The Inhibitory Effect of Adrenomedullin in the Rat Ileum: Cross-Talk with β₃-Adrenoceptor in the Serotonin-Induced Muscle Contraction

The hypotensive peptide, adrenomedullin (AM), was originally purified from both adrenal medulla and pheochromocytoma (Kitamura et al., 1993; Kitamura and Eto, 1997; Jougasaki and Burnett, 2000). Two unique structures, a 6-amino acid ring with a disulfide bond and a C-terminal amide in the molecule of AM (Kitamura et al., 1993; Hinson et al., 2000; Jougasaki and Burnett, 2000), are likely to determine its vasorelaxation properties. AM shares structural homology with the calcitonin gene-related peptide (CGRP), indicating that AM probably belongs to the CGRP superfamily (Kitamura and Eto, 1997; Hinson et al., 2000; Poyner et al., 2002). The signal transduction pathways have been studied in the greatest detail in the vasculature. A large body of experimental data shows AM synthesis in smooth muscle cells (Kitamura and Eto, 1997; Nishimura et al., 1997) and suggests that AM-induced vasodilation may be mediated by a specific receptor functionally coupled to adenylate cyclase (Eguchi et al., 1994a; Kitamura and Eto, 1997; Nussdorfer et al., 1997; Jougasaki and Burnett, 2000; Poyner et al., 2002). AM-specific receptors are not only found in smooth muscle cells, but also in endothelium cells (Kamitani et al., 1999). It has been reported that the binding of AM to the endothelial receptor can trigger the nitric oxide (NO)-cGMP pathway (Ikeda et al., 1996). Therefore, it is now widely accepted that at least two mechanisms appear to underlie the AM-induced relaxation in vascular muscles: 1) a direct action on vascular smooth muscle cells to increase cAMP (Eguchi et al., 1994a,b; Shimekake et al., 1995; Kitamura and Eto, 1997; Yoshimoto et al., 1998) and 2) an indirect action on endothelial cells to stimulate NO synthesis (Shimekake et al., 1995; Hinson et al., 2000; Jougasaki and Burnett, 2000).

The results of mRNA blot analysis reported 10 years ago (Sakata et al., 1993) and later confirmed (Nishimura et al., 1997; Cameron and Fleming, 1998; Sakata et al., 1998) imply that rat AM mRNA can also be expressed in visceral smooth muscle cells of the gastrointestinal tract. Immunoreactive AM (ir-AM) was detected in different regions of the gastrointestinal system: stomach, duodenum, jejunum, ileum, cecum, and colon (Mulder et al., 1996; Sakata et al., 1998; Kiyomizu et al., 2001). However, in contrast to vascular muscles, the contribution of AM to the regulation of contractile responses of the visceral muscles have not been much investigated. Only a single report indicates that AM is able to modify ion transport and inhibit muscle contraction in the rat colon (Fukuda et al., 1998). To obtain further information about the role of AM in the gut, the contents of preproadrenomedullin mRNA, the level of AM, and the effects of AM on the rat ileum precontracted with a natural regulator of smooth muscle tone, serotonin, have been studied. The effect of AM on serotonin-treated preparation is especially relevant in view of the colocalization of AM and serotonin in the enterochromaffin cells of the ileum (Mulder et al., 1996).

Materials and Methods

Determination of ir-AM in the Ileum

All studies were performed according to protocols approved by the Committee on the Use of Laboratory Animals for Teaching and Research, the Faculty of Medicine, the University of Hong Kong and in accordance with the Guide for the Care and Use of Laboratory Animals (National Academy of Sciences).

Male adult Sprague-Dawley (SD) rats (310-420 g) were sacrificed by decapitation; the ilea were dissected, frozen in dry ice, and stored at -70°C until use. The tissue was homogenized in 2 N acetic acid for 1 min and boiled in a water bath for 10 min to inactivate the proteases. After lyophilization, the residue was dissolved in RIA buffer and subjected to RIA as described (Hwang and Tang, 1999, 2000). Rat AM and AM antiserum were purchased from Peninsula Laboratories (Belmont, CA). Iodine¹²⁵AM was prepared in our laboratory by the Chloramine T method and purified by Biogel P₄ column. Protein contents of tissue samples were determined using the kit from Bio-Rad (Hercules, CA). Samples of stomach, duodenum, colon, and pancreas were also measured for AM contents.

Measurement of AM mRNA

The rat ilea (200-300 mg) were homogenized in 2 to 3 ml of TRIZOL reagent (Invitrogen, Carlsbad, CA) by polytron and further processed according to the protocol detailed previously (Wu et al., 1998). The RNA pellet was air-dried, dissolved in Tris-EDTA buffer (pH 7.5), and stored at -70°C until assay.

Details of the solution hybridization RNase protection assay have been reported earlier (Hwang and Tang, 1999, 2000). The plasmids containing cDNA for AM (613 base pairs in length) and β-actin (387 base pairs in length) were gifts from Dr. Autelitano (Prahran, Australia) and were subcloned into pGEM-4Z and pGEM-3Z, respectively. Restriction enzymes BamHI and EcoRI were used to linearize AM cDNA to generate the templates for the standard and the probe, respectively, whereas restriction enzymes EcoRI and HindIII were used for β-actin cDNA. Standards or tissue samples were incubated with 100,000 cpm of [³²P]AM or β-actin probe in hybridization buffer (80% formamide, 400 mM NaCl, 1 mM EDTA, 40 mM PIPES, pH 6.7) at 45°C overnight, after preincubation at 85°C for 5 min. After digestion by RNase, the hybrids were precipitated with isopropanolol at -20°C overnight and subjected to 4% polyacrylamide gel (19:1 acrylamine Bis) at 160 V and 34 mA for approximately 1 h. The hybrids were visualized by exposing them to X-ray film at -70°C in a cassette for 2 days, and the bands were cut out for liquid scintillation counting.

Contractile Experiments

Animals and Tissue Preparation. Male adult SD rats (310-420 g) were killed promptly by stunning and decapitation. The ileal strips were prepared as described by Hoey et al. (1996). Briefly, approximately 16 cm of ileum were removed and placed into physiological salt solution (PSS) (132 mM NaCl, 21.5 mM NaHCO₃, 1.1 mM NaH₂PO₄, 5.1 mM KCl, 0.95 mM MgSO₄, 2.5 mM CaCl₂, and 11 mM glucose at 22°C and pH 7.43). The ileum was trimmed of any fat and cut into strips 22 to 26 mm long, which were then cleared of their contents by a very gentle flushing with PSS. One end of the strip was connected to a hook made of stainless steel, and a silk thread at the other end connected the tissue to a force displacement transducer (Grass F-10) coupled to a Grass model 7H polygraph recorder (Grass Instruments, Quincy, MA). Each strip was placed in an organ bath containing PSS bubbled with 95% O₂ and 5% CO₂, and the temperature was maintained constant at 22°C. The preparations were suspended with a preload of 2 g, which was maintained for 70 min.

Protocols. Preliminary results have demonstrated that AM alone had no effect on the basal ileal tone. As serotonin is a natural constrictor of ileum (Mulder et al., 1996), further experiments were performed in serotonin precontracted rat ileum maintained at room temperature (22°C) to suppress spontaneous contraction. The dose of serotonin to get 50% of the maximal response (EC₅₀) was calculated, and 68 nM serotonin, which is equal to the EC₅₀, was used to evoke a sustained contractile response in the rat ileum. The inhibitory effects of ZM 226600 (an opener of the ATP-dependent K⁺ channel), BRL 37344 (a β₃-adrenoceptor agonist), and AM were confirmed before more elaborate studies were performed. In some experiments the effect of AM was studied on ileal strips precontracted with acetylcholine (100 nM) and histamine (10 μM).

To ascertain the usefulness of the smooth muscle preparations, the equilibrated ileal strips were tested for their ability to contract in response to 40 mM KCl. Within 30 min after washing by PSS, the ileal strips were treated by one of the following drug inhibitors of AM receptors and CGRP receptors: inhibitors of NO, inhibitors of K⁺ channels, d-cAMP, antagonist of β-AR, inhibitors of PKA or the same volume of deionized water (control) (for details, please refer to the figure legends). The ileum preparations were equilibrated with these drugs for 30-60 min before they were contracted by the addition of 68 nM serotonin. After a further 9 to 15 min to make sure that the contraction was stable, a concentration-response curve for AM was generated. In view of the slow onset and long duration of the responses to both AM and BRL 37344 (Fig. 3, B and C), each dose of these drugs was tested on the separate ileal strip. Activities of AM and the β₃-AR agonist were expressed as an IC₅₀ value, i.e., the concentration of either AM or the β₃-AR agonist that produced a relaxant response that was 50% of the maximal effect. β₃-Antagonist activities were determined by comparing the IC₅₀ values obtained from AM- and agonist-response curves in the absence of the antagonist to that obtained in the presence of the antagonist and calculated as concentration ratios. In some cases, a plot of log (antagonist concentration ratio - 1) versus log [antagonist] was constructed. When the slope of the regression was not significantly different from unity, the competition was considered to be competitive (Arunlakshana and Schild, 1959). Where the antagonistic effect was shown to be noncompetitive, the pK_B values were derived from the equation: pK_B = log (antagonist concentration ratio - 1) - log [antagonist], according to the method of Furchgott (1972).

Measurement of Intracellular cAMP Generation

The ileal strips were stretched and equilibrated in PSS according to aforementioned protocol for contractile experiments. Within 12 to 15 min after application either of 1 nM AM or of the equal volume of water (control) to the serotonin precontracted ileal strips bathed in PSS (room temperature), the tissues were collected and quickly frozen at -70°C. The frozen samples were later homogenized in PSS containing 10% trichloroacetic acid. After centrifugation at 1500g for 20 min, trichloroacetic acid in the supernatant was removed by washing with water-saturated ether, and cAMP was assayed with a radioimmunoassay kit (Amersham Biosciences UK, Ltd., Little Chalfont, Buckinghamshire, UK). The concentration of the intracellular cAMP was expressed in picomoles per milligram of protein. The protein content of each sample was determined by using the kit from Bio-Rad with bovine serum albumin as the standard.

Statistics, Data Analysis, and Drug Sources

The results are given as the mean ± S.E.M. A Student's t test was used to estimate the significance of the results, and a P value less than 0.05 was considered significant.

The EC₅₀ and IC₅₀ values were obtained from concentration-effect curves, using commercial software (GraphPad PRISM; GraphPad Software Inc., San Diego, CA).

AM_1-52,AM_22-52, CGRP_8-37, and CGRP were purchased from Peninsula Laboratories and Bachem California (Torrance, CA). Serotonin, acetylcholine, histamine, apamin, iberiotoxin, dendrotoxin, l-NAME, methylene blue, d-cAMP, tetrodotoxin (TTX), SR 59230A, and H-7 were from Sigma-Aldrich (St. Louis, MO). Xanthine amine congener and glibenclamide were purchased from ICN Pharmaceuticals (Costa Mesa, CA). The other agonists and antagonists of β₃-ARs as well as KT 5720 were from Tocris Cookson Inc. (Bristol, UK).

Results

Distribution of ir-AM in the Gastrointestinal Tract

Using the RIA, we detected AM immunoreactivity throughout the gastrointestinal tract of SD rats. In the stomach, colon, and ileum the concentrations of ir-AM were found to be comparably high (288 ± 27, 252 ± 18, and 301 ± 17 pg/mg protein, n = 6, respectively) as was the level of AM in the pancreas (252 ± 16 pg/mg protein, n = 6), whereas duodenum contained just a trace amount of AM (Fig. 1).

Expression of AM mRNA in Rat Ileum

The preproadrenomedullin mRNA was obtained to be highly expressed in the rat ileum (162 ± 15 fg/pg actin mRNA, n = 5). The X-ray recording of the separation of the hybrids by polyacryamide gel electrophoreis is shown in Fig. 2.

Organ Bath Experiments

Effects of ZM 226600 (an Opener of ATP-Dependent K⁺-Channels), BRL 37344 (a Selective β₃-Adrenoceptor Agonist), and AM on Serotonin-Induced Contraction. AM inhibited contraction of ileal strips precontracted with serotonin, acetylcholine, and histamine in a concentration-dependent manner, with IC₅₀ values of 0.85 ± 0.12 nM (n = 24), 0.080 ± 0.006 nM (n = 4), and 4.8 ± 0.2 (n = 4), respectively. The serotonin-induced contraction was completely inhibited (Fig. 3A) by 5 μM ZM 226600, a potent opener of ATP-dependent K⁺ channels (Grant et al., 1994), suggesting a pivotal contribution of these channels to the mechanism of ileal relaxation. The lack of effect of 1 μM TTX on the serotonin-induced contraction (Fig. 3) implies that the contractile response is not caused by neural inputs but may be related to the properties of smooth muscle itself. Both BRLK 37344 (Fig. 3B) and AM (Fig. 3C) were also inhibitory, and their actions were slow in onset and long-lasting. It is pertinent to note that all the experiments mentioned above were performed at room temperature. However, despite the strong spontaneous contractions, similar data were also obtained at 37°C. As more prominent effects were observed at room temperature (where the spontaneous responses are minimized), this temperature was chosen for subsequent studies.

Influence of AM on Smooth Muscle Tone: Receptor Analysis.Figure 4 demonstrates that AM-evoked relaxation of serotonin precontracted ileal strips was not blocked by CGRP_8-37, a specific antagonist of CGRP receptors (Eguchi et al., 1994a) (Fig. 4A), or by AM_22-52, a specific antagonist of AM receptors (Eguchi et al., 1994b) (Fig. 4B), or by xanthine amine congener, an inhibitor of adenosine receptors (Sabates et al., 1997) (Fig. 4C).

Figure 5A demonstrates that neither a specific inhibitor of β₁-AR (bisoprolol) (Hieble et al., 1995) nor a very selective β₂-AR (ICI 118,551) (Bilski et al., 1983), were able to prevent AM-induced relaxation. By contrast, SR 59230A, which is a selective β₃-AR antagonist for the gut (Manara et al., 1995; Rathi et al., 2003), inhibited the effect of AM in a dose-dependent manner (Fig. 5B). Further experiments revealed that when BRL 37344 was used as the agonist of β₃-AR (Roberts et al., 1999), SR 59230A (0.1-10 μM) elicited a competitive antagonism of the relaxant responses (Fig. 5C). An Arunlakshana-Schild plot of the data revealed a pA₂ value of 7.9, with the slope of the regression line not significantly different from unity (0.79, Fig. 5C, top). However, the antagonistic effect of SR 59230A versus adrenomedullin did not appear to be competitive (Fig. 5B) as the slope of the Arunlakshana-Shild plot was significantly different from unity (0.18, Fig. 5B, top). The pK_B value derived from the concentrations SR 59230A used was 7.6.

Effect of AM on the Level of Intracellular cAMP: Contribution of PKA to AM-Induced Relaxation. It should be noted that, in contrast to previous reports where the cAMP generation in cells has been evaluated after direct administration of AM to the relaxed smooth muscles cells and tissues (Eguchi et al., 1994a,b; Ishizaka et al., 1994; Yoshimoto et al., 1998), we determined the influence of AM on cAMP in the rat ileum precontracted with serotonin. We did not expect a large difference between the basal and AM-stimulated cAMP levels as the control cAMP might be already modified by serotonin (Meulemans et al., 1993). Application of AM (1 nM) to the serotonin precontracted aortic rings increased the level of the intracellular cAMP by 30% (p < 0.01) (Fig. 6A). Figure 6B shows that the inhibitors of PKA, H-7 (5 μM) and KT 5720 (1 μM), were able to reverse partially the AM-induced relaxation. On the other hand, 50 μM d-cAMP (the activator of PKA), which had no effect on serotonin contractile responses, potentiated the effect of 1 nM AM by 57% (Fig. 6C).

Effects of Inhibitors of NO and Modualtors of Various K⁺ Channels on AM-Induced Relaxation. As was shown in Fig. 7A, neither inhibitor of synthesis of NO, l-NAME (100 μM), nor inhibitor of soluble guanylate cyclase activity, methylene blue (3 μM), were capable of preventing AM-induced relaxation in serotonin precontracted ileal strips. The experiments on the K⁺-channels, which might also contribute to relaxation, revealed that glibenclamide (1 μM), an antagonist of the ATP-dependent K⁺-channels, considerably attenuated the influence of AM (1 nM) in serotonin precontracted rat ileum. Further screening of the inhibitors of the other K⁺-channels has revealed that apamin (100 nM) and iberiotoxin (100 ng ml^-1), the blockers of small conductance Ca²⁺-activated K⁺-channels and high conductance Ca²⁺-activated K⁺-channels, respectively, and dendrotoxin (10 nM), an antagonist of the voltage-gated K⁺-channels, failed to exert any effect (Fig. 7B).

Discussion

This study for the first time demonstrated that AM elicits relaxation of the rat ileum precontracted with serotonin (Figs. 3, 4, 5). Probably this is a general action of AM on the activated ileal tissue, since we have also revealed that AM was able to relax both acetycholine and histamine precontracted ileal strips. In addition, there is expression of AM mRNA in rat ileum (Fig. 2) indicative of AM synthesis, as has been reported for the pig and rat gastrointestinal tract (Nishimura et al., 1997; Cameron and Fleming, 1998), and the high level of ir-AM concentration in the rat ileum (Fig. 1) is consistent with a recent report of Kiyomizu et al. (2001). Collectively, these findings strongly suggest that AM is a local hormone, which, in contrast to AM in the pig (Nishimura et al., 1997), regulates the ileal tone of the rat.

In the second part of our work, we attempted to delineate the mechanism of inhibitory action of AM in ileal smooth muscle. AM is known to produce vasorelaxation through receptor-mediated mechanisms (Eguchi et al., 1994a,b; Kitamura and Eto, 1997; Hinson et al., 2000). However, it is still unclear what kind of receptors are responsible for AM-induced smooth muscle relaxation. They can be classified into three groups (Nishimura et al., 1997; Jougasaki and Burnett, 2000): (i) an AM receptor similar to CGRP receptor; (ii) an AM receptor which is distinct from CGRP receptor; or (iii) a nonspecific AM receptor which binds to both AM and CGRP with different affinities. Apparently, the receptor diversity is a result of the differential transport of calcitonin receptor like receptor, which is required for binding of both CGRP and AM, to the plasma membrane by different single transmembrane domain proteins termed receptor activity modifying proteins (RAMPs). RAMP1 coupled with calcitonin receptor like receptor will make the receptor into a CGRP receptor, while a combination of RAMP2 or RAMP3 will result in an AM receptor (McLatchie et al., 1998; Poyner et al., 2002). The studies with specific AM and CGRP receptor antagonists revealed that AM causes relaxation neither through AM-specific receptors nor CGRP receptors (Fig. 4, A and B). This nonreceptor-mediated action of AM was not surprising as Sabates et al. (1997) reported that AM can activate an adenosine receptor. However, a lack of effect of xanthine amine congener, an inhibitor of adenosine receptors, in our experiments (Fig. 4C) indicates that adenosine receptors are unlikely to be involved in this AM-induced relaxation. The other receptor, which may be responsible for the relaxation in the ileal strips, is a β-AR (Growcott et al., 1993; Roberts et al., 1999). In rat ileum β₁-, β₂-, and β₃-AR mRNA were detected (Roberts et al., 1999). Apparently, β₃-AR are the predominant β-AR subtype mediating rat ileal relaxation by different compounds, since the functional studies of rat ileum relaxation have shown that stimulation by selective β₃-AR agonists evokes a relaxation response in the presence of β₁- and β₂-AR blockades (Growcott et al., 1993; Hoey et al., 1996). The attenuation of the β₃-AR-dependent relaxation in serotonin precontracted ileal strips with the antagonist of β₃-AR, SR 59230A (Fig. 5C) supports this argument. The lack of effects of the antagonists of β₁- and β₂-ARs (bisoprolol and ICI 118,551, respectively) on AM-evoked relaxation shows that these subtypes of β-receptors are unlikely to be targeted by AM (Fig. 5A). In contrast, the attenuation of AM action with SR 59230A (Fig. 5B) imply that the relaxation may be mediated through the activation of β₃-AR. Since AM was suggested to release catecholamines from chromaffin and nervous cells (Nussdorfer et al., 1997; Fukuda et al., 1998), it is possible the indirect contribution of β-AR agonists to the AM induced relaxation. To prevent this pathway, all experiments were performed in the present of TTX. The noncompetitive inhibition of AM-induced relaxation by SR 59230A indicates that there is no release of β-AR agonists by AM (otherwise a competitive relationship should be observed).

The β-AR sensitive relaxation in the smooth muscles is thought to be initiated either through a G-protein-dependent generation of cAMP leading to a subsequent activation either of PKA or synthesis and release of NO (Rebich et al., 1995; von-der-Weid, 1998; Werstiuk and Lee, 2000). Thus, in order to clarify the next step in the mechanism of action of AM on muscular tone in ileum, it was required to investigate: 1) the influence of AM on cAMP level in ileal strips and 2) the contribution of PKA and NO to the AM-induced relaxation. As the AM-induced relaxation was associated with an enhanced cAMP level (Fig. 6A), it was suggested that cAMP is essential for activation of either the PKA or the NO machinery. A lack of effect of the inhibitor of NO synthesis (l-NAME) and inhibitor of soluble guanylate cyclase activity (methylene blue) on AM-induced relaxation (Fig. 7A) suggests that NO may not be involved. On the other hand, H-7 and KT 5720, the antagonists of PKA, markedly diminished the inhibitory effect of AM (Fig. 6B), suggesting the involvement of PKA in the signal transduction mechanism. In support of this, the synergistic influence of a subthreshold dose of d-cAMP on AM-induced relaxation was demonstrated (Fig. 6C). The screening of different K⁺-channels with various antagonists (Fig. 7B) suggests that the target for PKA activation by AM in rat ileum is likely to be the ATP-dependent K⁺-channels just like in the vascular smooth muscle cells (Sabates et al., 1997).

In conclusion, we have provided evidence that ir-AM in the rat ileum is important for the regulation of smooth muscle tone. From our results, it is not possible to pinpoint what kind of cells are responsible for synthesis and release of AM from the rat ileum. Some other reports indicate that AM is localized in a subpopulation of the enterochromaffin (serotonin-containing) cells of the gastrointestinal tract (Mulder et al., 1996). This colocalization of AM with serotonin means that after secretion of both substances, the ileal tone will be determined by the balance of free AM and serotonin outside the plasma membrane (paracrine action). On the other hand, as there is also AM synthesis in the ileal smooth muscle cells themselves (Nishimura et al., 1997), it is likely that the release of AM from smooth muscle cells may also contribute to the regulation of ileal tone (autocrine action).