Life Sciences, Vol . 23, pp . 351-356 Printed in the U .S .A .
Pergamon Press
Dpna~~a~en VA3CULAR PR08TACYCLIIQ IH 1~II~ifPàL DIABETJ~ Heather B . Harrison, Ann H . Reece and Maloolm Johnson Department of Pharmacology, Imperial Chemical Industries, èlderley Park, Macclesfield, Cheshire, >>hgland . (Received in final form June 12, 1978) Prostacyclin is a potent inhibitor of platelet aggregation . Its release from the aorta of streptoaotooin-diabetic rate ie reduced . High rates of prostacyclin production appear to be incompatible with high blood glucose levels . It is possible that decreased prostaoyclin release may be related to vascular complications in diabetes . Patients with diabetes mellitus develop miorovascular camplicatione,(1) and have an increased susceptibility to atheroeclerosis (2~ and thrombosis (3) . Disorders of blood coagulation and platelet ftimctian in diabetes include decreased fibrinolytic activity (4), increased platelet reactivity (5,6) toward ~, collagen, adrenaline and araohidonic acid, increased spontaneous aggregation (7) and decreased platelet survival (8) and increased turnover (9) . Such abnormalities are particularly marked in diabetics with vascular complicatia~ne (10,11 . Platelet hyper-reactivity may be related to increased activity of the prostaglandin synthetase system (6) and a greater production of the pro-aggregating prostaglandin endoperoxidee G2 and H2 and thrombox$ae ~ . It has recently been reported that vascular endotHelium generates proetacyc in (PGI 2 ), a potent, but unstable, inhibitor of platelet aggregation (12,13) . A decreased production of PGI2 in diabetics could also be ezpected to contribute to the hypercoagulable state . To investigate this possibility we have estimated PGI 2 release from the aorta of rats with experimentally-induced diabetes . MEPHODS Male rats (Alderley Park strain, 90-120 g body weight were made diabetic by i .v . injection of streptozotocin (80 mg~kg), 1-3 months prior to use . Gluoosuria was canfixmed (~Clinistix~~ just before each ezperiment . Sex and age-matched rate were used ae ca~ntrols . Where indicated, soluble insulin (~Novo, 9otrapid MCP) was given e .c . (q00 II~rg) to some animals and food withdrawn simultaneously, 2~ hours before testing. In fasting ezperiments, food was withdrawn for 18 or 24 hrs before testing . The animals were anaesthetised with ether, and a blood sample (0 .5 ml~ taken for glucose estimation (14~, by ~Teohnioon Autoanalyser~ . The aorta was rapidly ezcieed, and following removal of the adventitial layer, was immersed in Srebe buffer at 4'C, containing 200 mg~] .00 ml glucose (unless otherwise stated . The tissue was weighed, cut into fine rings (1-2 mg) (12~ and incubated (60 mg~200 pl~ in Srebe buffer at 22 ° C for 3 min . 6liquote of the supernatant (1-lA W1~ were added to rat citrated platelet-rich plasma (15~, 30 sec before the addition of 0300.-1653/ 78 / 07240351502 , 00/ 0 Copyright © 1978 Pergamon Press
352
Proatacyclin is Diabetic Rate
Vol . 23, No . 4, 1978
a submaximal concentration of adenosine-5~-diphoephate (A .D .P ., 2 Wmol~l) . The activity of the supernatant in inhibiting platelet aggregation was measured in a double channel ag~egometer (~Paytan~) . The quantity of PGI2 was estimated from a standard ourve using authentic PGI 2 (sodium salt), and expressed as ng,/mg wet weight of tissue . RESUIfPS
Control
Diabetic
FIG . 1 Proatacyclin (ng~mg wet weight) released by the aorta of fed diabetic (blood glucose 412 .1 ± 15 .1 mg%100m1) and control (blood glucose 76 .7 ± 5 .0 mg~100m1) rata . I~bera of animals in each group are shown in parentheses . Values represent mean ± S .E, and p ~ 0 .02 where indicated (*) .
Vaeoular rings, incubated in vitro for 3 mina at room temperatw-.e released a potent inhibitor of ~~-induced platelet aggregation . This in~, t bitory activity was confirmed to be due to PGI by (a) loos of effect after boiling for 15 seas (16) and (b) relaxation ôf bovine coronary artery (17) . The production of PG2 2 by the aorta of fed diabetic rata was significantly depressed (p ~ 0 .02) when compared with that of control animals (Figure 1) . This was confirmed using aortic rings fra~m 24 hour fasted diabetic and oontrol rate where the amount of PGI2 released was 0 .28 ± 0 .05 and 0 .45 ± 0 .04 ng~mg respectively (p ~ 0 .05) .
Vol . 23, No . 4, 1978
Proatacyclia is Diabetic Rate
353
Prostacyolin was still significantly (p r 0 .05) reduced (0 .05 ± 0 .01 ~mg) when aortas from diabetic rate were incubated in Srebs buffer caataining levels of glucose similar to those found in ezperimental diabetes 4500 ml) and oompared with control aortas (0 .15 ± 0 .03 ng~mg) in OOml glucose . Proetacyclin relesée was estimated in a number of 70 ezperimente in which aortic rings were taken from fed and fa.eted rats and its relationship to blood glucose levels ezamined (Figure 2) .
Ioo
O lOD-
0
"ô .l 0
"
9~
" ~, o " °
"
.2 0
O
"
i8 "" ° o
ô 0
0.5 Q3 .01 Prostuyclin Irplmg i"t w~IgM ~orhl
"
ô o r06 .
Q7
FIG . 2 Aortio prostacyclin production (ng~mg wet weight) and blood glucose (mg%1OOm1) in diabetic fed ( " ) and fasted (Q ) and control fed ( " ) and fasted (0 ) rate .
High rates of PGI production did not appear to be compatible with high blood glucose . Thé effect of an acute, insulin-induced fall in blood glucose was therefore investigated . Figure 3 demonstrates that a reduction in blood glucose from 341 .0 ± 22 .6 mg~100m1 to 178 .9 ± 22 .6 m$/100m1 in diabetic rats did not restore PGI2 to control levels (control blood glucose 77 .0 ± 2 .1 mgß.00m1) . DISCIISSIO~P 7~e results indicate that the production of proetacyolin is reduced in the aorta of etreptosotocin.diabetic rats . Decreased vascular prostacyclin could reflect (1) endothelial cell loss or damage, (2) decreased . PGI 2 syutheeis, perhaps due to reduced absolute levels or availability of arachidonic acid, or decreased activity of ayclo-o~genase or proetacyclin
354 .
Proetacyclin is Diabetic Rats
Vol . 23, No . 4, 1978
synthetase, or (3) increased PGI 2 degradation . These defects msy be a consequence of the diabetic state or a specific effect of streptozotooin .
Dlsb~lk Disbstk Gmtrol + + + S~Iin Insulin Sallru
FIG .
3
Prostacycli.n (ng~mg wet weight) released by the aorta of (a) insulin-treated diabetic rats, (b) saline-treated diabetic and (o) saline-treated caatrol rate . l~bere of animals in each group are shown in parentheses . Values represent mean ± S .E, and p ~ 0 .01 where indicated (*) .
Fjyperglycaemia, or a conca~mitant metabolic abnormality of diabetes, may depress prosta.cyclin production ae it has been shown that increased glucose concentration (18) and osmolality (19) decrease proetaglandin " synthesis . Our finding that PGI is depressed is animals with high blood glucose tends to support this suggéstion,althougb. acute insulin treatment failed to restore PG22 to control values . The influence of chronic insulin administraticm ie currently under investigation . Our experiments have not rued out the possibility of a specifio effect of streptozotocin . This would have important consequenoes as this agent is widely used in experimental studies of the metabolio and. vascular camplioatia~ae of diabetes . However, the finding that proetacyolin ie depressed 12 months after a single dose of etreptozotocin (unpublished
Vol . 23, No . 4, 1978
Prostacyclin in Diabetic Rate
355
observations) suggests that a direot effect is unlikely . Erperimente are being oarried out in animals with epontaaeous diabetes to further investigate this possibility. The relevanoe of our fiadinge in experimental animals to the vascular complications of diabetes canaot be assessed until proetacyclin hoe been measured in vessels of humaa diabetics and in diabetic tissues, such ae retina and kidney, which are susceptible to microangiopathy . ACSNOWLPDGENlENTS We are indebted to Dr . S. Gibean, Chemistry Department, for synthetic prostacyolin and to Mise 3. Bradley and Mr . J . Clayton for technical aesieteace . R~tSPCS3 1. 2.
3. 4"
5. 6. 7" 8. 9. ]A . 11 . 12 . 13 " 14 . 15 . 16 . 17 "
le .
19 .
H. FAT and J. JARRETT (Hde), Complications of Diabetes , Arnold, London (1975) " H. ~P, Post~ad.Med .J . `~2 445-451 (1976) . W.R . TT ~~~, J.D . WARD, F .E . PBESTOA, T . DUCHWORTH,and B.C . O+MALLEY, Diabetologia 12 237-243 (1976) . G.R . F~!~~~ , R. CHAHÜABATI, and P .R . AVIS, Br .Med .J . _1 921-923 (1963) " H.C . gülAdH, J.A . COLWELL, S. CRUL, H. 3QWAHWELA, and J.G . DO~HBIE, J .Lab .Clin.Med . 8~ 236-246 (1972) . P.V . HALUSHKA, D. LIIRIE, and J.A . COLWELL, Hew Eng.,~.Med . 297 1306-1310 (1977) " g. BREÛDIH, H. GRDZP, H.R . HRZYWANEg, and W.P . 3CEIiErfl~, Thromb .Haemoetae . ~ 669-691 (1976) . J.C . FERGII80N, H. MACSAY, J.A.D . PHILIP, and D.J . SQtßdElt, C11n .Sci .Mo1 . Med. ~ 115-120 (1975) . J .A . COLWBLL, J . SAGEL, L. CROOS, A. CHAMBBR.S, and M. LAII~IIHS, Metabolism ~ 279-285 (1976) . H. H6A'fH, W.D . BRIGDEI~, J .V . CAHEVER, J . POLLOCg, P.R. HIIPTR$, J. SSLSSY, and A. BLO(H~S, Diabetoloaia 3 308-315 (1971) . B .C . O+MALLEY, W.R . mII+O~L~, J .D . WARD, H.R . PORTER, and F.E . PRBSPOH, Lancet 2 1274-1276 (1975) " S . M7HCADA, E .A. HIGGS, and J.R . VAHE, Lancet 1 18-21 (1977) " s. MoHCADA, R. GRYGL , s . BmaTnvG, aaa J .B. vAxE, Hature ~ 663-665 (1976) . D. BARHAM and P. ~~~, Anolyst ~ 142-145 (1972) . M. JC~PSOH, E. RAMEY, and P.~[ RAMFIEL?", Am .J .Phyeiol . ~ H381-H385 (1977} S. Nl7HCADA, R. GRYGLÉWSBI, S . EIIATIHG, and J .R . VAHE, Prostaglandine _12 715-736 (1976) . G .J . DQSTIHG, S . MCQPCADA, and J.R . VAHE, Proetaglandine ~ 3-15 (1977) " J. TAtar~AIIM, A .s . HzELS, and g. AIILS~ROOg, Fea .Proc. ~ 223 (1976) . D. VAH PRAGG and S .J . FAHB~, Fed .Proc . ~ 223 (1976 .
Pergamon Press
Dpna~~a~en VA3CULAR PR08TACYCLIIQ IH 1~II~ifPàL DIABETJ~ Heather B . Harrison, Ann H . Reece and Maloolm Johnson Department of Pharmacology, Imperial Chemical Industries, èlderley Park, Macclesfield, Cheshire, >>hgland . (Received in final form June 12, 1978) Prostacyclin is a potent inhibitor of platelet aggregation . Its release from the aorta of streptoaotooin-diabetic rate ie reduced . High rates of prostacyclin production appear to be incompatible with high blood glucose levels . It is possible that decreased prostaoyclin release may be related to vascular complications in diabetes . Patients with diabetes mellitus develop miorovascular camplicatione,(1) and have an increased susceptibility to atheroeclerosis (2~ and thrombosis (3) . Disorders of blood coagulation and platelet ftimctian in diabetes include decreased fibrinolytic activity (4), increased platelet reactivity (5,6) toward ~, collagen, adrenaline and araohidonic acid, increased spontaneous aggregation (7) and decreased platelet survival (8) and increased turnover (9) . Such abnormalities are particularly marked in diabetics with vascular complicatia~ne (10,11 . Platelet hyper-reactivity may be related to increased activity of the prostaglandin synthetase system (6) and a greater production of the pro-aggregating prostaglandin endoperoxidee G2 and H2 and thrombox$ae ~ . It has recently been reported that vascular endotHelium generates proetacyc in (PGI 2 ), a potent, but unstable, inhibitor of platelet aggregation (12,13) . A decreased production of PGI2 in diabetics could also be ezpected to contribute to the hypercoagulable state . To investigate this possibility we have estimated PGI 2 release from the aorta of rats with experimentally-induced diabetes . MEPHODS Male rats (Alderley Park strain, 90-120 g body weight were made diabetic by i .v . injection of streptozotocin (80 mg~kg), 1-3 months prior to use . Gluoosuria was canfixmed (~Clinistix~~ just before each ezperiment . Sex and age-matched rate were used ae ca~ntrols . Where indicated, soluble insulin (~Novo, 9otrapid MCP) was given e .c . (q00 II~rg) to some animals and food withdrawn simultaneously, 2~ hours before testing. In fasting ezperiments, food was withdrawn for 18 or 24 hrs before testing . The animals were anaesthetised with ether, and a blood sample (0 .5 ml~ taken for glucose estimation (14~, by ~Teohnioon Autoanalyser~ . The aorta was rapidly ezcieed, and following removal of the adventitial layer, was immersed in Srebe buffer at 4'C, containing 200 mg~] .00 ml glucose (unless otherwise stated . The tissue was weighed, cut into fine rings (1-2 mg) (12~ and incubated (60 mg~200 pl~ in Srebe buffer at 22 ° C for 3 min . 6liquote of the supernatant (1-lA W1~ were added to rat citrated platelet-rich plasma (15~, 30 sec before the addition of 0300.-1653/ 78 / 07240351502 , 00/ 0 Copyright © 1978 Pergamon Press
352
Proatacyclin is Diabetic Rate
Vol . 23, No . 4, 1978
a submaximal concentration of adenosine-5~-diphoephate (A .D .P ., 2 Wmol~l) . The activity of the supernatant in inhibiting platelet aggregation was measured in a double channel ag~egometer (~Paytan~) . The quantity of PGI2 was estimated from a standard ourve using authentic PGI 2 (sodium salt), and expressed as ng,/mg wet weight of tissue . RESUIfPS
Control
Diabetic
FIG . 1 Proatacyclin (ng~mg wet weight) released by the aorta of fed diabetic (blood glucose 412 .1 ± 15 .1 mg%100m1) and control (blood glucose 76 .7 ± 5 .0 mg~100m1) rata . I~bera of animals in each group are shown in parentheses . Values represent mean ± S .E, and p ~ 0 .02 where indicated (*) .
Vaeoular rings, incubated in vitro for 3 mina at room temperatw-.e released a potent inhibitor of ~~-induced platelet aggregation . This in~, t bitory activity was confirmed to be due to PGI by (a) loos of effect after boiling for 15 seas (16) and (b) relaxation ôf bovine coronary artery (17) . The production of PG2 2 by the aorta of fed diabetic rata was significantly depressed (p ~ 0 .02) when compared with that of control animals (Figure 1) . This was confirmed using aortic rings fra~m 24 hour fasted diabetic and oontrol rate where the amount of PGI2 released was 0 .28 ± 0 .05 and 0 .45 ± 0 .04 ng~mg respectively (p ~ 0 .05) .
Vol . 23, No . 4, 1978
Proatacyclia is Diabetic Rate
353
Prostacyolin was still significantly (p r 0 .05) reduced (0 .05 ± 0 .01 ~mg) when aortas from diabetic rate were incubated in Srebs buffer caataining levels of glucose similar to those found in ezperimental diabetes 4500 ml) and oompared with control aortas (0 .15 ± 0 .03 ng~mg) in OOml glucose . Proetacyclin relesée was estimated in a number of 70 ezperimente in which aortic rings were taken from fed and fa.eted rats and its relationship to blood glucose levels ezamined (Figure 2) .
Ioo
O lOD-
0
"ô .l 0
"
9~
" ~, o " °
"
.2 0
O
"
i8 "" ° o
ô 0
0.5 Q3 .01 Prostuyclin Irplmg i"t w~IgM ~orhl
"
ô o r06 .
Q7
FIG . 2 Aortio prostacyclin production (ng~mg wet weight) and blood glucose (mg%1OOm1) in diabetic fed ( " ) and fasted (Q ) and control fed ( " ) and fasted (0 ) rate .
High rates of PGI production did not appear to be compatible with high blood glucose . Thé effect of an acute, insulin-induced fall in blood glucose was therefore investigated . Figure 3 demonstrates that a reduction in blood glucose from 341 .0 ± 22 .6 mg~100m1 to 178 .9 ± 22 .6 m$/100m1 in diabetic rats did not restore PGI2 to control levels (control blood glucose 77 .0 ± 2 .1 mgß.00m1) . DISCIISSIO~P 7~e results indicate that the production of proetacyolin is reduced in the aorta of etreptosotocin.diabetic rats . Decreased vascular prostacyclin could reflect (1) endothelial cell loss or damage, (2) decreased . PGI 2 syutheeis, perhaps due to reduced absolute levels or availability of arachidonic acid, or decreased activity of ayclo-o~genase or proetacyclin
354 .
Proetacyclin is Diabetic Rats
Vol . 23, No . 4, 1978
synthetase, or (3) increased PGI 2 degradation . These defects msy be a consequence of the diabetic state or a specific effect of streptozotooin .
Dlsb~lk Disbstk Gmtrol + + + S~Iin Insulin Sallru
FIG .
3
Prostacycli.n (ng~mg wet weight) released by the aorta of (a) insulin-treated diabetic rats, (b) saline-treated diabetic and (o) saline-treated caatrol rate . l~bere of animals in each group are shown in parentheses . Values represent mean ± S .E, and p ~ 0 .01 where indicated (*) .
Fjyperglycaemia, or a conca~mitant metabolic abnormality of diabetes, may depress prosta.cyclin production ae it has been shown that increased glucose concentration (18) and osmolality (19) decrease proetaglandin " synthesis . Our finding that PGI is depressed is animals with high blood glucose tends to support this suggéstion,althougb. acute insulin treatment failed to restore PG22 to control values . The influence of chronic insulin administraticm ie currently under investigation . Our experiments have not rued out the possibility of a specifio effect of streptozotocin . This would have important consequenoes as this agent is widely used in experimental studies of the metabolio and. vascular camplioatia~ae of diabetes . However, the finding that proetacyolin ie depressed 12 months after a single dose of etreptozotocin (unpublished
Vol . 23, No . 4, 1978
Prostacyclin in Diabetic Rate
355
observations) suggests that a direot effect is unlikely . Erperimente are being oarried out in animals with epontaaeous diabetes to further investigate this possibility. The relevanoe of our fiadinge in experimental animals to the vascular complications of diabetes canaot be assessed until proetacyclin hoe been measured in vessels of humaa diabetics and in diabetic tissues, such ae retina and kidney, which are susceptible to microangiopathy . ACSNOWLPDGENlENTS We are indebted to Dr . S. Gibean, Chemistry Department, for synthetic prostacyolin and to Mise 3. Bradley and Mr . J . Clayton for technical aesieteace . R~tSPCS3 1. 2.
3. 4"
5. 6. 7" 8. 9. ]A . 11 . 12 . 13 " 14 . 15 . 16 . 17 "
le .
19 .
H. FAT and J. JARRETT (Hde), Complications of Diabetes , Arnold, London (1975) " H. ~P, Post~ad.Med .J . `~2 445-451 (1976) . W.R . TT ~~~, J.D . WARD, F .E . PBESTOA, T . DUCHWORTH,and B.C . O+MALLEY, Diabetologia 12 237-243 (1976) . G.R . F~!~~~ , R. CHAHÜABATI, and P .R . AVIS, Br .Med .J . _1 921-923 (1963) " H.C . gülAdH, J.A . COLWELL, S. CRUL, H. 3QWAHWELA, and J.G . DO~HBIE, J .Lab .Clin.Med . 8~ 236-246 (1972) . P.V . HALUSHKA, D. LIIRIE, and J.A . COLWELL, Hew Eng.,~.Med . 297 1306-1310 (1977) " g. BREÛDIH, H. GRDZP, H.R . HRZYWANEg, and W.P . 3CEIiErfl~, Thromb .Haemoetae . ~ 669-691 (1976) . J.C . FERGII80N, H. MACSAY, J.A.D . PHILIP, and D.J . SQtßdElt, C11n .Sci .Mo1 . Med. ~ 115-120 (1975) . J .A . COLWBLL, J . SAGEL, L. CROOS, A. CHAMBBR.S, and M. LAII~IIHS, Metabolism ~ 279-285 (1976) . H. H6A'fH, W.D . BRIGDEI~, J .V . CAHEVER, J . POLLOCg, P.R. HIIPTR$, J. SSLSSY, and A. BLO(H~S, Diabetoloaia 3 308-315 (1971) . B .C . O+MALLEY, W.R . mII+O~L~, J .D . WARD, H.R . PORTER, and F.E . PRBSPOH, Lancet 2 1274-1276 (1975) " S . M7HCADA, E .A. HIGGS, and J.R . VAHE, Lancet 1 18-21 (1977) " s. MoHCADA, R. GRYGL , s . BmaTnvG, aaa J .B. vAxE, Hature ~ 663-665 (1976) . D. BARHAM and P. ~~~, Anolyst ~ 142-145 (1972) . M. JC~PSOH, E. RAMEY, and P.~[ RAMFIEL?", Am .J .Phyeiol . ~ H381-H385 (1977} S. Nl7HCADA, R. GRYGLÉWSBI, S . EIIATIHG, and J .R . VAHE, Prostaglandine _12 715-736 (1976) . G .J . DQSTIHG, S . MCQPCADA, and J.R . VAHE, Proetaglandine ~ 3-15 (1977) " J. TAtar~AIIM, A .s . HzELS, and g. AIILS~ROOg, Fea .Proc. ~ 223 (1976) . D. VAH PRAGG and S .J . FAHB~, Fed .Proc . ~ 223 (1976 .
