Characterization of I2 Imidazoline and s Binding Sites in the Rat and Human Stomach

Radioligand binding experiments were carried out to identify and characterize nonadrenoceptor [H]idazoxan binding sites and [H](1,2-di-(2-tolyl)guanidine) binding sites in the rat and human stomach. Furthermore, we examined two selected aspects of their potential functional significance. Binding of [H]idazoxan (Kd 5 11.1 nM and 12.4 nM, respectively) and [H]DTG (Kd 5 932 nM and 242 nM, respectively) to cell membranes from rat and human stomach was rapid, reversible, specific and saturable. In rat stomach, binding of the radioligands was inhibited by imidazolines and by nonimidazoline s-site ligands, respectively, at different rank orders of affinity, which suggests the existence of I2-imidazoline binding sites as well as s2-sites. In two functional models, the direct effects of I2-site ligands and s2-site ligands on gastric smooth muscle and glands were investigated. (1) Cirazoline, clonidine and 4-chloro-2-(2-imidazolin-2-ylamino)-isoindoline (BDF 6143) failed to contract the longitudinal muscle of the rat stomach fundus; BDF 6143 also failed to induce relaxation of this preparation when it was precontracted with 30 mM KCl. (2) Clonidine, idazoxan, BDF 6143, 1,2-di-(2-tolyl)guanidine, agmatine and (R)-3-(3-hydroxyphenyl)-N-propylpiperidine up to 100 mM did not induce acid secretion from rabbit isolated gastric glands. Our data provide evidence that the rat stomach is endowed with s2 sites and I2 binding sites in addition to the previously identified non-I1/non-I2 [ H]clonidine binding sites. Our experiments also offer basic evidence of the existence of I2 and s binding sites in the human stomach. Neither the I2 and [H]clonidine binding sites nor the s sites in rat stomach are directly related to a postsynaptic effect on gastric smooth muscle or to acid release from isolated gastric glands. Nonadrenoceptor IBS are recognized with high to moderate affinity by imidazolines and related compounds, but not by catecholamines. At least two classes of IBS exist, I1-IBS and I2-IBS, which can be labeled by [ H]clonidine and [H]idazoxan, respectively (for review, see Regunathan and Reis, 1996; Molderings, 1997). Binding experiments with [H]clonidine and [H]idazoxan in membranes from guinea pig (Houi et al., 1987), rabbit (Tesson et al., 1992) and rat gastric tissue (Molderings et al., 1995) provided basic evidence that IBS are also present in the stomach. Interestingly, it has been shown that in the rat stomach, s-receptor ligands exhibited a remarkably high affinity for nonadrenoceptor [H]clonidine binding sites (Molderings et al., 1995). Moreover, s-like sites were recently identified in the porcine gastric mucosa (Harada et al., 1994), but not in rat and human gastric tissue. On the basis of these findings, the first aim of the present study was to identify and characterize [H]idazoxan and [H]DTG binding sites in the rat and human stomach and to investigate whether a relationship exists between nonadrenoceptor [H]clonidine and [H]idazoxan binding sites on the one hand and s binding sites on the other. Therefore, we determined and compared the affinity of key ligands for IBS and s sites in rat stomach membranes labeled with [H]clonidine, [H]idazoxan or [H]DTG (a radioligand for s sites; Weber et al., 1986). In previous in vivo studies, imidazolines such as clonidine exerted a dual action on gastric acid secretion; at low concentrations, these compounds reduced acid secretion (Del Tacca et al., 1982; Bhandare et al., 1991; Blandizzi et al., 1995; Carlisle et al., 1995; Glavin and Smyth, 1995). This inhibitory effect on acid secretion was prevented by alpha-2 adrenoceptor antagonists (Del Tacca et al., 1982; Bhandare et al., 1991; Blandizzi et al., 1995), which suggests that it is mediated mainly by activating presynaptic alpha-2 adrenoceptors on cholinergic nerves innervating the stomach. Additionally, evidence has been presented that peripheral I1-imidazoline receptors might also contribute to the antisecretory and anReceived for publication June 30, 1997. ABBREVIATIONS: U46619, 9,11-dideoxy-11a,9a-epoxy-methano-prostaglandin F2a; BDF 6143, 4-chloro-2-(2-imidazolin-2-ylamino)-isoindoline; (1)-3-PPP, (R)-3-(3-hydroxyphenyl)-N-propylpiperidine; DTG, 1,2-di-(2-tolyl)guanidine; IBS, imidazoline binding sites; CDS, clonidine-displacing substance. 0022-3565/98/2851-0170$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 285, No. 1 Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 285:170–177, 1998 170 at A PE T Jornals on O cber 5, 2017 jpet.asjournals.org D ow nladed from tiulcer effects of imidazoline derivatives (Carlisle et al., 1995; Glavin and Smyth, 1995). At higher concentrations, several imidazolines and agmatine stimulated acid secretion in vitro and in vivo (Medgett and McCulloch, 1979; Del Tacca et al., 1982; Houi et al., 1987; Bhandare et al., 1991; Glavin et al., 1995). The stimulatory effect was not due to activation of alpha-2 adrenoceptors, because it was not mimicked by the alpha-2 adrenoceptor agonist a-methylnoradrenaline and it was not counteracted by yohimbine (Houi et al., 1987). Some have speculated that the stimulatory effect of the imidazolines may be due to activation of imidazoline receptors in stomach tissue (Houi et al., 1987; Bhandare et al., 1991; Glavin et al., 1995). Therefore, the second aim of this study was to investigate whether ligands with high affinity for IBS and/or s sites could induce acid secretion from isolated rabbit gastric glands, which is the standard preparation for investigating acid secretion in vitro. Finally, it was demonstrated that CDS, a putative endogenous ligand at IBS (Regunathan and Reis, 1996) induced a contraction of gastric smooth muscle via an unknown mechanism of action (Felsen et al., 1987). It has been proposed that imidazoline receptors on gastric smooth muscle may be involved. Hence the third aim of the present study was to examine whether other ligands at imidazoline binding sites mimic the effect of CDS and, if so, whether binding of drugs at [H]clonidine, [H]idazoxan and [H]DTG binding sites directly contract rat stomach smooth muscle cells in a vagal nerve-independent manner. Parts of this study have been presented at scientific meetings. Materials and Methods Membrane preparation. Fresh stomachs were obtained from Wistar Kyoto rats immediately after killing. Segments of macroscopically normal human stomach were obtained from male or female patients undergoing gastric surgery. The study was approved in all respects by the local ethics committee. All steps of the preparation procedure were performed on ice. The rat glandular stomach and the segments of the mucosal layer from human stomach were prepared and cut into small fragments that were placed in 40 ml of buffer solution containing sucrose 270 mM, ascorbic acid 0.6 mM and Trissulfate 10 mM (pH 7.4), minced by means of an Ultraturax (five times for 20 s each) and homogenized using a glass-Teflon homogenizer (three times for 30 s each). The homogenates were centrifuged (5 min, 1200 3 g, 4°C). The supernatant was filtered through four layers of gauze, diluted to 420 ml with HEPES buffer (HEPES-Na 5 mM, EGTA 0.1 mM, PMSF 0.3 mM, pH 7.4; buffer I) and recentrifuged (20 min, 40,000 3 g, 4°C). The pellet was washed twice and then resuspended in buffer I, homogenized, diluted to give a protein concentration of about 2 mg/ml and stored at 280°C until use. Before use, the membranes were centrifuged (20 min, 40,000 3 g, 4°C), resuspended in the incubation buffer (HEPES-Na 5 mM, EGTA 0.5 mM, MgCl2 0.5 mM, ascorbic acid 0.1 mM, pH 7.4; buffer II), homogenized by ultrasonics and diluted to a final protein concentration of