Opioid Receptor Reserve in Normal and Morphine-Tolerant Guinea Pig Ileum
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Proc. Natl. Acad. Sci. USA Vol. 81, pp. 7253-7257, November 1984 Pharmacology Opioid receptor reserve in normal and morphine-tolerant guinea pig ileum myenteric plexus (tolerance) CHARLES CHAVKIN* AND AVRAM GOLDSTEINt Addiction Research Foundation and Department of Pharmacology, Stanford University, Palo Alto, CA 94304 Contributed by Avram Goldstein, July 23, 1984 ABSTRACT We have measured the opioid receptor re- selectively tolerant to the 8 agonist [D-Ala2,-D-Leu5]enke- serve in the guinea pig ileum myenteric plexus by means of the phalin; then [Leulenkephalin, which previously had acted on site-directed alkylating agent, 8-chlornaltrexamine. Treat- the 8 receptor, now exerted its full effect (with potency re- ment of the tissue with low (<10 nM) concentrations of /3- duced by a factor of 100) through the As receptor, as indicated chlornaltrexamine caused a parallel shift of the log concentra- by a change in its naloxone sensitivity (see below). The first tion-response curves for both normorphine and dynorphin A- goal of the present study was therefore to determine what (1-13). Analysis of the resulting curves indicated that the Kd types of spare receptors are present in the guinea-pig myen- values were 1.5 ± 0.5 x 10-6 and 10 ± 4 x 10-9, respectively. teric plexus. Using the naloxone Ke to distinguish between the ,u and K re- Our initial demonstration of the presence of spare opioid ceptors in this tissue, we found that the receptor selectivities of receptors in an intact tissue preparation (3) had two impor- normorphine and dynorphin A-(1-13) were unchanged after a tant implications. The first was that the sensitivity of a neu- maximum parallel shift, thus demonstrating that there are ron to an opioid could be controlled by the magnitude of the both spare IL and spare K receptors present. The spare-recep- opioid receptor reserve. This follows from the classical work tor fraction for both receptor types was about 90%. In mor- on spare receptors by Stephenson (4), Ariens et al. (5), and phine-tolerant preparations (chronic pellet implantation), Furchgott (9) showing that the greater the excess of function- there was an apparent reduction in the fraction of spare IL al receptors present, the lower the concentration of agonist receptors without any change in the apparent affinity of nor- required for effect. The second was the suggestion that toler- morphine. Reduction in the spare receptor fraction does not ance to an opioid could be the consequence of a reduced necessarily imply reduction in the number of binding sites. We opioid receptor reserve (10). In binding studies neither affini- suggest that this reduction in receptor reserve is the basis of ty nor site number in the guinea pig ileum myenteric plexus opioid tolerance, since the agonist concentration needed to is affected by chronic morphine administration (11). The ob- produce a given effect is expected to increase as the receptor served reduction in sensitivity of the tolerant tissue to reserve decreases. opioids therefore implies that a higher fractional receptor oc- cupancy is required for a given effect, as would be the direct The irreversible opioid receptor antagonist, 3-chlornaltrexa- consequence of a reduced spare-receptor fraction. The hy- mine (f-CNA) is a site-directed alkylating agent synthesized pothesis that opioid tolerance is associated with a reduction and characterized by Portoghese and coworkers (1, 2). We of receptor reserve can be tested by the ,B3CNA technique. A showed previously (3) that in vitro treatment of the myen- preliminary account of our results was presented at the 1982 teric plexus-longitudinal muscle preparation from guinea pig International Narcotic Research Conference (10); Porreca ileum with low concentrations of 3-CNA results in a parallel and Burks (12) later provided additional experimental sup- shift to the right of the log concentration-response curves port for this idea. for [Leulenkephalin, normorphine, and dynorphin A-(1-13). Higher concentrations of f3CNA result in a reduced maxi- MATERIALS AND METHODS mum response with concomitant decrease in slope. These Guinea pig ileum longitudinal muscle strips were prepared findings provide classical evidence that there are spare from male Hartley guinea pigs (300-450 g) (Simonsen Labo- opioid receptors (4, 5) in the myenteric plexus. ratories, Gilroy, CA) as described (13). Strips (5 cm) in oxy- With spare receptors present, the EC50 in the pharmaco- genated Krebs bicarbonate buffer [composition (mM); NaCl, logic preparation should be lower than the Kd measured by 118; KCl, 4.75; CaCl2, 2.54; KH2PO4, 1.19; MgSO4, 1.20; radioreceptor binding techniques. Yet when Creese and Sny- NaHCO3, 25; glucose, 11; choline chloride, 0.02; pyrilamine der (6) and later Cox et al. (7) compared the Kd and EC50 maleate, 1.25 x 10-4] under 1-g tension were stimulated values of various opioid agonists in the guinea pig ileum maximally (field stimulation, 90 V, 0.1 Hz, 0.5 msec) with preparation, they found good agreement between these two platinum electrodes, and the isometric contractions were re- parameters. Thus, our result was in sharp conflict with corded by a force-displacement transducer (Grass FT 0.03C) theirs. To rationalize the discrepancy, we hypothesized that and a Grass polygraph. Before testing, the muscle strip was the spare receptors we had observed with ,u agonists were of stimulated for 1-2 hr, with repeated changes of the bath solu- the K type and those observed with K agonists were of the ,u tion. Opioid inhibition of the stimulated muscle twitch was type. Thus, with each type of agonist, EC50 and Kd values measured as the ratio of minimum twitch amplitude in the might still agree. On this model, if the preferred receptor for presence of drug to that immediately prior to drug addition. an agonist were inactivated, that agonist would then exert its Muscle contractions were allowed to return to pre-drug am- effects (at higher concentration) through another type of plitude before the next dose was tested. Preparations not in- opioid receptor. Cox and Chavkin (8) showed that this phe- hibited at least 95% by 1 ,M normorphine were discarded. nomenon occurred after the mouse vas deferens was made Abbreviation: -CNA, ,3-chlornaltrexamine. The publication costs of this article were defrayed in part by page charge *Present address: Department of Basic and Clinical Research, Re- payment. This article must therefore be hereby marked "advertisement" search Institute of Scripps Clinic, La Jolla, CA 92037. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 7253 Downloaded by guest on September 28, 2021 7254 Pharmacology: Chavkin and Goldstein Proc. Natl. Acad Sci. USA 81 (1984) Drugs and peptides were diluted in methanol/0.1 M HCl, 1:1 (vol/vol), which, in amounts <25 pl added to the 5-ml tissue bath, had no effect on the amplitude of contraction. To measure the apparent naloxone equilibrium dissocia- tion constant (Ke), the agonist EC50 was first determined with at least two concentrations bracketing 50% inhibition of the twitch amplitude. Then, naloxone (50 nM final concen- tration) was added to the organ bath and, after a 20-min equilibration period, the agonist EC50 was determined. The naloxone Ke is given by c/(dr - 1), where c is the concentra- tion of naloxone (here 50 nM) and dr, the dose ratio, is the ratio of agonist EC50 in the presence of naloxone to that in its absence (14). Schild plots (15) showed that the naloxone Ke is independent of the naloxone concentration used (unpub- lished observations). log concentration (M) P-CNA was dissolved in 0.1 M HCl in ethanol and stored at -70'C. Muscle strips were treated in the organ bath at 370C for 20 min with freshly prepared dilutions of -CNA. For the selective protection experiments, 3 nM P-CNA was used for 20 min in the presence of either 10 ,gM DADLE or 100 nM dynorphin A-(1-13) added 1 min prior to addition of ,B-CNA. These treatment conditions have been shown to confer selective protection on the ,u and K receptors, respec- tively, while allowing f3-CNA to inactivate the unprotected receptor type (3, 16). To prepare morphine-tolerant ilea, guinea pigs were im- planted subcutaneously under ether anesthesia with mor- phine pellets (75 mg of base each), four on day 1 and six on day 3. This method was previously shown to induce mor- phine tolerance (11, 13). Animals were killed on day 6, and ileum longitudinal muscle strips were set up in the organ bath, then allowed to equilibrate for 2 hr before testing. The effect of P-CNA treatment on the agonist dose-response re- lationship was evaluated by the method of Furchgott (9) us- (1/A') X 106 ing the equation 1/[A] = [(1 - q)/qKd] + 1/q[A']. Here [A] and are concentrations of agonist before FIG. 1. Effects of f3-CNA on normorphine log concentration- [A'] equieffective response curves. (a) Effects of different concentrations of 3-CNA and after receptor alkylation. The fraction of receptors re- on normorphine potency in guinea pig ileum. Each point represents maining after ,l3CNA treatment is defined as q; and Kd is the a mean, with the SEM shown as vertical bars. The following num- equilibrium dissociation constant of the agonist. bers of independent determinations were made on separate muscle [D-Ala2,D-Leu5]enkephalin, dynorphin A (heptadecapep- strips: untreated (C), 31; f3CNA at 1 nM, 6; at 3 nM, 6; at 10 nM, 9; tide), and dynorphin A-(1-13) were purchased from Peninsu- at 30 nM, 8. (b) Furchgott analysis of the equieffective normorphine la Laboratories, San Carlos, CA; normorphine hemihydrate, concentrations before (1/A) and after (1/A') 3-CNA treatment.