Characterization of opioid receptors on smooth muscle cells from guinea pig stomach L. ZHANG,

Z. F. GU, T. PRADHAN,

R. T. JENSEN,

AND

P. N. MATON

Digestive Diseases Branch, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892 Zhang, L., 2. F. Gu, T. Pradhan, R. T. Jensen, and P. N. Maton. Characterization of opioid receptors on smooth muscle cells from guinea pig stomach. Am. J. PhysioZ. 262 (Gastrointest. Liver Physiol. 25): G461-G469, 1992.-On the basis of opioid-stimulated contraction of dispersed gastric smooth muscle cells it has been suggestedthat these cells possessopioid receptors of three subtypes: kappa (K), mu (p), and delta (6). We have usedselective peptidase-resistantradioligands, agonists and antagonists, to examine receptor subtypes on dispersedgastric smooth musclecellsfrom guineapigs prepared by collagenasedigestion. The K-agonist U-50488H, the p-agonist [D-Ala2,N-Me-Phe4,G1y5-ollenkephalin(DAGO), and the a-agonist [D-Pen2,Pen5]enkephalin (DPDPE) each caused muscle contraction. The concentrations required to caused half-maximal contraction were U50488H (6 PM) > DAGO (13 PM) > DPDPE (6 nM). The abilities of these agonists to inhibit binding of [3H]U-69593 (K-preferring) by 50% were U50488H (43 nM) > DAGO (43 PM) > DPDPE (200 PM). Their abilities to inhibit binding of [3H]naloxone (ppreferring) by 50% were DAGO (0.2 PM) > U50488H (10 PM) > DPDPE (X00 PM). No binding could be detected with the a-selective ligand [3H]DPDPE. The K-preferring antagonist Mr2266 (10 nM) preferentially inhibited contraction stimulated by the K-agonistU50488H, and naltrexone (10 nM) (p-selective antagonist) preferentially inhibited contraction stimulated by the p-agonist DAGO. ICI 174864(200 PM; a-selectiveantagonist) had no effect on contraction stimulated by p-, K-, or 6agonists.Contraction stimulated by the a-agonistDPDPE was inhibited by both K- and p-receptor antagonists.Studies on the effect of the antagonistson binding of [3H]naloxone and [3H]U69593also provided evidencefor K - and p-sites but not for 6sites. These data demonstratethat gastric smooth musclecells possessK- and p-receptors, occupation of which causesmuscle contraction, but no &receptors could be detected by these methods. agonist; antagonists;peptides; musclecontraction HAVE NUMEROUS complex effects on gut motility. In the whole animal, opioids delay gastric emptying, inhibit gastric contractions, or both and delay whole gut transit (9,10,22,24,32). In tissue preparations receptors for opioids have been found on neurones of the myenteric and submucosal plexuses in the gut, and opioids are thought to act mainly on neurones by inhibiting the release of acetylcholine (26,37). In addition, on the basis of exposing dispersed muscle cells to various opioids and measuring contraction, opioid receptors have been postulated to be present on gastric and intestinal muscle cells (3, 4). Characterization of opioid receptors has proved difficult because of the occurrence of multiple opioid receptor subtypes (5, 23, 35), tissue heterogeneity (12, 24, 25, 30), the susceptibility of opioids to tissue peptidases (27, 28), and, until recently, the lack of selective agonists, antagonists, and high affinity radiolabeled ligands for some receptor subtypes (19). OPIOIDS

In an attempt to overcome these problems we have sought in the present study to identify and characterize opioid receptor subtypes on gastric smooth muscle cells by using highly selective agonists and antagonists. Furthermore, we performed ligand binding studies in whole dispersed muscle cells under the same conditions that the effect of opioids on contraction was measured and not in membranes at 4°C in nonphysiological solutions as has often been done in the past (10, 11, 14, 20, 25,28, 29). We used dispersed smooth muscle cells for all of our studies because they are free of other tissue and neural elements; this allowed us to examine quantitatively the relationship between receptor occupation and biological activity. We used nonpeptide or modified peptide agonists and nonpeptide antagonists to avoid the problems of tissue peptidases. The selective agonists were chosen based on their actions in tissues known to possess only certain opioid receptor subtypes (7,8, 13, 14, 35, 36) and by binding studies, usually to brain membranes (11, 18, 20). Each of the selective agonists used is several hundredfold more potent at one opioid receptor subtype than others. We also used selective antagonists, but the antagonists are less selective than the agonists, and Mr2266 and naloxone have a

Characterization of opioid receptors on smooth muscle cells from guinea pig stomach.

On the basis of opioid-stimulated contraction of dispersed gastric smooth muscle cells it has been suggested that these cells possess opioid receptors...
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