in the cardiac effects of CGRP and amylin are still controversial. In the present study, we demonstrated that amylin caused dose-dependent increases in atrial contractility. The direct positive inotropic effect of amylin was blocked by receptor antagonist for CGRP1 [CGRP (8-37)] and receptor antagonist for salmon calcitonin (AC 187) but not by receptor for amylin [amylin (8-37), acetyl-amylin]. These results are consistent with other report showing amylin-induced positive contractile response blocked by rat a-CGRP (8-37) but not by amylin (8-37) [12,23]. The positive inotropic effect by amylin showed approximately 300-fold less potent than rat a-CGRP. The interaction of amylin with weak affinity at CGRP1 receptors coupled to a positive inotropic response is in agreement with studies in which the peptide was infused and found to be approximately 100-fold less potent as a peripheral vasodilator than CGRP [25]. The lower potency of amylin may be due to low affinity to CGRP1 receptor or differences in receptor density in species and tissues. These results suggest that the positive inotropic effect of amylin in isolated rat
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atria may be mediated by receptors for both CGRP1 and salmom calcitonin. The secretion of ANP is closely related to atrial hemodynamics, such as increased contractility and heart rate [21]. Our results show that amylin as well as CGRP had a suppressive effect of ANP release with increased contractility and its antagonists except AC187 had an accentuated effect of ANP release without changes in contractility. Positive inotropism is one of important factors, which influences ANP release. Our data are not consistent with the report showing the stimulation of ANP secretion by CGRP in rat beating atrial strips [13]. They show biphasic ANP secretory response to CGRP with increased atrial tension mediated by cAMP. The discrepancy may be partly due to differences in atrial model used for the study. Ca2 + and cAMP act as negative regulators of ANP secretion in isolated perfused atria [27,28] but as positive regulators in atrial strips [13,29] even though the exact reason is not clear. The relationship between Ca2 + and ANP secretion is still debatable. To identify the receptor subtypes responsible for the suppression of ANP release by amylin, we used 0.3 Amol/ l of antagonists, which did not show their own antagonistic effects. Amylin-induced suppression of ANP release was blocked by rat a-CGRP1 and AC187 but not by amylin (837) and acetyl-amylin. Our findings are in a good agreement with other report demonstrating the antagonistic effects of rat a-CGRP (8-37) and AC187 on the inhibitory effect of amylin in rat soleus muscle [18]. It has been reported that despite of little sequence homology, rat amylin and salmon calcitonin compete with high affinity for a single class of binding sites in the region of the rat brain [30]. As shown in Fig. 5, our results show that the inhibitory effect of increased pulse pressure by rat a-CGRP (8-37) seems to be more potent than by AC187. On the other hand, the blocking effect of suppression of ANP release by AC187 seems to be more potent than by rat a-CGRP (8-37). No appreciable inhibitory effects by amylin (8-37) and acetylamylin were found on those effects. These results may be due to not only differences in binding affinities of amylin to multiple receptors but also differences in receptor densities and distributions. These observations indicate that the effect of amylin on the ANP release is mediated by specific receptors for CGRP1 and salmon calcitonin. We demonstrated antagonism of endogenous ligand versus exogenous amylin in Figs. 4 and 5. None of the antagonists added to beating atria modulated contractility, however, the amylin and CGRP antagonists accentuate ANP release. On the other hand, if amylin is used as agonist, both CGRP and calcitonin receptor antagonist inhibit amylinmediated inotropic effects as well as ANP release effects. These results suggest that other ligand rather than amylin may be an endogenous ligand released from beating rat atria. It is highly probable that adrenomedullin released by the heart may be responsible for the antagonistic effects demonstrated in Fig. 4 because of a similarity of adreno-
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medullin to amylin. It has been shown that endogenous adrenomedullin inhibits ANP expression in cultured neonatal cardiomyocytes via a CGRP1 receptor [31]. The difference may be partly due to experimental materials or parameters studied. It has previously been shown that amylin is co-secreted with insulin [3] and that hyperinsulinemic states are associated with hyperamylinaemia [5]. In various animal models of insulin resistance with obesity, glucose tolerance and hypertension, amylin concentrations are markedly elevated [14]. Ogawa et al. [16] showed a loss of amylin secretion after treatment with streptozotocin because of destruction of pancreatic h-cells. It is possible that the response of ANP secretion to amylin may be different depending on the secretory function of pancreatic h-cells. The present study shows an attenuation of responsiveness of ANP secretion to amylin and amylin (8-37) without differences in pulse pressure in streptozotocin-treated DM. It may be due to changes in receptor density, or sensitivity. More studies are needed to define the pathophysiological involvement of amylin in the development of hypertension with different types of DM.
Acknowledgements The authors thank Dr. Mie-Jae Lim for her valuable critics and comments. The authors also thank Kyong-Sook Kim for her secretarial support. This work was supported by Korea Science and Engineering Foundation (98-0403-1001-5) and the Korea Research Foundation (2000-015FP0023), Republic of Korea.
References [1] Cooper GJS, Willis AC, Clark A, Turner RC, Sim RB, Teid KBM. Purification and characterization of a peptide from amyloid-rich pancreases of type-2 diabetic patients. Proc Natl Acad Sci U S A 1987; 84:8628 – 32. [2] Westermark P, Wernstedt A, Wilander E, Hayden DW, O’Brien TD, Johnson KH. Amyloid fibrils in human insulinoma and islets of Langerhans of the diabetic cat are derived from neuropeptide-like protein also present in normal islet cell. Proc Natl Acad Sci U S A 1987;84: 3881 – 5. [3] Fehmann HC, Weber V, Goke R, Goke B, Arnold R. Cosecretion of amylin and insulin from isolated rat pancreas. FEBS Lett 1990;262: 279 – 81. [4] Inoue K, Hisatomi A, Umeda F, Nawata H. Release of amylin from perfused rat pancreas in response to glucose, arginine, beta-hydroxybutyrate, and gliclazide. Diabetes 1991;40:1005 – 9. [5] Cooper GJ. Amylin compared with calcitonin gene-related peptide: structure, biology, and relevance to metabolic disease. Endocrine Rev 1994;15:163 – 201. [6] Chantry A, Leighton B, Day AJ. Cross-reactivity of amylin with calcitonin-gene-related peptide binding sites in rat liver and skeletal muscle membranes. Biochem J 1991;277:139 – 43. [7] Rink TJ, Beaumont K, Koda J, Young A. Structure and biology of amylin. TIPS 1993;14:113 – 8. [8] Deems RO, Cardinaux F, Deacon RW, Young DA. Amylin or CGRP
166
[9] [10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
F.L. Piao et al. / Regulatory Peptides 117 (2004) 159–166 (8-37) fragments reverse amylin-induced inhibition of 14C-glycogen accumulation. Biochem Biophys Res Commun 1991;181:116 – 20. Edwards BJ, Morley JE. Amylin. Life Sci 1992;51:1899 – 912. Wookey PJ, Tikellis C, Du HC, Qin HF, Sexton PM, Cooper ME. Amylin binding in rat renal cortex, stimulation of adenylyl cyclase, and activation of plasma renin. Am J Physiol 1996;270:F289 – 94. Wookey PJ, Cooper ME. Amylin: physiological roles in the kidney and a hypothesis for its role in hypertension. Clin Exp Phamacol Physiol 1998;25:653 – 60. Bell D, McDermott BJ. Activity of amylin at CGRP1-preferring receptors coupled to positive contractile response in rat ventricular cardiomyocytes. Regul Pept 1995;60:125 – 33. Schiebinger RJ, Santora AC. Stimulation by calcitonin gene-related peptide of atrial natriuretic peptide secretion in vitro and its mechanism of action. Endocrinology 1989;124:2473 – 9. Gill AM, Yen TT. Effects of ciglitazone on endogenous plasma islet amyloid polypeptide and insulin sensitivity in obese-diabetic viable yellow mice. Life Sci 1991;48:703 – 10. Kautzky-Willer A, Thomaseth K, Pacini G, Clodi M, Ludvik B, Streli C, et al. Role of islet amyloid polypeptide secretion in insulin-resistant humans. Diabetologia 1994;37:188 – 94. Ogawa A, Harris V, McCorkle SK, Unger RH, Luskey KL. Amylin secretion from the rat pancreas and its selective loss after streptozotocin treatment. J Clin Invest 1990;85:973 – 6. Chiba T, Yamaguchi A, Yamatani T, Nakamura A, Morishita T, Inui T, et al. Calcitonin gene-related peptide receptor antagonist human CGRP-(8-37). Am J Physiol 1989;256:E331 – 5. Beaumont K, Pittner RA, Moore CX, Wolfe-Lopez D, Prickett KS, Young AA, et al. Regulation of muscle glycogen metabolism by CGRP and amylin: CGRP receptors not involved. Br J Pharmacol 1995;115:713 – 5. Cho KW, Seul KH, Kim SH, Seul KM, Ryu H, Koh GY. Reduction volume dependence of immunoreactive atrial natriuretic peptide secretion in isolated perfused rabbit atria. J Hypertens 1989;7: 371 – 5. Han JH, Cao C, Kim SZ, Cho KW, Kim SH. Decreases in ANP
[21]
[22]
[23]
[24]
[25]
[26] [27]
[28]
[29]
[30] [31]
secretion by lysophosphatidylcholine through PKC. Hypertension 2003;41:1380 – 5. Cho KW, Seul KH, Kim SH, Seul KM, Koh GY. Atrial pressure, distension, and pacing frequency in ANP secretion in isolated perfused rabbit atria. Am J Physiol 1991;260:R39 – 46. Cho KW, Seul KH, Kim SH, Koh GY, Seul KM, Hwang YH. Sequential mechanism of atrial natriuretic peptide secretion in isolated perfused rabbit atria. Biochem Biophys Res Commun 1990;172: 423 – 31. Ole Saetrum O, de Vries R, Tom B, Edvinsson L, Saxena PR. Positive inotropy of calcitonin gene-related peptide and amylin on porcine isolated myocardium. Eur J Pharmacol 1999;385:147 – 54. Ole Saetrum O, Kasbak P, de Vries R, Saxena PR, Edvinsson L. Positive inotropy mediated via CGRP receptors in human myocardial trabeculae. Eur J Pharmacol 2000;397:373 – 82. van Rossum D, Menard DP, Fournier A, St-Pierre S, Quirion R. Autoradiographic distribution and receptor binding profile of [125I]Bolton Hunter-rat amylin binding sites in the rat brain. J Pharmacol Exp Ther 1994;270:779 – 87. Kim D. CGRP activates the muscarinic-gated potassium current in atrial cells. Eur J Physiol 1991;418:338 – 45. Cho KW, Kim SH, Seul KH, Hwang YH, Kook YJ. Effect of extracellular calcium depletion on the two-step ANP secretion in perfused rabbit atria. Regul Pept 1994;52:129 – 37. Cui X, Wen JF, Jin JY, Xu WX, Kim SZ, Kim SH, et al. Protein kinase-dependent and Ca2 +-independent cAMP inhibition of ANP release in beating rabbit atria. Am J Physiol Regul Integr Comp Physiol 2002;282:R1477 – 89. Schiebinger RJ, Li Y, Cragoe Jr EJ. Calcium dependency of frequency-stimulated atrial natriuretic peptide secretion. Hypertension 1994;23:710 – 6. Beaumont K, Kenney MA, Young AA, Rink TJ. High affinity amylin binding sites in rat brain. Mol Pharmacol 1993;44:493 – 7. Autelitano DJ, Ridings R. Adrenomedullin signalling in cardiomyocytes is dependent upon CRLR and RAMP2 expression. Peptides 2001;22:1851 – 7.