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Reversal of the antilipolytic effect of prostaglandin E2 by an oxazepine derivative (SC-19220)
Life Sciences Vol. 10, Part I, pp . 1281.1285, 1971 . Printed in Great Britain
Pergamon Preen
RSV~t3AL 0f TRB AlITILIPOLYTIC R~~.T OF PROB'DAGLAIiDIlf BY AN O7UZ~PINB DDEtIVATIVS (9C-19220) Frederick K. Radeialowski and Leila Novak Division of Hiooheaiosl Researoh, G. D. 9earle & Co. P .O. Hoa 5110, Chioago, Illinois 60680 (Received 17 August 1971 ; in final form 8 October 1971) 9ussary This study shows that SC-19220 can antagonise PG~ inhibdtian of epinephrine stisulsted lipolysis in the isolated fat cell. The aatagoniaa appears direct and is not due to the stiaulating effect of the coapouad oa lipolysis or its potantiation of the lipoljrtic notion of epinephrine. Proatag~ "wa~r ", a p~oup of natural~r occurring fatty acid derivstiws, haw recently been shown to possess a aide apeot=vs of potent pharsaoologio aotivit, (1) including the ability to inhibit the actions of the lipolytio ho~rscnes in adipose tissue (2~). 31noe prostaglandins are derived Eros cer4O.Lfi fatty acids, are present in adipose tissue and are released following horsonal and aerw
etiaulation, they have been postulated to be iavolwd is a physiological feedback secherties contro lling lipolyeis (5) "
Reoent~r, 3anaer showed that the oospound, 1-acetyl-2-(8~hloro-10,11-dityr-
drodibeas(b,f)(1,4) o:asspiae-l0-oarbogyl) hydrssine (SC-19220) ras able to
spscifioal~ block oontractione of guinea pig ileum induced ~ pxosta8laadia In riex of these observations it was of interest to detersine whether the
inhibitory action of 9C-19220 on PG1~ is restricted to either ssooth snsole or to the stiaulatory effects of PCSrt. This question xas e:asined ~ evaluating the sfFecte of 3C-19220 on ep~iasphrine etiaulated sad PGF~ iahibàted lipolyrsie
in the isolated fst cell preparation. It was found that 8C-19220 does reveres PCB2 1nhl bition of epinephrine stimulated lipolysis sad the effect le ooaoentra" tics dependent. ll~rther, SC-19220 does not affect either basal lipolysis or potentists epinephrine suggesting a specific notion on PCB. 1281
1282 Yol. Antagonism a~f PGE 2 10, No. 22 Methods Male a1Mno Charls Aiwr rata (iso-too g) xere e.ployed tk~rou~ont the study. Aniaals xare alloxed free access to food sad xatar until the tine of saarifioe. Anisals rare sacrificed by oarvioal dislocation, the epididyaal fat pads xare reacted and s suspension of free fat ceue xsre prepared using the sethod of Rodbeu (~).
The osus (100 sB tissue/al) rare suspended is I~ebs-
Phosphate Huffier (pH 7 .4) containing one-half the usual aaount of calatua and 496 x/v borins albuain, Fraatios V, fatty acid poor type (Pantez) .
Mixtures ooa-
taming 1 et of oeu suspension sad different ooabinatlans of drugs xere incubated is a Dubnoff ehaldag inaabator st ~oC far 2 hours under as stsosphere of air.
The cospouads tested sad in the firu~l oaaaratrations term
(2xi0~M), ~`L (2x10~M) sad sc-19¢zo (1~ao~`M to izl.o 7M) 1.
eplnephriae rdpo~rata xae
detsrsined by asssuraaeat of the releas of firs fatty acids end glycerol . Fatty acids xere assured using the aethod of Dole and Meiaerts (6).
Glycerin
xas detarsiasd by either the ohxwsotropic acid iethod of Labert and Neigh (9) as sodifiad by ]Corn (10) or bpr as adaptation of the Nosh (11) reaatloa .
In this
adaptation the aqueous phase r~+n+n g attar phase separation Sn the Dole sad Meiaert$ procedure (8 ) xas wntrifuged .
Qie siuiliter of the dear superna-
tant xas si~esd xith 0.1 u of 0.05 M sodiui periodate sad alloxed to stand at roux teaperature for 5 sin.
Thaw 0.1 sl of 0.5 M eodina areenite solution xaa
added, nixed sad alloxed to stead far 5-10 sin at roux teaparature.
Txo aiui-
litars of aster sad 2 al of Naeh reagent xare added, the aixttae xaa heated far 45 sin at 60° C, the eaapls xere cooled and abeorbeace detsx~insd spsctrophotosetriaauy at 410 as.
The aethod using Nosh reagent, xhile less sansitiw
than the chroaotropic acid ~edure, elisinates the need to xorY xith the light seasitiw, oarrosiw chroaotropio acid regent sad solar farsatias taiae place at a bast teapsrat~ne. i
PGF,~ xae prsPared 1~ Dr . P. 8. ~-.o,..~ sad Mr. Fred Fago and the 9C-19Q20
xaa synthesised by Dr. W. >ß . Coyne.
Vol . 1283 10, No. 22 Antago~sm ouf PGE 2 Results amd Discussion The data pa~ssented in !!gars 1 shox that 3C-19220 has the abdlity to rwerse tM inhibition of PGLY~ an epinephrine stisulated lipolysis .
This afieot is ooa-
oe~ntratioa dependent .
Drug heafmenh FIG. 1
effect of various concxntrstiona oY SC-19220 on proataglaadin F~ (PGâ~) inhibition of epinephrine stisulated lipolysis in the isolated adipocyte . ate of lipolysis xas deterained b7 swearing the release of free fatty aside . Dupli oste ~ ss and fiw anirsle per group xsre used . Response to epinephrine (2~d0 M xu considexsd 10096. The black lines xithin the bare are the etaadard axzor values .
1284
Antagonism od PGE 2
Vol . 10, No. 22
A phatsau in ths eßßeativenees oß 9C-19220 xae appsrently reaohed betxssn oonoentrstians of
Sx1Ô5!!
and lx10~!!.
This lifitation is not understood but
aq be dus to the sttain~ent of saxLrna penetration rates through the cell ~brace or poor wlnbility oß the aosPoumd st these oonwntxations in the inaubafion fixture. The data presented in Table 1 shox that 3c-19220 dose not etisalats lipol,sie and does cot inhi Mt the lipolytio aotia~n oß spinephriae.
Shoes data elifi-
hate the possihility that sc-19~o cats tp potentiating the et'feat of epinephrine. TA.H18 1 Sffsot oß 3~iasphrins, Pros +~~~n H2 and 3C-19220 on Lipo~sis in the Isolated Adipooyte.
Treatsente spinephrine
fires !atty Release (+ 9.5 .)
Release (+ s.g)
ioo
100
Control (11)
5 ± 0.3
? ± 0.8
sC-19220 (6)
4 + 0.6+
7 + o.y+
Rpiaephrine ~ 8C-19220 (8)
94 + 1.4
g~ + 2.6
S~inephrine ~ PCdr~ (9)
55 ± 2.3
60 ± 1 .4
3~inephrine + 9C-19220 ~ PGi2 (lo)
89 + 5"2~
91 + 5,4~
f
Ausba oß ~±~ used is given in parenthesis aada~17~ is xae done ia dnplioate. ~6 oaaoentratione xere~ S~inephrins 2x10 -°I!, Prostaglandin $2 (PC~) 2z10~l1 and 3c-19220 ix10-5M . e+ Lipolsrtio activity ie eapxesesd rslatirs to epinephrine - 1009i. + xot signißioeatl~r düßerent iror oantrol gr0up dilYexent ~ Rpin phrl.n. aaa Proetaglaadia ~2 ( G~p1( ) pt0
O1)~~i~~
She rever.al e~f PfR2 inhi bitiom is observed xhethaa~ releeee od ßree fatty acids or g1,Toesol L used as s aeasure oß lipolrsie (Tabls 1) .
Thus, the inoreaae in
re].eaee of tYee ßstty acids oennot be due to a bl,ookads of ths triglyceride e~ynthefis aeohaaisas finoe glyeerol release is alao inamseaed.
Sinoe glyosrol ie
Antagonism ad PGE 2 Vol . 1285 10, No. 22
not naad fa~ rs-~st~xifioatian in tM fst oall (12) it xould not bs eigoifioaat1T aff~atad b7 flnotvatiana in tha rata of fattf aoid ra~ataifioation .
Rafnynoaa i.
9 . B~xBstroa sad H . Saaueleson, Proo~ad . 2ad Nobal ~Yap ., ALquiat aad üiolaall, SteokhoL, 1967 .
2.
D . BEdnbasg, x . Vausim, P. J . Nastal and 3 . H~zgst~roa, Bioohaa. R~araao . 12, 7d+ (1963) "
3.
R . Paolatti, R. L Iwatati and Z . goro7l~axios in S . Hagatroa aad B . 9aanaLson (Dditoxa), Proowd . 2nd Fobal 9yap . . Alaquiat and Yiola~ll, 3tooidtola, 1967, p . iW1 .
4.
R . Y. Hntoha aad
j.
J . ~. 3hax aad P. U . Raaxell, J . Biol. Chea . ~ 1498 (1968) .
6.
J . H. 9aaaa~, Axbh . Int . PharraAOdm . 180 . 4 b (1969) .
x.
Y . 8nthaLad, A~ . 11. Y . Aoad. 9ai . 13Q, 8r+9 (1967) .
x . Rodball, J . HLol . Gàg. 2~Q, 37$ (19ä+) . 8.
Y . P. Dola aad H. xainaxts, J . Biol. Chaa . ~, 2595 (1960) .
9.
x . Labaxt aad A. C . lfaish, Caa . J . Rasaaroh ~ 83 (1950) "
10.
x . D . Rorn, J . Hiol . G7~~ . 2~ i (1955) "
11 .
T . xash, Bioohaa. J . ~, 416 (1953) "
12 .
J . A . fain, A . Rad aad R . 3aparetain, J . Biol . Cfian . 242 . 188 7 (1967) "
Pergamon Preen
RSV~t3AL 0f TRB AlITILIPOLYTIC R~~.T OF PROB'DAGLAIiDIlf BY AN O7UZ~PINB DDEtIVATIVS (9C-19220) Frederick K. Radeialowski and Leila Novak Division of Hiooheaiosl Researoh, G. D. 9earle & Co. P .O. Hoa 5110, Chioago, Illinois 60680 (Received 17 August 1971 ; in final form 8 October 1971) 9ussary This study shows that SC-19220 can antagonise PG~ inhibdtian of epinephrine stisulsted lipolysis in the isolated fat cell. The aatagoniaa appears direct and is not due to the stiaulating effect of the coapouad oa lipolysis or its potantiation of the lipoljrtic notion of epinephrine. Proatag~ "wa~r ", a p~oup of natural~r occurring fatty acid derivstiws, haw recently been shown to possess a aide apeot=vs of potent pharsaoologio aotivit, (1) including the ability to inhibit the actions of the lipolytio ho~rscnes in adipose tissue (2~). 31noe prostaglandins are derived Eros cer4O.Lfi fatty acids, are present in adipose tissue and are released following horsonal and aerw
etiaulation, they have been postulated to be iavolwd is a physiological feedback secherties contro lling lipolyeis (5) "
Reoent~r, 3anaer showed that the oospound, 1-acetyl-2-(8~hloro-10,11-dityr-
drodibeas(b,f)(1,4) o:asspiae-l0-oarbogyl) hydrssine (SC-19220) ras able to
spscifioal~ block oontractione of guinea pig ileum induced ~ pxosta8laadia In riex of these observations it was of interest to detersine whether the
inhibitory action of 9C-19220 on PG1~ is restricted to either ssooth snsole or to the stiaulatory effects of PCSrt. This question xas e:asined ~ evaluating the sfFecte of 3C-19220 on ep~iasphrine etiaulated sad PGF~ iahibàted lipolyrsie
in the isolated fst cell preparation. It was found that 8C-19220 does reveres PCB2 1nhl bition of epinephrine stimulated lipolysis sad the effect le ooaoentra" tics dependent. ll~rther, SC-19220 does not affect either basal lipolysis or potentists epinephrine suggesting a specific notion on PCB. 1281
1282 Yol. Antagonism a~f PGE 2 10, No. 22 Methods Male a1Mno Charls Aiwr rata (iso-too g) xere e.ployed tk~rou~ont the study. Aniaals xare alloxed free access to food sad xatar until the tine of saarifioe. Anisals rare sacrificed by oarvioal dislocation, the epididyaal fat pads xare reacted and s suspension of free fat ceue xsre prepared using the sethod of Rodbeu (~).
The osus (100 sB tissue/al) rare suspended is I~ebs-
Phosphate Huffier (pH 7 .4) containing one-half the usual aaount of calatua and 496 x/v borins albuain, Fraatios V, fatty acid poor type (Pantez) .
Mixtures ooa-
taming 1 et of oeu suspension sad different ooabinatlans of drugs xere incubated is a Dubnoff ehaldag inaabator st ~oC far 2 hours under as stsosphere of air.
The cospouads tested sad in the firu~l oaaaratrations term
(2xi0~M), ~`L (2x10~M) sad sc-19¢zo (1~ao~`M to izl.o 7M) 1.
eplnephriae rdpo~rata xae
detsrsined by asssuraaeat of the releas of firs fatty acids end glycerol . Fatty acids xere assured using the aethod of Dole and Meiaerts (6).
Glycerin
xas detarsiasd by either the ohxwsotropic acid iethod of Labert and Neigh (9) as sodifiad by ]Corn (10) or bpr as adaptation of the Nosh (11) reaatloa .
In this
adaptation the aqueous phase r~+n+n g attar phase separation Sn the Dole sad Meiaert$ procedure (8 ) xas wntrifuged .
Qie siuiliter of the dear superna-
tant xas si~esd xith 0.1 u of 0.05 M sodiui periodate sad alloxed to stand at roux teaperature for 5 sin.
Thaw 0.1 sl of 0.5 M eodina areenite solution xaa
added, nixed sad alloxed to stead far 5-10 sin at roux teaparature.
Txo aiui-
litars of aster sad 2 al of Naeh reagent xare added, the aixttae xaa heated far 45 sin at 60° C, the eaapls xere cooled and abeorbeace detsx~insd spsctrophotosetriaauy at 410 as.
The aethod using Nosh reagent, xhile less sansitiw
than the chroaotropic acid ~edure, elisinates the need to xorY xith the light seasitiw, oarrosiw chroaotropio acid regent sad solar farsatias taiae place at a bast teapsrat~ne. i
PGF,~ xae prsPared 1~ Dr . P. 8. ~-.o,..~ sad Mr. Fred Fago and the 9C-19Q20
xaa synthesised by Dr. W. >ß . Coyne.
Vol . 1283 10, No. 22 Antago~sm ouf PGE 2 Results amd Discussion The data pa~ssented in !!gars 1 shox that 3C-19220 has the abdlity to rwerse tM inhibition of PGLY~ an epinephrine stisulated lipolysis .
This afieot is ooa-
oe~ntratioa dependent .
Drug heafmenh FIG. 1
effect of various concxntrstiona oY SC-19220 on proataglaadin F~ (PGâ~) inhibition of epinephrine stisulated lipolysis in the isolated adipocyte . ate of lipolysis xas deterained b7 swearing the release of free fatty aside . Dupli oste ~ ss and fiw anirsle per group xsre used . Response to epinephrine (2~d0 M xu considexsd 10096. The black lines xithin the bare are the etaadard axzor values .
1284
Antagonism od PGE 2
Vol . 10, No. 22
A phatsau in ths eßßeativenees oß 9C-19220 xae appsrently reaohed betxssn oonoentrstians of
Sx1Ô5!!
and lx10~!!.
This lifitation is not understood but
aq be dus to the sttain~ent of saxLrna penetration rates through the cell ~brace or poor wlnbility oß the aosPoumd st these oonwntxations in the inaubafion fixture. The data presented in Table 1 shox that 3c-19220 dose not etisalats lipol,sie and does cot inhi Mt the lipolytio aotia~n oß spinephriae.
Shoes data elifi-
hate the possihility that sc-19~o cats tp potentiating the et'feat of epinephrine. TA.H18 1 Sffsot oß 3~iasphrins, Pros +~~~n H2 and 3C-19220 on Lipo~sis in the Isolated Adipooyte.
Treatsente spinephrine
fires !atty Release (+ 9.5 .)
Release (+ s.g)
ioo
100
Control (11)
5 ± 0.3
? ± 0.8
sC-19220 (6)
4 + 0.6+
7 + o.y+
Rpiaephrine ~ 8C-19220 (8)
94 + 1.4
g~ + 2.6
S~inephrine ~ PCdr~ (9)
55 ± 2.3
60 ± 1 .4
3~inephrine + 9C-19220 ~ PGi2 (lo)
89 + 5"2~
91 + 5,4~
f
Ausba oß ~±~ used is given in parenthesis aada~17~ is xae done ia dnplioate. ~6 oaaoentratione xere~ S~inephrins 2x10 -°I!, Prostaglandin $2 (PC~) 2z10~l1 and 3c-19220 ix10-5M . e+ Lipolsrtio activity ie eapxesesd rslatirs to epinephrine - 1009i. + xot signißioeatl~r düßerent iror oantrol gr0up dilYexent ~ Rpin phrl.n. aaa Proetaglaadia ~2 ( G~p1( ) pt0
O1)~~i~~
She rever.al e~f PfR2 inhi bitiom is observed xhethaa~ releeee od ßree fatty acids or g1,Toesol L used as s aeasure oß lipolrsie (Tabls 1) .
Thus, the inoreaae in
re].eaee of tYee ßstty acids oennot be due to a bl,ookads of ths triglyceride e~ynthefis aeohaaisas finoe glyeerol release is alao inamseaed.
Sinoe glyosrol ie
Antagonism ad PGE 2 Vol . 1285 10, No. 22
not naad fa~ rs-~st~xifioatian in tM fst oall (12) it xould not bs eigoifioaat1T aff~atad b7 flnotvatiana in tha rata of fattf aoid ra~ataifioation .
Rafnynoaa i.
9 . B~xBstroa sad H . Saaueleson, Proo~ad . 2ad Nobal ~Yap ., ALquiat aad üiolaall, SteokhoL, 1967 .
2.
D . BEdnbasg, x . Vausim, P. J . Nastal and 3 . H~zgst~roa, Bioohaa. R~araao . 12, 7d+ (1963) "
3.
R . Paolatti, R. L Iwatati and Z . goro7l~axios in S . Hagatroa aad B . 9aanaLson (Dditoxa), Proowd . 2nd Fobal 9yap . . Alaquiat and Yiola~ll, 3tooidtola, 1967, p . iW1 .
4.
R . Y. Hntoha aad
j.
J . ~. 3hax aad P. U . Raaxell, J . Biol. Chea . ~ 1498 (1968) .
6.
J . H. 9aaaa~, Axbh . Int . PharraAOdm . 180 . 4 b (1969) .
x.
Y . 8nthaLad, A~ . 11. Y . Aoad. 9ai . 13Q, 8r+9 (1967) .
x . Rodball, J . HLol . Gàg. 2~Q, 37$ (19ä+) . 8.
Y . P. Dola aad H. xainaxts, J . Biol. Chaa . ~, 2595 (1960) .
9.
x . Labaxt aad A. C . lfaish, Caa . J . Rasaaroh ~ 83 (1950) "
10.
x . D . Rorn, J . Hiol . G7~~ . 2~ i (1955) "
11 .
T . xash, Bioohaa. J . ~, 416 (1953) "
12 .
J . A . fain, A . Rad aad R . 3aparetain, J . Biol . Cfian . 242 . 188 7 (1967) "