Life Sciences,Vol. 42, pp. Printed in the U.S.A.
Pergamon Press
2217-2222
EVIDENCE FOR A NON-OPIOID SIGMA BINDING SITE IN THE GUINEAPIG MYENTERIC PLEXUS
Francois
Roman, Xavier Pascaud, Daniel VauchC and Jean-Louis Junien
JOUVEINAL Laboratoires - I rue des Moissons 94263 Fresnes Cedex - FRANCE (Received
in final
form March 28, 1988)
Summary
The presence of a binding site to (+)-(3H)SKF 10,047 was demonstrated in a guinea-pig myenteric plexus (MYP) membrane preparation. Specific binding to this receptor was saturable, reversible, linear with protein concentration and consisted of two components, a high affinity site (KD = 46 + 5 nM ; Bmax = 3.4 + 0.5 pmole/g wet weight) and a low affinity site (KD= = 342 ?r 72 nM ; Bmax = 22 + 3 pmole/g wet weight). Morphine and naloxone lo-4M were unable to displace (+I-(3H)SKF 10,047 binding. Haloperidol, imipramine, ethylketocyclazocine and propranolol were among the most potent compounds to inhibit this specific binding. These results suggest the presence of a non-opioid haloperidol sensitive sigma receptor in the MYP of the guinea-pig. Martin et al. (1976) have first hypothesized a sigma receptor subtype based on the unique effects of the benzomorphan SKF 10,047 in the chronic spinal dog, including mydriasis, tachypnea, tachycardia and mania. Among the pharmacological effects of SKF 10,047 and cyclazocine, some of them are not blocked by opioid antagonists e.g. naloxone or naltrexone (Iwamoto, 1981 ; Vaupel , 1983 ; Young and Khazan, 1984) suggesting that these drugs interact at sites other than the classical mu, delta or kappa receptors. Furthermore, on the basis of in vitro harmacological and autoradiographic studies Largent et al. (1986) have shown that (+)_(gH)SKF 10,047 labels two sites in rat and guinea-pig brain : a high affinity site stereo-selective for the (+)-isomer SKF 10,047 which shows the characteristics of the putative sigma receptor sites labeled by (3H)(3-(3hydroxyphenyl)-N-(1-propyl)piperidine (fPPP) and a low affinity site identified as the phencycline (PCP) site with (3H)l-(2-thienyl)cyclohexylpiperidine (TCP). Little is known about the presence and the role of such sigma receptors in peripheral tissues. No evidence for the presence of sigma receptors in the gastro-intestinal tract using ligand-receptor interaction has been published so far. The present report was designed in view of characterizing the binding site for I+)-(3H)SKF 10,047 in a guinea-pig myenteric plexus (MYP) membrane preparation.
Materials
and Methods
Membrane preparation : Male guinea-pigs (tricoloured, 350-400 g, COB LABO, FRANCE) were sacrificed by a blow on the head and bled. Small intestine was removed and transfered in a Krebs-Tris solution (NaCl 118 mM, KC1 4.75 mM, CaC12 2.54 mM, MgS04 1.2 mM,Tris-HCl 25 mM) pH 7.4 maintained at 37°C. The longitudinal muscle with 0024-3205188 $3.00 + .OO Copyright (c) 1988 Pergamon Press
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attached myenteric plexus was removed by the method of Ambache as described by Rang (1964) and placed in ice-cold Tris-HCI 50 mM pH 7.4 buffer. Weighed muscle strips were minced and then homogeneized using a glass homogeneizer with a teflon pestle at 1,000 rpm. The homogenate was centrifuged at 1,000 g for 10 min ; the supernatant was saved and the pellet was resuspended in the same volume of Tris-HCI 50 mM buffer and was pooled with the previous one centrifuged again at 1,000 g for 10 min ; the supernatant and centrifuged at 49,000 g for 10 min. The supernatant was discarded and the pellet was resuspended in Tris-HCI 50 mM buffer and centrifuged again at 49,000 g for 10 min. The pellet was resuspended in Tris-HCl 50 mM in such a volume that the protein concentration was between 1 and 2 mg/ml and was stored at -20°C until assay. Binding assay : Membrane suspension was thawed at 4”C, centrifuged at 49,000 g for 10 min and resuspended in the same volume of Tris-HCl 5 mM pH 7.4. 240 pl fractions were incubated at 25°C in a total volume of 300 I containing (+I-(3HlSKF 10,047 France). Thkl Incubation . was terminated by (30.3 Ci/mmole New England Nuclear, filtration under reduced pressure through Whatman glass-fiber circles (GF/B) that had been soaked for at least 4 h in a solution of Tris-HCl 5 mM containing polyethyleneimine 0.1 %. Filters were washed 2 times with 5 ml of cold Tris buffer and placed in microvials with 5 ml “Scintillator 299” (Packard) added. Radioactivity was determined by scintillation counting in a PACKARD 4000 spectrometer. Non specific binding was obtained with haloperidol 10 -6M or PCP lo-4M as indicated in the results section. Data analysis : Saturation curves were analysed using computer assisted fitting program “LIGAND” described by Munson et al. (19801. For analysis curves the program “EBDA” (MC Pherson, 1983) was used.
iterative curve of competition
Results When guinea-pig MYP membranes were incubated with increasing concentrations of (+)-(3HlSKF 10,047, specific binding defined as that portion of binding inhibited by 1x10-4M PCP represented 70% of total binding and showed saturability. Two components of binding were readily apparent (Fig. 1). Analysis of saturation data by the Scatchard plot revealed two affinity components and this result was confirmed by the LIGAND program. The high affinity component had a KD of 46 + 5 nM and a Bmax of 3.4 + 0.5 pmol/g wet weight ; the low affinity site had a KD of 342 + 72 nM and a Bmax of 22 + 3 pmol/g wet weight fn = 6). This specific binding was linear with protein concentration up to 2 mg/ml (Fig. 1). Equilibrium was reached within 30 to 40 minutes (Fig. 2). The amount bound was stable up to at least 2 h. When 1x10-6M haloperidol was added to the medium, dissociation occured and was completed in 15 to 20 min. From the lines shown in Fig. 2A and Fig. 2B it is possible to calculate the second order rate constant Kl = (Kobs-K21/T(+)-(3HlSK~ 10,047 where Kobs is the observed rate constant for the reversible first order reaction of association, K2 is the rate constant for the reverse reaction. The ratio K2/Kl = 27.9 nM is a kinetically derived estimate of the KD for the reaction of (+I-(3HlSKF 10,047 with its binding site. This value is comparable to the KD (46 nM) determined by saturation studies. The affinities of several compounds for the f+)-(3HlSKF 10,047 receptor were evaluated by competitive displacement. The concentration of f+)-(3H)SKF 10,047 used was 7 nM in order to investigate the high affinity component of the binding. The results
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loo (+I[
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i?. 4 Protein concentration (mglml )
3H] SKF 10,047 CONCENTRATION (rhl )
Figure 1
Saturation isotherm of specific (+I-(3H)SKF 10,047 binding to guineapig myenteric plexus (A). Membranes were incubated for 60 min at 25OC with increasing concentrations (2-500 nM) of f+)-(3H)SKF 10,047. Non specific binding was obtained in the presence of 1x10-4M PCP. The assay was determined as described under Materials and Methods section. Results are from a typical experiment and values are the average of triplicate determinations. B : Scatchard analysis of the binding data. C : Dependence of binding on protein concentration.
(Table 1) show that among the compounds tested, haloperidol UC50 = 14.4 + 4.3 nM) was the most potent. Several other compounds were able to display such a displacement with IC5O’s ranging between IO’6M and lo-7M. Their rank potency order was imipramine > ethylketocyclazocine > chlorpromazine = propranolol. Phencyclidine had a lower affinity whereas the opiate naloxone and morphine were only weak competitors at concentrations as high as 10-4 M.
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X
10-s M
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DISSOCIATION
9
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100
140
TIME
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Figure 2
Dependence of binding of (+I-(3H)SKF 10,047 on incubation time (A). (+I-(3H)SKF 10,047 (3 nM) was incubated with guinea-pig MYP membranes for the indicated times at 25°C and total binding was determined as described under “Materials and Methods”. At t = 90 min a large excess of (1x10-6M) was added. B: First order kinetic plot of halo eridol (+)-( e,H)SKF 10,047 specific binding after correction of total binding by non specific binding determined in presence of haloperidol (1x10-6M). C : First order rate plot of the dissociation of the receptor-ligand complex. Discussion
The present study demonstrates that (+)-(3H)SKF 10,047 labels two sites in guineapig myenteric plexus with dissociation constants very close to that reported in brain by Largent et al. (1986) : KD = 42 vs 46 nM for the high affinity site, and 615 vs 342 nM for the low affinity site in brain and myenteric plexus respectively. The fact that the binding of (+)-(3H)SKF 10,047 is dependent upon protein concentration and that dissociation occurs when an excess of haloperidol is added to the incubation medium are strong
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TABLE 1 IC50 values for drugs competing against (+)-(3H)SKF 10,047 for sigma binding sites.
Compound
IC5OW
Haloperidol Imipramine Chlorpromazine Propranolol Ethylketocyclazocine Phencyclidine Naloxone Morphine
1.44 + 0.43 5.73 + 1.71 1.34 + 0.34 1.41 + 0.44 7.46 + 2.82 ;.9”,;277 >
10-4
n x x x x x x
10-8 10-7 10-6 10-6 10-7 10-6
(5) (4) (5) I:; :f; (2)
Guinea-pig MYP membranes were incubated 60 min at 25°C with increasing concentrations of unlabeled ligands and (+)_(3H)SKF 10,047 (7 nM). Non specific binding was determined with PCP 1x10-4M. Values are the mean + SEM for the number (n) of determinations. arguments for rejecting the possibility of an artefactual result. It is also noteworthy that in both preparations the best affinity was shown with haloperidol, while a total lack of activity was recorded with morphine and naloxone. Furthermore compounds as different as ethylketocyclazocine, imipramine, chlorpromazine or propranolol were active in our preparation. Su (1982) has obtained similar results in brain preparations using racemic SKF 10,047. Another important finding is that the low affinity component for the binding of I+)-(3H)SKF 10,047which was assumed to be due to specific PCP receptor site in the brain is also present in the MYP. Nevertheless PCP appeared to be much less active in the MYP than it was described in the brain (Largent et al. 1986). From our data it is not possible to conclude to a quantitative difference between binding parameters in the brain and in the MYP and we need further characterization of both high and low affinity components to compare (+)-(3H)SKF 10,047binding sites in both tissues. The only result reported so far in the literature using binding assay with peripheral tissue and suggesting the presence of such sigma/PCP rece tors is that of Samovilova et al. (1985) with showed a competition between PCP and (+)-( 3 H)SKF 10,047 in rat liver membranes. In this report the binding characteristics obtained with (+)-(3H)SKF 10,047 in such a preparation were close to that described in the brain. Interestingly enough in such a preparation affinity described for PCP (IC50 = 1.3x10-6M) is very close to that we have observed in the MYP preparation . To our knowledge, no other (+)-(3H)SKF 10,047 binding assay using peripheral tissue has been published so far. Finally the results reported herein give direct evidence for the presence of (+)-(3H)SKF 10,047 specific binding sites in guinea-pig MYP. Such a presence had already been hypothesized in guinea-pig ileum by Su et al. (1981) and Kromer et al. (1982) using isolated organ experiments. A functional role of these receptors in the modulation of intestinal motility was suggested from the blocking effect of SKF 10,047 on kappa and on a lesser extent on mu opioid agonists and from the inhibition of acetylcholine release induced by electrical stimulation of the MYP longitudinal muscle preparation. However, in these in vitro experiments, the racemic SKF 10,047 was used and it has been shown by several authors (Pert et al., 1976 ; Aceto and May, 1983) that (-)-(3H)SKF 10,047 interacts with naloxone sensitive opiate sites. As a consequence, it is very difficult to make the difference between opioid and sigma effects when using the racemic SKF 10,047 as a pharmacological probe. In connection with these few in vitro experiments, the demonstration of the presence of a binding site for (+)-(3H)SKF 10,047 in
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the guinea-pig MYP reported here raises the possibility of the physiological importance of sigma “haloperidol-sensitive” receptors in this organ and suggest a putative role of these receptors at the gastro-intestinal tract level.
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