Life Sai ces, vol . 17, P8 . Printed ~ the U.S .A.
633-640
OPIATE RECEPTORS IN MICE :
Pergamon Presa
GENETIC DIFFEßSNCES
Avi Baraa, Louis Shueter, Basil E. Eleftheriou* and Donald W. Bailey* Department of Biochemistry a~ Pharmacology Tufts üniveraity School of Medicine Bonton, Maenachusette 02111 (II.S .A .) and The Jackson Laboratory* Bar Harbor, haine 04609 (D .S .A .) (Received in final form July 23, 1975) SDMKARY The concentration of opiate receptors in the brains of mice was determined by means of a aaloaone-binding assay . The attains of mice used is these ezperimeats were C57BL/6By, BALB/cBy, their reciprocal F1 hybrids, and 7 recombinant-inbred strains derived by inbreeding from the F2 generation . These strains could be divided into 3 groups on the basis of the number of opiate receptors : high (CSBH) ; low (CRBR) ; and intermediate (all the other strains) . The difference in atereoapecific binding of nalozone reflects a difference is the total number of receptor sites rather than in the affinity for the drug . The recombinantinbred strains also differ in their analgesic response to morphine, The differences as previously determined by the tail-flick assay. in the number of opiate receptors are not enough to account for the genetic difference in analgesic respoaeivenese . Both these parameters appear to be under different genetic control, and at least 2 genetic determinants may be involved in regulating the level of opiate receptors . Tereaius (1), Simon et _al . (2) and Pert and Snyder (3) have independently described narcotic receptors in the central nervous system, barred on an assay The affinity of for stereospecific binding introduced by Goldstein et al .(4) . different narcotic drugs for these opiate receptors parallels their pharmacological potency. Binding is greatest in those areas of the brain is which micro-injection of morphine most effectively elicits analgesia, such as different portions of the limbic system (5,6) . However, no correlation has been discerned se yet between the level of narcotic receptors in the braise of different species of animals and their analgesic response to narcotic drugs (7) . The development of tolerance to the analgesic effect of morphine is not accompanied by a matching increase in the number of narcotic receptors (8,9) . Recently, we have used recombinant-inbred strains of mice (10) to investigate the genetic basis for variability of the analgesic response to morphine (11) . An o=A~{++p tioa of the brains of these animals suggests that only a portion of the difference in analgesic response nay be due to a difference in the number of narcotic receptors .
634
Genetic Difference in Opiate Receptors
Vol. 17, No . 4
Methode The recoabiaant-inbred (GI) strains used in these experiments were derived under full-sib mating fros a cross between C57BL/6By and BALB/cBy as described by Bailey (10) . Male mice, 2 to 3 months old, of the progenitor atr~ina, reciprocal Fl hybrids, and the 7 RI strains were tested . The binding of H-nalozone to opiate receptors is brain homogenates was detarsiaed by a modification of the sathod of Pert and Snyder (12) . Brains without the cerebellum were homogenized at 4°C in 6 ml of 0 .05 M Tris-HC1 buffer, pH 7 .4, containing 5 mM NaCl, using 8 strokes of a glaseglaes tissue grinder, drives by a motor . The hooogenata of each brain was di luted to 65-72 ml with the sass buffer solutiçn containing disodium ethylene diamine tetraacetic acid, (Na2-SDTA), 8 a 10 M. The concentration of protein in these homogenates, as determined by the method of Lowry at al . (13), was 0.7 to 0 .8 mg per ml . Baactly 1.76 ml of hosogenata was incubated for 20 siautas at 35 °C together with either 0.8 UM levorphaaol or 0.8 VM deatrorphan, and 7 nM 3 H-nalozone (New $nglaad 1Quclear Corp ., 23 .6 Ci per insole) . All solutions were prepared is 0.05 M Trie-C1, pH 7 .4, containing 5 ~ NaCl and 8 z 10-4 M EDTA . 3H-nalozone gave a single radioactive spot identical to that of unlabeled nalozone when co-chromatographed on silica gel plates vith the solvent system : n-butaaol-glacial acetic acid-H20 4 :2 :1 . The nalozone was added immediately after the levorphanol or dearorphan without preincubation . Samples were cooled and filtered, and the glass fiber-GF/B filters washed as described by Pert a~ Snyder (12) . The filters were dried under as infra-red lamp and counted is 5 sl of toluene scintillation cocktail is a Packard Tricarb scintillation spectrometer . All samples were run in triplicate . Washed particles ware prepared by centrifuging the homogenates, prepared in sodius free 0.05 M Trie-C1 pH 7 .4, at 20,000 g for 10 mix., díecarding the supernatant fluids, washing once with the same buffer sad reconstituting the pellets is the original voluse of 0.05 M Tris-C1, pH 7 .4, containing different concentrations of NaCl . Analgesic response was determined by the tail-flick assay of D'Amour and Smith (14), using a time limit of 8 seconds . The baseline latency of response was between 1 .5 and 2 .5 seconds. The increase is latency was determined at 30, 60, 90 sad 120 minutae after intraperitoaeal injection of 5 sg per kg sorphine-sulfate . The analgesic ire: is the area is minutes-seconds under the curve obtained by plotting increase is latency versus time after sorphine injection (11) . Data were analyzed statistically by a number of one- sad twoway analyses of variance, Student-Nesman-Reins multiple range test, and Tukey's W sensate (15) . Additionally, regression coefficients were estimated for Scatchard plots of the nalozone binding for the various groups . Analyses of variance were conducted on z-azis intercepts a~ slopes of these regression lines . ßesults Naloaone Binding sad_ Anal~a ic Gesponse Table 1 compares nalozoae binding a~ analgesic responsiveness . There was little difference bat~rean either the two progenitor strains or the reciproThe GI strains can be cal Fl hybrids with respect to these two parameters . divided into 3 major statistical groups on the basis of nalozone-binding the other strains) . activity : high (CRBH) ; low (CRBR) ; intermediate For the purpose of binding classification, the Student-Newmn-Rein's (SNR) This is a less conservative teat than some (for multiple range test was used . exasple, Tukey's W estimate, d.f . 154 ; w ~ 123.38) which fail to distinguish
(all
635
Genetic Difference in Opiate Receptors
Vol . 17, No . 4
C%HH in a separate class. However, wa chose to rely oer the SIB test based on additional genetic studies, soon to be published, which indicate that CSBH strata ie genetically different in its receptor binding when compared to the imoediate nezt class. The protein content of the brain without the cerebellum Strata C%BR, which had was approzimtely the seas for the different strains . the lowest binding, also ezhibited the lowest analgesic response . The other 10 strains together exhibited a positive but etatístically not eignificsat corra].~tion between binding sad analgesia (r ~ 0 .48, d .f . ~ 9) . TAHLE 1 Nalozone Binding and Analgesic Response ier the Recombinant Inbred Strains, Their Progenitor Strains (C57BL/6By ; BALB/cBy) and F1 Hybrids of the Progenitor Strains. Bierding Classification
Mouse Strain
Stereospecific Nalozoere Binding (cpm/mg protein + 5.8 .)
High
CSBH
Intermediate
C57BL/6By C%BJ BALB/cBy CEBI CSBE B6CF 1 CB6F1 CSBD CSBG
968 952 948 940 934 923 915 893 844
Low
CSR
609 + 30
Analgesic Besponse (min . z sec + S .E .)
1044 + 33* + + ± + + + + + ±
25 27 26 27 25 22 27 24 21
326 + 22 (12) * * * * * * * * *
655 326 520 512 511 623 613 607 289 *
+ 7 + 38 + 24 + 10 + 25 + 10 + 7 + 12 ± 12
(12) (12) (12) (9) (12) (11) (11) (12) (12)
166 + 37 (12)
Binding of nalozoae to opiate receptors was assayed as described in the tent, using 15 ice of each strain . 1 picomole equals 8740 cps. Class mesas for binding values are reeked from high to low according to Student-NaYmaa-Reins mltiple range test, with overlapping asterisks indicating non-significant differences. The analgesic response is the area in minute-seconds under the curve obtained by plotting increase ier tail-flick latency versus the interval after iertraperitoneal injection of morphine sulfate, 5 mg/kg. The numbers in parentheses refer to the number of mice used for analgesic testing.
The presence of soluble inhibitors or activators of nalozone binding is the brains from different RI strains was ruled out by perforaing several mizing ezperimmte . The particulate fraction of a brass homogenate, obtained by centrifugation at 20,000 g for 10 aiwites, was resuspended with the supernatant solution obtained from either the sage strain or a different strain . !lore than 95Z of the binding activity waa associated with the particulate fraction . The extent of bring was deteroi.aed solely by the genetic origin of the particulate fraction sad was ueraffected by changing the source of the supernatant solution . Sffact of lialoxone Concentration on Hiadiag Tha stereospecific binding of 3H-nalozone was determined as a function of eralozona concentration for brain homogenates free 2 mice of each of the 11
63 6
Genetic Difference
in Opiate Receptors
Vol . 17, No . 4
strains. The results were grouped according to the dívisioa shown in Table 1 - i.e . CXBH, CXBR and the average of the remaining strains (Fig . 1,A) . This figure suggests that the difference in stereospecific binding between the 3 groups reflects a difference is the total number of receptor sites rather than is the affinity for the drug . When these data were used to make a Scatchard plot (Fig . 1,B), it was found that all three groups had the same RD (2 nM) for etereospecific nalozone binding . Analyses of variance for a-azia intercepts and elopes of the 3 regression lines show that the elopes for CSHH (-0.45), C%BR (-0.41) and the remaining strains (-0 .50) are not significantly different from the mean elope for all the strains together (-0 .49) at the 0 .05 level (adj . total d .f . ~ 19, F = 0 .394) . The a-axis intercepts for CSBH (160 .76), CRBZ (97 .37) a~ the mining strains (131 .57) are significantly different at least at the 0.01 level (d .f . ~ 19, F ~ 8 .046) . The maa+~1 stereospecific binding of naloaone for the high, intermediate and low groups was 16 .1, 13 .1 a~ 9 .6 z 10-14 moles per mg protein, respectively . (corresponding to 21, 17 .1 and 12 .3 pmoles per brain) . The number of respective receptor FIG. 1 A. $ffect of Naloaone Concentration on Stereoepecific Binding of 3H-Nalozone to Brain Homogenates from Different Mouse Strains . B . Scatchard Flot of 3H-Naloaone Bioding to Brain Homgenates from Different Mouse Strains.
The concentration of 3H-naloaone was varied between 0 .5 and 10 nlS. The concentration of levorphanol sad deztrorphan was kept at 0.8 u!I thr ghout. All other ezperimental conditions were as described is the test . ~-naloaone binding is ezpressed ae moles per mg protein, and free naloaoae ae moles per liter . o) . c~H (~` ~) ; cnx (e--- n) , au other strains (o
Vol .
63 7
Genetic Difference in Opiate Receptors
17, No . 4
sites calculated fras these results, assuming that each nalozone solecule interacts with one receptor unit, would be 12 .8, 10 .4 and 7 .5 x 10 12 per g of brain tissue (calculated by multiplying soles/g by Avogadro'e nusber) . These values are of the same order [9 to 10 : 10-14 soles dihydromorphina per s8 protaia (16) ; 8-16 picomolee nalozone bound per g brain (17)] as those reported by other workers for the binding of narcotics and narcotic antagonists to rat brain. However, it should be esphasised that this is the first report of statistically significant differences in biadiag for different strains within a given species. Influeaca of NaCl on Binding of 3H-nalosona to Mouse Brain Bosogaaates In view of a recent report that receptor binding of tritisted nalozona is enhanced by sodium ion (17), va tested receptor binding activity in the two progenitor strains a~ strains C%BH and CEB$, in the absence of added sodivv ions and in the presence of various concentrations of sodius chloride up to 150 nti. These ezperiseate were done using eaeasively washed particles. Binding at 6.5, 20, 50 100 sèi NaCl vas increased by a factor of 1.75 over the binding without sodium (Table 2) . Binding at these concentrations of NaCl vas the sass for the 4 strains tested as that found previously with 6 .6 mM sodium (5 mM NaCl + 0.8 n!i Na~-ETA) (Table 1) . Binding at 150 mM NaCl vas decreased slightly cospared to binding at 6.5, 50, or 100 mM NaCl . The main etimm~lation of binding by NaCl was in the range between 0-6 .5 mti. 1.5 mM NaCl produced an increase of 40-60x while 5 mli produced as increase of 50-75Z . Binding is 6 .5 m!I NaCl was equal to binding is 5 sK NaCl, 0.8 Nat-SDTA . These results suggest that the stimulating effect observed with this concentration of SDTA vas saialy a sodium effect . When the data on binding at 50 mM NaCl with different coacentratioae of nalazone were used to sake a Scatchard plot, it was found that RD for all 4 strains vas the ease as at 6 .6 mM Na+ (2 . 0 n!i) . TABLE 2 3H-Naloaone Binding at Different NaCl Concentrations
Strain
(a)
HALB/cBy C57BL/6By CEBH CRBH
(3) (3) (3) (3)
Stereospecific binding, cpm/sg protein + 5.8 . NaCl concentration (sue 100 0 6 .5 50 552 576 354 540
± 43 + 72 + 31 + 32
940 952 639 992
+ 23 + 1 + 5 ± 14
935 934 632 968
+ 31 + 8 + 18 + 15
946 934 613 991
+ + + +
28 57 33 28
All details are as is Table 1, ezcept that binding was assayed in a suspension of washed particles, with different concentrations of NaCl . Discussion The results presented here suggest that there are genetically-controlled differences in the amount of narcotic receptors in the brain. The statistical separation of binding values into 3 classes, together with the observation that both progenitor strains and the reciprocal Fl hybrids were within the same
63 8
Genetic Difference in Opiate Receptors
Vol. 17, No . 4
group, suggests that 2 or mre genetic determinants are involved . Bidelberg et _al . (18) have recently measured the nusbar of opiate receptors in several purebred strains of mice . They found the soma number of receptors in both BA.LB/cJ and C57BL/6J mice . Additional evidence that the difference in the amount of opiate receptors are geantically controlled was obtained by measuring opiate receptor binding in the brains of offspring from crosses of both CSBH and CEBR to each of strains C57BL/6By, BALB/cBy, CSBD and CSBG . It was found that the values for receptor binding were intercediate between those of the parental strains of these crosses. flesulte of these ezperiseats will appear in a subsequent paper dealing with a mre detailed genetic analysis . The number of opiate receptors appears to be only one factor in the analgesic response to narcotic drugs . In the case of the CZBR strain, low binding was accaspanied by a very low analgesic response : This observation alone would suggest a staple relationship between the amount of narcotic raceptore in the brain of an animal a~ its pharmacological response to mrphine . However, other strains did not show such a relationship . C~H mice, with the highest amunt of nalozone binding, did not ezhibit the greatest analgesic response . Considering all the strains tested, the correlation between the amount of narcotic receptors and analgesic response was positive but statistically sot significant . The relationship between narcotic binding and analgesic response depends to some extent on the method that is used to censure analgesia. Oliverio et _al . (19) have used the hot-plate method .to measure the analgesic response of the saue BI strains described here . They found that strain CEBH and BALB/cBy showed the greatest analgesic response, strains C%BR a~ CSBB showed the least response, while all the other strains, including C57BL/6By, were in the intercediate range. The correlation between their results and ours was significantly positive (r ~ 0.77 ; d.f . ~ 9; p < 0 .01), but there were some striking differences . It is possible that the hot-plate assay is affected by the increased rotor activity produced by mrphine . Some of the genetic determinants for the running response to sorphiae seem to differ from those for the analgesic response (11) . There are additional indications that the relationship between the amount of narcotic receptors and the analgesic response is not simple and direct . Preliminary analgesic tests have been carried out with crosses of CRBR to the two progenitor strains . Both C%BR x C57BL/6By a~ C%BR z BALB/cBy mice have approzicately the sane number of narcotic receptors, an amount that is intermediate between the values of the two parental lines. The analgesic response of the offspring of CSBR z C57BL/6Bq is also intercediate between that of CSBR and C57BL/6By. However, the analgesic response of CBBR z BALB/cBy mice is very law, eves lower than that of C%BR mice . Rlee and Streaty (9) have measured opiate receptors in the brains of rats made tolerant to morphine . There was a marked tolerance to the analgesic effect of morphine, but no change in either the number of narcotic receptors or their affinity for dihydrosorphine . We did not find any genetic differences in the affinity of narcotic receptors for nalozane . Appropriate mining experiments have ruled out the presence of soluble activators or inhibitors as a possible explanation for the observed differences in the amunt of receptors . The amunt of Na+ ions used in our assays was foul to produce complete activation, and there were no interstrain differences in responsiveness to Na+. It is of interest that we were unable to discern any activation by Na2EDTA independent of its content of Na+
Vol. 17, No . 4
Genetic Difference in Opiate Receptors
63 9
ion. We found that 6.5 mM Na+ produced complete activation of nalozone binding. Oa the other head, Pert and Snyder (17) have reported that ~_+~~ binding of aalozone (1 nM) to washed rat brain membraaee tine attained at 50 nM Na+. How ever, these authors also showed in the same paper that the increase is binding of 1 .5 nM nalozoae produced by 5 ~ Na+ roe SOx of that achieved at 50 u!i Na+. In a previous paper, Pert and Snyder, using rhole homogenates and a nalozone concentration of 8 nM, reported that the addition of physiological caacentrations of Na+ had no affect on the binding (12) . We used a nalozone coaceatration of 7 nli. The possibility ezi.sts that the ezteat of activation by lw concentrations of Na+ say vary with the concentration of nalozone that ie used for binding assays . The physiological role of narcotic receptors is still nnkaown, although Sharma et al . (20) have recently observed an association betreen the presence of opiate receptors a~ the inhibition of adenyl cyclase by uorphine in cul tured neuroblaetoma-glioua hybrid cells . Collier a~ Roy (21) had previously shown that proataglaadin-stisulated adeayl cylase in rat brain is inhibited by lw concentrations of sorphine . These findings suggest that it may be fruitful to easmine adenyl cyclase in the brains of RI strains of mice that we have been using. Analysis of genetic factors that codify the analgesic response of human patients to narcotic drugs is obviously complicated by the role of psychological reactions to pain and treatment . Hwever, it is of interest to note that in carafullq-conducted double-blind studies about 25x of post-operative patients treated rith 15 mg of morphine sulfate par 70 Rg body weight, did not experience any relief of pain (22) . Acknowledgments This wrk was supported by PHS Grants DA-00022 and DA-00323 fron the National Institute of Drug Abuse, HD05860 from the Institute of Child Health and Devlopuent and GM 15574 from the Division of General Medical Sciences, National Institutes of Health . The Jackson Laboratory and Tufts IIniversíty School of Medicine are fully accredited by the American Association for the Accreditation of Laboratory Animal Care . References 1. 2. 3. 4. 5. 6. 7.
8. 9. 10 . 11 . 12 .
L. TERENIIIS, Acta m,A*,~ ..ol . Tozicol . 32 317-320 (1973) . E . J. SIMON, J. M. HILLER and I . EDELMAN, Proc . Nati . Aced . Sçi . II .S .A . 70 1947-1949 (1973) C . B. PERT and S . H . SNYDSR, Science _179 1011-1014 (1973) . A . GOLDSTEIN, L . I . LOWNEY and B . R. PAL, Proc . Nati . Aced . Sçi . II .S .A . _68 1742-1747 (1971) . J . M. HILLSR, J . PEARSON and E . J. SIMON, Rea . Commua . Chem . Pathol . Pharmacol. 6 1052-1062 (1973) . M. J. RIIHAR, C. B. PERT and 5 . H . SNYDER, Nature 245 447-450 (1973) . C. B . PERT, D. APOSHIAN and S . H . SNYDER, Brain Rea . 75 356-361 (1974) .
C. B . PERT, G. PASTSRNAR and S . H. SNYDER, Science 182 1359-1361 (1973) . W. A. RLES and R. A. STREATY, Nature 248 61-63 (1974) . D . W. BAILEY, Traasvlaaatation 11 325-328 (1971) . L. SHIISTEB, G. YII, G . W. WSBSTERand B . E. ELSFTHSBIOII, PeychoPharmacolo i~a 42 249-254 (1975) . C. B . PERT and S . H . SNYDER, Proc . Nati . Aced . Sçi. II .S .A . 70 2243-2247 (1973) .
640 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 .
Genetic Difference in Opiate Receptors
vol . 17, No . 4
0 . H . LOWRY, N . J . ROSEBROÜGH, A . L . FARR and R . J . RAND_~T~i~ , J . Biol . Chew . _193 265-275 (1951) . F . F . D'AMOUR and D . L . SMITH, J . Phnruacol . ~. Ther . 72 74-79 (1941) . B . J . WINER, Statistical Principles in Eayeriseatal Des ia, pp . 46-104, 140-227, McGra~-Hill, New York (1962) . D . T . WONG and J . S . HORNG, Life Sci . 13 1543-1556 (1973) . C . B . PERT and S . H . SNYDER, _Mol . Pharnacol . 10 868-879 (1974) . E . EIDELBERG, R . ERSPAMER, C . J . RAEIIiICR and J . HARRIS, Personal Co~uaication . A . OLIVERIO, C . CA,ç~rer .Taxn a~ B . E . ~_EF7~T OII, PsychopharmacoloAia in press . S . R . SHABMA, M . NIRSNBERG and W . A . KLEE, Proc . Natl . Aced . Sçi . U .S .A . 7 2 590-594 (1975) . H. 0 . J . COLLIER and A . C . ROY, Nature 248 24-27 (1974) . L . T .açeraas and H . R . BSECHSR, J . Axer . Med . Ass . 15 6 230-234 (1954) .
633-640
OPIATE RECEPTORS IN MICE :
Pergamon Presa
GENETIC DIFFEßSNCES
Avi Baraa, Louis Shueter, Basil E. Eleftheriou* and Donald W. Bailey* Department of Biochemistry a~ Pharmacology Tufts üniveraity School of Medicine Bonton, Maenachusette 02111 (II.S .A .) and The Jackson Laboratory* Bar Harbor, haine 04609 (D .S .A .) (Received in final form July 23, 1975) SDMKARY The concentration of opiate receptors in the brains of mice was determined by means of a aaloaone-binding assay . The attains of mice used is these ezperimeats were C57BL/6By, BALB/cBy, their reciprocal F1 hybrids, and 7 recombinant-inbred strains derived by inbreeding from the F2 generation . These strains could be divided into 3 groups on the basis of the number of opiate receptors : high (CSBH) ; low (CRBR) ; and intermediate (all the other strains) . The difference in atereoapecific binding of nalozone reflects a difference is the total number of receptor sites rather than in the affinity for the drug . The recombinantinbred strains also differ in their analgesic response to morphine, The differences as previously determined by the tail-flick assay. in the number of opiate receptors are not enough to account for the genetic difference in analgesic respoaeivenese . Both these parameters appear to be under different genetic control, and at least 2 genetic determinants may be involved in regulating the level of opiate receptors . Tereaius (1), Simon et _al . (2) and Pert and Snyder (3) have independently described narcotic receptors in the central nervous system, barred on an assay The affinity of for stereospecific binding introduced by Goldstein et al .(4) . different narcotic drugs for these opiate receptors parallels their pharmacological potency. Binding is greatest in those areas of the brain is which micro-injection of morphine most effectively elicits analgesia, such as different portions of the limbic system (5,6) . However, no correlation has been discerned se yet between the level of narcotic receptors in the braise of different species of animals and their analgesic response to narcotic drugs (7) . The development of tolerance to the analgesic effect of morphine is not accompanied by a matching increase in the number of narcotic receptors (8,9) . Recently, we have used recombinant-inbred strains of mice (10) to investigate the genetic basis for variability of the analgesic response to morphine (11) . An o=A~{++p tioa of the brains of these animals suggests that only a portion of the difference in analgesic response nay be due to a difference in the number of narcotic receptors .
634
Genetic Difference in Opiate Receptors
Vol. 17, No . 4
Methode The recoabiaant-inbred (GI) strains used in these experiments were derived under full-sib mating fros a cross between C57BL/6By and BALB/cBy as described by Bailey (10) . Male mice, 2 to 3 months old, of the progenitor atr~ina, reciprocal Fl hybrids, and the 7 RI strains were tested . The binding of H-nalozone to opiate receptors is brain homogenates was detarsiaed by a modification of the sathod of Pert and Snyder (12) . Brains without the cerebellum were homogenized at 4°C in 6 ml of 0 .05 M Tris-HC1 buffer, pH 7 .4, containing 5 mM NaCl, using 8 strokes of a glaseglaes tissue grinder, drives by a motor . The hooogenata of each brain was di luted to 65-72 ml with the sass buffer solutiçn containing disodium ethylene diamine tetraacetic acid, (Na2-SDTA), 8 a 10 M. The concentration of protein in these homogenates, as determined by the method of Lowry at al . (13), was 0.7 to 0 .8 mg per ml . Baactly 1.76 ml of hosogenata was incubated for 20 siautas at 35 °C together with either 0.8 UM levorphaaol or 0.8 VM deatrorphan, and 7 nM 3 H-nalozone (New $nglaad 1Quclear Corp ., 23 .6 Ci per insole) . All solutions were prepared is 0.05 M Trie-C1, pH 7 .4, containing 5 ~ NaCl and 8 z 10-4 M EDTA . 3H-nalozone gave a single radioactive spot identical to that of unlabeled nalozone when co-chromatographed on silica gel plates vith the solvent system : n-butaaol-glacial acetic acid-H20 4 :2 :1 . The nalozone was added immediately after the levorphanol or dearorphan without preincubation . Samples were cooled and filtered, and the glass fiber-GF/B filters washed as described by Pert a~ Snyder (12) . The filters were dried under as infra-red lamp and counted is 5 sl of toluene scintillation cocktail is a Packard Tricarb scintillation spectrometer . All samples were run in triplicate . Washed particles ware prepared by centrifuging the homogenates, prepared in sodius free 0.05 M Trie-C1 pH 7 .4, at 20,000 g for 10 mix., díecarding the supernatant fluids, washing once with the same buffer sad reconstituting the pellets is the original voluse of 0.05 M Tris-C1, pH 7 .4, containing different concentrations of NaCl . Analgesic response was determined by the tail-flick assay of D'Amour and Smith (14), using a time limit of 8 seconds . The baseline latency of response was between 1 .5 and 2 .5 seconds. The increase is latency was determined at 30, 60, 90 sad 120 minutae after intraperitoaeal injection of 5 sg per kg sorphine-sulfate . The analgesic ire: is the area is minutes-seconds under the curve obtained by plotting increase is latency versus time after sorphine injection (11) . Data were analyzed statistically by a number of one- sad twoway analyses of variance, Student-Nesman-Reins multiple range test, and Tukey's W sensate (15) . Additionally, regression coefficients were estimated for Scatchard plots of the nalozone binding for the various groups . Analyses of variance were conducted on z-azis intercepts a~ slopes of these regression lines . ßesults Naloaone Binding sad_ Anal~a ic Gesponse Table 1 compares nalozoae binding a~ analgesic responsiveness . There was little difference bat~rean either the two progenitor strains or the reciproThe GI strains can be cal Fl hybrids with respect to these two parameters . divided into 3 major statistical groups on the basis of nalozone-binding the other strains) . activity : high (CRBH) ; low (CRBR) ; intermediate For the purpose of binding classification, the Student-Newmn-Rein's (SNR) This is a less conservative teat than some (for multiple range test was used . exasple, Tukey's W estimate, d.f . 154 ; w ~ 123.38) which fail to distinguish
(all
635
Genetic Difference in Opiate Receptors
Vol . 17, No . 4
C%HH in a separate class. However, wa chose to rely oer the SIB test based on additional genetic studies, soon to be published, which indicate that CSBH strata ie genetically different in its receptor binding when compared to the imoediate nezt class. The protein content of the brain without the cerebellum Strata C%BR, which had was approzimtely the seas for the different strains . the lowest binding, also ezhibited the lowest analgesic response . The other 10 strains together exhibited a positive but etatístically not eignificsat corra].~tion between binding sad analgesia (r ~ 0 .48, d .f . ~ 9) . TAHLE 1 Nalozone Binding and Analgesic Response ier the Recombinant Inbred Strains, Their Progenitor Strains (C57BL/6By ; BALB/cBy) and F1 Hybrids of the Progenitor Strains. Bierding Classification
Mouse Strain
Stereospecific Nalozoere Binding (cpm/mg protein + 5.8 .)
High
CSBH
Intermediate
C57BL/6By C%BJ BALB/cBy CEBI CSBE B6CF 1 CB6F1 CSBD CSBG
968 952 948 940 934 923 915 893 844
Low
CSR
609 + 30
Analgesic Besponse (min . z sec + S .E .)
1044 + 33* + + ± + + + + + ±
25 27 26 27 25 22 27 24 21
326 + 22 (12) * * * * * * * * *
655 326 520 512 511 623 613 607 289 *
+ 7 + 38 + 24 + 10 + 25 + 10 + 7 + 12 ± 12
(12) (12) (12) (9) (12) (11) (11) (12) (12)
166 + 37 (12)
Binding of nalozoae to opiate receptors was assayed as described in the tent, using 15 ice of each strain . 1 picomole equals 8740 cps. Class mesas for binding values are reeked from high to low according to Student-NaYmaa-Reins mltiple range test, with overlapping asterisks indicating non-significant differences. The analgesic response is the area in minute-seconds under the curve obtained by plotting increase ier tail-flick latency versus the interval after iertraperitoneal injection of morphine sulfate, 5 mg/kg. The numbers in parentheses refer to the number of mice used for analgesic testing.
The presence of soluble inhibitors or activators of nalozone binding is the brains from different RI strains was ruled out by perforaing several mizing ezperimmte . The particulate fraction of a brass homogenate, obtained by centrifugation at 20,000 g for 10 aiwites, was resuspended with the supernatant solution obtained from either the sage strain or a different strain . !lore than 95Z of the binding activity waa associated with the particulate fraction . The extent of bring was deteroi.aed solely by the genetic origin of the particulate fraction sad was ueraffected by changing the source of the supernatant solution . Sffact of lialoxone Concentration on Hiadiag Tha stereospecific binding of 3H-nalozone was determined as a function of eralozona concentration for brain homogenates free 2 mice of each of the 11
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strains. The results were grouped according to the dívisioa shown in Table 1 - i.e . CXBH, CXBR and the average of the remaining strains (Fig . 1,A) . This figure suggests that the difference in stereospecific binding between the 3 groups reflects a difference is the total number of receptor sites rather than is the affinity for the drug . When these data were used to make a Scatchard plot (Fig . 1,B), it was found that all three groups had the same RD (2 nM) for etereospecific nalozone binding . Analyses of variance for a-azia intercepts and elopes of the 3 regression lines show that the elopes for CSHH (-0.45), C%BR (-0.41) and the remaining strains (-0 .50) are not significantly different from the mean elope for all the strains together (-0 .49) at the 0 .05 level (adj . total d .f . ~ 19, F = 0 .394) . The a-axis intercepts for CSBH (160 .76), CRBZ (97 .37) a~ the mining strains (131 .57) are significantly different at least at the 0.01 level (d .f . ~ 19, F ~ 8 .046) . The maa+~1 stereospecific binding of naloaone for the high, intermediate and low groups was 16 .1, 13 .1 a~ 9 .6 z 10-14 moles per mg protein, respectively . (corresponding to 21, 17 .1 and 12 .3 pmoles per brain) . The number of respective receptor FIG. 1 A. $ffect of Naloaone Concentration on Stereoepecific Binding of 3H-Nalozone to Brain Homogenates from Different Mouse Strains . B . Scatchard Flot of 3H-Naloaone Bioding to Brain Homgenates from Different Mouse Strains.
The concentration of 3H-naloaone was varied between 0 .5 and 10 nlS. The concentration of levorphanol sad deztrorphan was kept at 0.8 u!I thr ghout. All other ezperimental conditions were as described is the test . ~-naloaone binding is ezpressed ae moles per mg protein, and free naloaoae ae moles per liter . o) . c~H (~` ~) ; cnx (e--- n) , au other strains (o
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sites calculated fras these results, assuming that each nalozone solecule interacts with one receptor unit, would be 12 .8, 10 .4 and 7 .5 x 10 12 per g of brain tissue (calculated by multiplying soles/g by Avogadro'e nusber) . These values are of the same order [9 to 10 : 10-14 soles dihydromorphina per s8 protaia (16) ; 8-16 picomolee nalozone bound per g brain (17)] as those reported by other workers for the binding of narcotics and narcotic antagonists to rat brain. However, it should be esphasised that this is the first report of statistically significant differences in biadiag for different strains within a given species. Influeaca of NaCl on Binding of 3H-nalosona to Mouse Brain Bosogaaates In view of a recent report that receptor binding of tritisted nalozona is enhanced by sodium ion (17), va tested receptor binding activity in the two progenitor strains a~ strains C%BH and CEB$, in the absence of added sodivv ions and in the presence of various concentrations of sodius chloride up to 150 nti. These ezperiseate were done using eaeasively washed particles. Binding at 6.5, 20, 50 100 sèi NaCl vas increased by a factor of 1.75 over the binding without sodium (Table 2) . Binding at these concentrations of NaCl vas the sass for the 4 strains tested as that found previously with 6 .6 mM sodium (5 mM NaCl + 0.8 n!i Na~-ETA) (Table 1) . Binding at 150 mM NaCl vas decreased slightly cospared to binding at 6.5, 50, or 100 mM NaCl . The main etimm~lation of binding by NaCl was in the range between 0-6 .5 mti. 1.5 mM NaCl produced an increase of 40-60x while 5 mli produced as increase of 50-75Z . Binding is 6 .5 m!I NaCl was equal to binding is 5 sK NaCl, 0.8 Nat-SDTA . These results suggest that the stimulating effect observed with this concentration of SDTA vas saialy a sodium effect . When the data on binding at 50 mM NaCl with different coacentratioae of nalazone were used to sake a Scatchard plot, it was found that RD for all 4 strains vas the ease as at 6 .6 mM Na+ (2 . 0 n!i) . TABLE 2 3H-Naloaone Binding at Different NaCl Concentrations
Strain
(a)
HALB/cBy C57BL/6By CEBH CRBH
(3) (3) (3) (3)
Stereospecific binding, cpm/sg protein + 5.8 . NaCl concentration (sue 100 0 6 .5 50 552 576 354 540
± 43 + 72 + 31 + 32
940 952 639 992
+ 23 + 1 + 5 ± 14
935 934 632 968
+ 31 + 8 + 18 + 15
946 934 613 991
+ + + +
28 57 33 28
All details are as is Table 1, ezcept that binding was assayed in a suspension of washed particles, with different concentrations of NaCl . Discussion The results presented here suggest that there are genetically-controlled differences in the amount of narcotic receptors in the brain. The statistical separation of binding values into 3 classes, together with the observation that both progenitor strains and the reciprocal Fl hybrids were within the same
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group, suggests that 2 or mre genetic determinants are involved . Bidelberg et _al . (18) have recently measured the nusbar of opiate receptors in several purebred strains of mice . They found the soma number of receptors in both BA.LB/cJ and C57BL/6J mice . Additional evidence that the difference in the amount of opiate receptors are geantically controlled was obtained by measuring opiate receptor binding in the brains of offspring from crosses of both CSBH and CEBR to each of strains C57BL/6By, BALB/cBy, CSBD and CSBG . It was found that the values for receptor binding were intercediate between those of the parental strains of these crosses. flesulte of these ezperiseats will appear in a subsequent paper dealing with a mre detailed genetic analysis . The number of opiate receptors appears to be only one factor in the analgesic response to narcotic drugs . In the case of the CZBR strain, low binding was accaspanied by a very low analgesic response : This observation alone would suggest a staple relationship between the amount of narcotic raceptore in the brain of an animal a~ its pharmacological response to mrphine . However, other strains did not show such a relationship . C~H mice, with the highest amunt of nalozone binding, did not ezhibit the greatest analgesic response . Considering all the strains tested, the correlation between the amount of narcotic receptors and analgesic response was positive but statistically sot significant . The relationship between narcotic binding and analgesic response depends to some extent on the method that is used to censure analgesia. Oliverio et _al . (19) have used the hot-plate method .to measure the analgesic response of the saue BI strains described here . They found that strain CEBH and BALB/cBy showed the greatest analgesic response, strains C%BR a~ CSBB showed the least response, while all the other strains, including C57BL/6By, were in the intercediate range. The correlation between their results and ours was significantly positive (r ~ 0.77 ; d.f . ~ 9; p < 0 .01), but there were some striking differences . It is possible that the hot-plate assay is affected by the increased rotor activity produced by mrphine . Some of the genetic determinants for the running response to sorphiae seem to differ from those for the analgesic response (11) . There are additional indications that the relationship between the amount of narcotic receptors and the analgesic response is not simple and direct . Preliminary analgesic tests have been carried out with crosses of CRBR to the two progenitor strains . Both C%BR x C57BL/6By a~ C%BR z BALB/cBy mice have approzicately the sane number of narcotic receptors, an amount that is intermediate between the values of the two parental lines. The analgesic response of the offspring of CSBR z C57BL/6Bq is also intercediate between that of CSBR and C57BL/6By. However, the analgesic response of CBBR z BALB/cBy mice is very law, eves lower than that of C%BR mice . Rlee and Streaty (9) have measured opiate receptors in the brains of rats made tolerant to morphine . There was a marked tolerance to the analgesic effect of morphine, but no change in either the number of narcotic receptors or their affinity for dihydrosorphine . We did not find any genetic differences in the affinity of narcotic receptors for nalozane . Appropriate mining experiments have ruled out the presence of soluble activators or inhibitors as a possible explanation for the observed differences in the amunt of receptors . The amunt of Na+ ions used in our assays was foul to produce complete activation, and there were no interstrain differences in responsiveness to Na+. It is of interest that we were unable to discern any activation by Na2EDTA independent of its content of Na+
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Genetic Difference in Opiate Receptors
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ion. We found that 6.5 mM Na+ produced complete activation of nalozone binding. Oa the other head, Pert and Snyder (17) have reported that ~_+~~ binding of aalozone (1 nM) to washed rat brain membraaee tine attained at 50 nM Na+. How ever, these authors also showed in the same paper that the increase is binding of 1 .5 nM nalozoae produced by 5 ~ Na+ roe SOx of that achieved at 50 u!i Na+. In a previous paper, Pert and Snyder, using rhole homogenates and a nalozone concentration of 8 nM, reported that the addition of physiological caacentrations of Na+ had no affect on the binding (12) . We used a nalozone coaceatration of 7 nli. The possibility ezi.sts that the ezteat of activation by lw concentrations of Na+ say vary with the concentration of nalozone that ie used for binding assays . The physiological role of narcotic receptors is still nnkaown, although Sharma et al . (20) have recently observed an association betreen the presence of opiate receptors a~ the inhibition of adenyl cyclase by uorphine in cul tured neuroblaetoma-glioua hybrid cells . Collier a~ Roy (21) had previously shown that proataglaadin-stisulated adeayl cylase in rat brain is inhibited by lw concentrations of sorphine . These findings suggest that it may be fruitful to easmine adenyl cyclase in the brains of RI strains of mice that we have been using. Analysis of genetic factors that codify the analgesic response of human patients to narcotic drugs is obviously complicated by the role of psychological reactions to pain and treatment . Hwever, it is of interest to note that in carafullq-conducted double-blind studies about 25x of post-operative patients treated rith 15 mg of morphine sulfate par 70 Rg body weight, did not experience any relief of pain (22) . Acknowledgments This wrk was supported by PHS Grants DA-00022 and DA-00323 fron the National Institute of Drug Abuse, HD05860 from the Institute of Child Health and Devlopuent and GM 15574 from the Division of General Medical Sciences, National Institutes of Health . The Jackson Laboratory and Tufts IIniversíty School of Medicine are fully accredited by the American Association for the Accreditation of Laboratory Animal Care . References 1. 2. 3. 4. 5. 6. 7.
8. 9. 10 . 11 . 12 .
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