Life Sciences Vol . 16, pp, Printed in the 17 .5 .!1 .
1875-1876
Pergamon Press
EFFECTS OF PROâTAßlA18flINS AND AAORPHINE ON BRAIN ADENYLYL CYCLASE" RA . Van ImwSwr, SJ. Strede, ard ß .A. Rabbon Deprtersnt of Plrrnrodooy, TM Unkrsnky of Taxe Medial School at Hosebn, Hornfon, Tors 7702b (Received in final form May 24,
1975)
Data implksrtirrp Inwlvwnerrt of cydk: nudeotkles M the pharrrrscology of narPhin b oompranhsd by corrtradk:taY data M tire Ifbntun . Far azampla, /n dtro morphine sulfata (AAS) ttirrwlafsd (1) and had no effaà (2) on bwl adanylyl cydar IACI in nt stratum, and inhibited AC of wvrel bNn cress (31 . Aab MS hijsctbn krawrd AC of rat stratum (1) end moues a+rebrai cortex (41, had no effect on AC of mouse rd nt oanlxal coron, cerebellum end hypotFralamw (4-8) and decreapd AC of moues bolo sbm (41 . Doprrrhr IDAI and sodium 1luafde IWFI stimulated AC of rat striatum wore rzrffectad by MS !n v/bo (1, 2, 7) or by sate k~sctbrr (1, 81, but carts injections rFrensd DA smilles AC 171 . Cydk: AMP !such irrcrenad in PIWiferY and sUbtum Iel, decnned In hypotlralarnw (3) a dkl not charge M cerebral axteor, anbellum or hypothalamus (8) sifter acufs hIS k>]actlon . Chronic MS adminhtration dsusard (4) and Incrwd 161 AC In moor cobol telex, but dkl not afhct AC In rat ambrai axtwr, cerebellum and hypothaamw (61 . Abetirrarrca withdrawal of sddkxed mie decnned AC of cerebral oortor, a+nbellum end hypoUnanzrs (81 . Hid Km CAMP phroephodiwbraw (PDE) of rat striatum was inhibiud bY AAS both lh vlbo and by aorte iMaction (11, but PDE In moues cerebellum, hyDahderrxr, cerebral axtaz, and brain slam wre not affected bY M8 in/ectlona (4, 81 . MS had ro sfhct on hbh Km PDE a stratum of eddlctad rata, but during abstlnsnce tM Vmax of hier Km PDE decreased 191 . Our studls ware deshprd to resoles soma of thsss corrtradictkrm. MATERIALS AND MET110DS: Adsnyhd cycar (101, CAMP phospFrodfeatrsar (11, 121, and Protaln (13) wars asryad as described . Reb (180 9m, mais Sprague-Dawlsy-Timoo) wore dsapitatad and tires Iwmogsnlzad In 40 mM Trb - CI pH 7.4; for AC awys the bufhr oormhud 2 mM methyl-hobutylarrRMrre and 1 mM EGTA. Aably inJscted ad MS addfeted Me (9) waro killed one hour after foal in/sctkm Il .p .l . "Abstlrrwwe withdrawal" reb nceNed fini MS irrjrrtfonf 1 hr and 20 arp/Irp Nebxorrs 1/2 hrr before aaffls. Nwrroblawornaylfoma hybrida (courtesy of Dr . M . Nfranberp) and BHK-21 d13 alb IAmsrian Typa Cukun Collection) wanarttund adssRbad (14, 161. Confluant al~ wan trypdnizsd, watlred, reeseded (3 x 10s alb per 100 x 20 mm dbh in ahan medium) and krarbated 24 M pria m eddkion of MS (10pAA) rd/a PGEt 13yAA1 . Aftx 10 min Irrabatlon, the madlun was dbarded, PCA (0 .4N) was added, and al~ ware scraped from the disfres for DNA (17) rd CAMP puriflcatlon and datwmlrrticn (181 . RESl1LTS : No corrfWnt affects of MS wan found it rlbo a /n vivo on basal, NeF stYrwlabd-, a DA stMaratad AC of striate of nNe, addicted, or addktad rata in whhdnwal (Tabla 11 . TM does reeporoa arwe for DA sürrrulRion of AC of stratum wan the cama for nlve, aartaly ajeMad (10 nrp/kol. addhxad, and addicted nab In ebstirrerrce a ptedpltated withdrawal . PDE In stricts from dl these trsatrrrerrta showed similar kinetin (total Vmax - 136 nM/min/mp and arodated Km - 0.3 - 0.4 mM ; Vmax octrapoated from krw corrcerrtratkrro of cAMP ~ 6A pm/min/mp, and wocated Km ~ ?lrMj . Prwtepandln ftimulation of AC esse reatiwly s,rreq (10~261i) t GTP In control a kdamethradn treated nb (Table 111. PGEr , PGEs, and 18,1fldlmedtyl PGEs gava dmlar resrrlb, but PGF rQ ard PGF~erd PGA 1 wan irreffecWe. Morphine dkl not anhponbw tfia rtimuatory affect of prosfeplerdlm rd/a GTP or DA or NeF. In one expsHment, PGEr (10pAA) stimuated pituitary AC epprozknetely twobid (Y .C. Cierrrsrrt-0ormisr, J. J . Fieindel, sd G . A . Rohbon, paraovrl oenrrnrvnkaöonh but hIS (100 pAll) had no effect an thb a on had activity . In epreertrent with opened data (181 . PGE r (3pM1 incrawd 10-fold the rI~MP aoarrrwletbn in naurabakomaglkzn hybrid alla, and MS (10pAA) markedly inhibited (70%) thb stimuatlon . PGE r irrcraesd 6dold the CAMP aaumuatbn In BHK alla, but MS bed Iktle a rro eifact an thb rtimuatbn . "Sr+Pt~ad M a pont (DA-00744) fiom Vre US . Puhrlk Heelth Servis.
1876
PGE
and Morphine on Adenylyl Cyclase
Vol . 16, No . 12
TABLE I: ADENYLYL CYCLASE OF STRIATUM. Basel - pm/mln/mg ; other are % basal. In vivo ~ 100 /rM Morphlns Sulfate (MS) in away; A# - Aorte ir~ectlon of # mg MS/kg; ADD . - addicted ; ASST . = abstinents wkhdrawal; PW - precipetstad wfthdnrwal ; DA - 100 /tM Dopemlne, NaF - 6 mM sodium fluoride, Nal - 100 /rM Nabxone, MS ~ 100 NM . CONTROL BASAL NaF DA MS Nri
110 190 190 108 106
IN VITRO 100 200 200 102
A-10
A-20
Aa0
99 190 180
120 210 200 _ -
116 206 216
110
-
ADD. 86 197 180 96 96
ASST.
PW .
110 180 180 102 106
84 180 170 102 100
TABLE 11 : IN V1TR0 EFFECTS OF MS ON CEREBRAL CORTEX ADENYLYL CYCLASE. Exp. rata had 20 mg/kg irdomethecin i .p . 24 and 2 hours before setxlfka. Basal activity - pm/min/mg; other -% boat . Data averaps of 3 axperimants . MS, DA, and NsF as in T~ble I ; naspinephriro (NEI, epinaphrlne (EPI), and fsoproterenol (ISO) - 50 pM ; PGEr - 100 /rM (same result with 10 /rMl : GTP = 20 kM guerasine tHphosphete. NAIVE NAIVE + Mg+ EXP. EXP. + M8+
BASAL
NE
EPI
98 99 103 101
204 166 -
206 -
DA
192 190 180 182
ISO
NaF
POET
ßTP
OTP+POE,
185 200 136 -
412 313 -
113 112 126 124
148 145 119 120
182 168 140 134
DISCUSSION : We wen able to mrrfirm the previously reported finding that morphine inhibits the prostaglandin-Induced rise In CAMP level: in skated neurobleatoms~lioma hybrkl cilla (18, 191. This effect of morphine does not occur In the psnrttal call litres, which lack morphine receptor, end neithx does k ocwr in BHK cilia, which presumably riso lack these receptor. To thh eucterrt our data support the hypothesis of Cdlbr end Roy 1201 that cAMP may be involved in the action of morphine . Unfortunately wa wero rot able to resolve the wntradictory data from norrcukurod brain cells, for ws wero unable to see arty consistent effect of maphins on AC or PDE from arty brain area, either In resporxs m morphine injected !n vlho or added In vlvo . This may reflect nothing more than the complexity of the brln and our inebllky m control the many factors whk:h aro known m effect AC and PDE In this tiaue, wch as Ions, nucleotkies, rteurotrarumktar, and other potential modulators. Continued studies of the mechanism by which morphine affects AC ecthrky In cukurod cells seem warranted and may eventually contribute m a better understanding of how morphine effects brain furrctbn In vlvo . REFERENCES : 1) Purl, S.K., J. Cochin and L. Vollcar, Lifs Sciences 16 : 769-788 (1976) ; 2) Tell, G.P G.W. Partsmek end P. Cuatradsss, FEES Letters 61 : 242-246 (19761 : 3) Iwstsubo, K. and D.H, Clouet, Fed. Pros. 3?: 538 Abe. (19731 ; 4) Chau, WS., A.KS. Ho and H.H . Loh, Proc. West . Pharnrecol . Soc. 1~1: 42-48 (19711 : 6) Nrito, K. and K. Kuriyama, Japsn J. Phermacol. 23 : 274278 (19731 : 8) Singhot, R.L ., S. Kacew, and R. Lafroniero, J. Pherm. PMrrnecol. 2b : 1022-1024 (19731 : 7) Iwatsubo, K., G.J. Gak! and D.H . Cbust, The Pharmaeologist 16 : 270 abs. (1974) ; 8) Costa, E., A. Caranzi, A. Guklottl and A. Rewrite, Frontier in Catecholamine Resdrch pp. 1003-1010. Pergemon Pre:, London . (19731 : 9) Volksar, L., S.K . Purl end J. Cochin, Adv. Cyclic Nuc. Rss. 5: 814 Abs. (1976) ; 10) Thompson, W.J., S.A . Littb and R.H. Willkma, Bbchemistry 12 : 1889-1~4 (19731 : 11) Thompson, W.J. and M.M . Appleman, Biochemistry 10: 311318 (1971) ; 12) Wsia, B., R. Lehne and S.J. Suada, Anal. Bbcham. I6: 222-236 (19721: 13) Schectarle, G,R. and R.L . Pollack, Anal . Blochern . S1 : 8546 (1973) ; 14) Klee, W.A. and M. Nirenberg, Proc. Nat. Aced . Scl. 71 : 34743477 (1974) ; 15) Pbdger, W.L, WJ. Thompsorr end S.J. Suada, J. Cycic Nuc. Res. In Prey (1976) ; 18) Helndri, J., R. Rothenbay, GA. Robison end A. Steinbanger, J. Cycic Nuc. Res. 1: x-79 (19761 : 17) Burton, K. Bbchem . J. 6? : 316323 (19681 : 18) Traber, J., K. Fischar, S. Latzin and B. HamprcM, Natur ?63: 120-122 (19761 : 19) Sharma, S.K ., M. Ninnberg, and W.A. Klee, Proc . Net. Add. Sci., 72 : 690584 (19761 : 20) Collier, H.O.J ., and A.C. Roy, Newn ë18: 24-27 (19741 .
1875-1876
Pergamon Press
EFFECTS OF PROâTAßlA18flINS AND AAORPHINE ON BRAIN ADENYLYL CYCLASE" RA . Van ImwSwr, SJ. Strede, ard ß .A. Rabbon Deprtersnt of Plrrnrodooy, TM Unkrsnky of Taxe Medial School at Hosebn, Hornfon, Tors 7702b (Received in final form May 24,
1975)
Data implksrtirrp Inwlvwnerrt of cydk: nudeotkles M the pharrrrscology of narPhin b oompranhsd by corrtradk:taY data M tire Ifbntun . Far azampla, /n dtro morphine sulfata (AAS) ttirrwlafsd (1) and had no effaà (2) on bwl adanylyl cydar IACI in nt stratum, and inhibited AC of wvrel bNn cress (31 . Aab MS hijsctbn krawrd AC of rat stratum (1) end moues a+rebrai cortex (41, had no effect on AC of mouse rd nt oanlxal coron, cerebellum end hypotFralamw (4-8) and decreapd AC of moues bolo sbm (41 . Doprrrhr IDAI and sodium 1luafde IWFI stimulated AC of rat striatum wore rzrffectad by MS !n v/bo (1, 2, 7) or by sate k~sctbrr (1, 81, but carts injections rFrensd DA smilles AC 171 . Cydk: AMP !such irrcrenad in PIWiferY and sUbtum Iel, decnned In hypotlralarnw (3) a dkl not charge M cerebral axteor, anbellum or hypothalamus (8) sifter acufs hIS k>]actlon . Chronic MS adminhtration dsusard (4) and Incrwd 161 AC In moor cobol telex, but dkl not afhct AC In rat ambrai axtwr, cerebellum and hypothaamw (61 . Abetirrarrca withdrawal of sddkxed mie decnned AC of cerebral oortor, a+nbellum end hypoUnanzrs (81 . Hid Km CAMP phroephodiwbraw (PDE) of rat striatum was inhibiud bY AAS both lh vlbo and by aorte iMaction (11, but PDE In moues cerebellum, hyDahderrxr, cerebral axtaz, and brain slam wre not affected bY M8 in/ectlona (4, 81 . MS had ro sfhct on hbh Km PDE a stratum of eddlctad rata, but during abstlnsnce tM Vmax of hier Km PDE decreased 191 . Our studls ware deshprd to resoles soma of thsss corrtradictkrm. MATERIALS AND MET110DS: Adsnyhd cycar (101, CAMP phospFrodfeatrsar (11, 121, and Protaln (13) wars asryad as described . Reb (180 9m, mais Sprague-Dawlsy-Timoo) wore dsapitatad and tires Iwmogsnlzad In 40 mM Trb - CI pH 7.4; for AC awys the bufhr oormhud 2 mM methyl-hobutylarrRMrre and 1 mM EGTA. Aably inJscted ad MS addfeted Me (9) waro killed one hour after foal in/sctkm Il .p .l . "Abstlrrwwe withdrawal" reb nceNed fini MS irrjrrtfonf 1 hr and 20 arp/Irp Nebxorrs 1/2 hrr before aaffls. Nwrroblawornaylfoma hybrida (courtesy of Dr . M . Nfranberp) and BHK-21 d13 alb IAmsrian Typa Cukun Collection) wanarttund adssRbad (14, 161. Confluant al~ wan trypdnizsd, watlred, reeseded (3 x 10s alb per 100 x 20 mm dbh in ahan medium) and krarbated 24 M pria m eddkion of MS (10pAA) rd/a PGEt 13yAA1 . Aftx 10 min Irrabatlon, the madlun was dbarded, PCA (0 .4N) was added, and al~ ware scraped from the disfres for DNA (17) rd CAMP puriflcatlon and datwmlrrticn (181 . RESl1LTS : No corrfWnt affects of MS wan found it rlbo a /n vivo on basal, NeF stYrwlabd-, a DA stMaratad AC of striate of nNe, addicted, or addktad rata in whhdnwal (Tabla 11 . TM does reeporoa arwe for DA sürrrulRion of AC of stratum wan the cama for nlve, aartaly ajeMad (10 nrp/kol. addhxad, and addicted nab In ebstirrerrce a ptedpltated withdrawal . PDE In stricts from dl these trsatrrrerrta showed similar kinetin (total Vmax - 136 nM/min/mp and arodated Km - 0.3 - 0.4 mM ; Vmax octrapoated from krw corrcerrtratkrro of cAMP ~ 6A pm/min/mp, and wocated Km ~ ?lrMj . Prwtepandln ftimulation of AC esse reatiwly s,rreq (10~261i) t GTP In control a kdamethradn treated nb (Table 111. PGEr , PGEs, and 18,1fldlmedtyl PGEs gava dmlar resrrlb, but PGF rQ ard PGF~erd PGA 1 wan irreffecWe. Morphine dkl not anhponbw tfia rtimuatory affect of prosfeplerdlm rd/a GTP or DA or NeF. In one expsHment, PGEr (10pAA) stimuated pituitary AC epprozknetely twobid (Y .C. Cierrrsrrt-0ormisr, J. J . Fieindel, sd G . A . Rohbon, paraovrl oenrrnrvnkaöonh but hIS (100 pAll) had no effect an thb a on had activity . In epreertrent with opened data (181 . PGE r (3pM1 incrawd 10-fold the rI~MP aoarrrwletbn in naurabakomaglkzn hybrid alla, and MS (10pAA) markedly inhibited (70%) thb stimuatlon . PGE r irrcraesd 6dold the CAMP aaumuatbn In BHK alla, but MS bed Iktle a rro eifact an thb rtimuatbn . "Sr+Pt~ad M a pont (DA-00744) fiom Vre US . Puhrlk Heelth Servis.
1876
PGE
and Morphine on Adenylyl Cyclase
Vol . 16, No . 12
TABLE I: ADENYLYL CYCLASE OF STRIATUM. Basel - pm/mln/mg ; other are % basal. In vivo ~ 100 /rM Morphlns Sulfate (MS) in away; A# - Aorte ir~ectlon of # mg MS/kg; ADD . - addicted ; ASST . = abstinents wkhdrawal; PW - precipetstad wfthdnrwal ; DA - 100 /tM Dopemlne, NaF - 6 mM sodium fluoride, Nal - 100 /rM Nabxone, MS ~ 100 NM . CONTROL BASAL NaF DA MS Nri
110 190 190 108 106
IN VITRO 100 200 200 102
A-10
A-20
Aa0
99 190 180
120 210 200 _ -
116 206 216
110
-
ADD. 86 197 180 96 96
ASST.
PW .
110 180 180 102 106
84 180 170 102 100
TABLE 11 : IN V1TR0 EFFECTS OF MS ON CEREBRAL CORTEX ADENYLYL CYCLASE. Exp. rata had 20 mg/kg irdomethecin i .p . 24 and 2 hours before setxlfka. Basal activity - pm/min/mg; other -% boat . Data averaps of 3 axperimants . MS, DA, and NsF as in T~ble I ; naspinephriro (NEI, epinaphrlne (EPI), and fsoproterenol (ISO) - 50 pM ; PGEr - 100 /rM (same result with 10 /rMl : GTP = 20 kM guerasine tHphosphete. NAIVE NAIVE + Mg+ EXP. EXP. + M8+
BASAL
NE
EPI
98 99 103 101
204 166 -
206 -
DA
192 190 180 182
ISO
NaF
POET
ßTP
OTP+POE,
185 200 136 -
412 313 -
113 112 126 124
148 145 119 120
182 168 140 134
DISCUSSION : We wen able to mrrfirm the previously reported finding that morphine inhibits the prostaglandin-Induced rise In CAMP level: in skated neurobleatoms~lioma hybrkl cilla (18, 191. This effect of morphine does not occur In the psnrttal call litres, which lack morphine receptor, end neithx does k ocwr in BHK cilia, which presumably riso lack these receptor. To thh eucterrt our data support the hypothesis of Cdlbr end Roy 1201 that cAMP may be involved in the action of morphine . Unfortunately wa wero rot able to resolve the wntradictory data from norrcukurod brain cells, for ws wero unable to see arty consistent effect of maphins on AC or PDE from arty brain area, either In resporxs m morphine injected !n vlho or added In vlvo . This may reflect nothing more than the complexity of the brln and our inebllky m control the many factors whk:h aro known m effect AC and PDE In this tiaue, wch as Ions, nucleotkies, rteurotrarumktar, and other potential modulators. Continued studies of the mechanism by which morphine affects AC ecthrky In cukurod cells seem warranted and may eventually contribute m a better understanding of how morphine effects brain furrctbn In vlvo . REFERENCES : 1) Purl, S.K., J. Cochin and L. Vollcar, Lifs Sciences 16 : 769-788 (1976) ; 2) Tell, G.P G.W. Partsmek end P. Cuatradsss, FEES Letters 61 : 242-246 (19761 : 3) Iwstsubo, K. and D.H, Clouet, Fed. Pros. 3?: 538 Abe. (19731 ; 4) Chau, WS., A.KS. Ho and H.H . Loh, Proc. West . Pharnrecol . Soc. 1~1: 42-48 (19711 : 6) Nrito, K. and K. Kuriyama, Japsn J. Phermacol. 23 : 274278 (19731 : 8) Singhot, R.L ., S. Kacew, and R. Lafroniero, J. Pherm. PMrrnecol. 2b : 1022-1024 (19731 : 7) Iwatsubo, K., G.J. Gak! and D.H . Cbust, The Pharmaeologist 16 : 270 abs. (1974) ; 8) Costa, E., A. Caranzi, A. Guklottl and A. Rewrite, Frontier in Catecholamine Resdrch pp. 1003-1010. Pergemon Pre:, London . (19731 : 9) Volksar, L., S.K . Purl end J. Cochin, Adv. Cyclic Nuc. Rss. 5: 814 Abs. (1976) ; 10) Thompson, W.J., S.A . Littb and R.H. Willkma, Bbchemistry 12 : 1889-1~4 (19731 : 11) Thompson, W.J. and M.M . Appleman, Biochemistry 10: 311318 (1971) ; 12) Wsia, B., R. Lehne and S.J. Suada, Anal. Bbcham. I6: 222-236 (19721: 13) Schectarle, G,R. and R.L . Pollack, Anal . Blochern . S1 : 8546 (1973) ; 14) Klee, W.A. and M. Nirenberg, Proc. Nat. Aced . Scl. 71 : 34743477 (1974) ; 15) Pbdger, W.L, WJ. Thompsorr end S.J. Suada, J. Cycic Nuc. Res. In Prey (1976) ; 18) Helndri, J., R. Rothenbay, GA. Robison end A. Steinbanger, J. Cycic Nuc. Res. 1: x-79 (19761 : 17) Burton, K. Bbchem . J. 6? : 316323 (19681 : 18) Traber, J., K. Fischar, S. Latzin and B. HamprcM, Natur ?63: 120-122 (19761 : 19) Sharma, S.K ., M. Ninnberg, and W.A. Klee, Proc . Net. Add. Sci., 72 : 690584 (19761 : 20) Collier, H.O.J ., and A.C. Roy, Newn ë18: 24-27 (19741 .
