Alterations of the prostacyclin-thromboxane ratio in streptozotocin induced diabetic rats

Prostaglandins Leukotrienes and Medicine 8:

93-103, 1982

ALTERATIONS OF THE PROSTACYCLIN-THROMBOXANE RATIO IN STREPTOZOTOCIN INDUCED DIABETIC RATS C.W. Karpen, K.A. Pritchard J r . , A.J. Merola and R.V. Panganamala, Department of Physiological Chemistry, The Ohio State University, Columbus, Ohio 43210. [Reprint requests to R.V.Po] ABSTRACT Thrombin induced thromboxane A2 and prostaglandin E2 production were s i g n i f i c a n t l y increased in platelets of streptozotocin induced diabetic rats as compared to non-diabetic control rats, while collagen induced thromboxane A2 production was decreased. Using exogenous arachidonic acid, prostaglandin E2 production, but not thromboxane A2 production, was increased in platelets from streptozotocin treated animals. Prostacyclin production in the diabetic aorta was s i g n i f i c a n t l y lowered; however, control levels of prostacyclin production resulted after incubation of the tissue with d i p y r i damole. Diabetic animals demonstrated a f i v e f o l d decrease in the endogenous arterial prostacyclin/platelet thromboxane A2 r a t i o when thrombin or ADP was used to induce thromboxane A2 production. This elevated r a t i o could be a c o n t r i buting factor to the vascular complications of diabetes. Dipyridamole, due to i t s a b i l i t y to p a r t i a l l y normalize this r a t i o , may be useful as a therapeutic agent i~ this and related vascular diseases. INTRODUCTION Increased deacylation of phospholipids (1), increased thrombin and ADP induced aggregation (2), and increased thrombin induced release of serotonin (2) have been reported in association with platelets from rats with streptozotocin induced diabetes. Vascular tissue from these rats has been shown to generate decreased amounts of prostacyclin (1,3,4). This decreased prostacyclin (PGI2) production, however, was normalized by the administration of insulin (5), by the antithrombotic drug Bay 6575 (3), and by pancreatic transplantation ( I ) . The present investigation, using radioimmunoassay (RIA) and 14C-incorporation techniques, complements and extends the above studies by demonstrating the changes in platelet thromboxane A2 (TxA2),

93

prostaglandin E2 (PGE2), and a r t e r i a l PGI2 in rats rendered diabetic with streptozotocin. MATERIALS AND METHODS Streptozotocin, bovine thrombin, bovine albumin (BA), bovine achilles tendon collagen, and adenosine diphosphate (ADP) were purchased from Sigma Chemical Co. Keto-Diastix urine t e s t s t r i p s were purchased from Ames D i v i s i o n , Miles Laboratories, Inc. Soluble calfskin collagen was from Worthington Biochemical Corp. Prostaglandins were a kind g i f t from Dr. John Pike (UpJohn), and arachidonic acid (AA) was purchased from Nu Chek Prep. 1-14C-arachidonic acid (sp. act. = 52.7 mCi/mmol), JH-thromboxane B2 (sp. act. = 150 Ci/mmol), JH-PGE2, (sp. act. = I00 Ci/mmol), and 3H-6-keto-PGFl~ (sp. act. = 100 Ci/mmol) were purchased from New England Nuclear Corp, Dipyridamole (Persantine) was kindly supplied by Boehringer Ingelheim L t d . , Ridgef i e l d , CT. ADP and thrombin were dissolved in calcium-free Krebs buffer. Arachidonic acid, thromboxane B2 (TxB2), PGE2 ,and 6-keto-PGFl~ were dissolved in ethanol, stored at -70°C, and d i l u t e d with TRIS(5On~I)-BA(O.I%) buffer (pH = 7.6) for RIA. PGI 2 in TRIS(5OnC~)-NaCl (150mM) buffer (pH 9.0) was stored at -70°C. Bovine achilles tendon collagen suspension was prepared according to Nakanishi et al. (6), and the collagen concentration was determined using the me~od--of Lowry et al. (7), with soluble calfskin collagen as the standard, Animals Male Sprague-Dawley rats (lO0-120gm) were injected through a t a i l vein with streptozotocin (80 mg/kg) in 0.05 M c i t r a t e buffer (pH 4.5) or with c i t r a t e buffer alone. Glucosuria was evident w i t h i n 5 days and persisted throughout the study. G!ucose Assay Plasma glucose was measured using a commercial k i t supplied by Bio Dynamics/ BMC. The assay u t i l i z e s a glucose oxidase reaction coupled to a peroxidase reaction with spectrophotometric q u a n t i t a t i o n using 4-aminophenazone as the color reagent (8). ! n s u l i n Assay Plasma i n s u l i n was measured with RIA using a guinea pig anti-porcine i n s u l i n antiserum that is 100% cross-reactive with rat i n s u l i n . Rat i n s u l i n standards were used f o r c a l i b r a t i o n . Bound and free immunoreactive i n s u l i n were separated by polyethylene glycol p r e c i p i t a t i o n (9). Platelet Oxygenation of Arachidonic Acid Washed platelets were prepared as described e a r l i e r (10), and suspended in TRIS(5OmM)-NaCI(15On~M)-EDTA(ImM) buffer (pH 7.4). The p l a t e l e t count (phase contrast microscopy) was adjusted to 2 x lOm/O.5 ml incubation volume. Platelets were s t i r r e d at 37°C for one minute after the addition of 1.0 nmole 1-14C-arachidonic acid, and the reaction terminated with 200 ~I 1N HCI. The products were extracted twice with 3 ml diethyl ether, the ether

94

evaporated under N2, and the residue dissolved in methanol. Recovery of r a d i o a c t i v i t y was over 90%. The extract and authentic prostaglandins were spotted on a Whatman LK6D t h i n layer chromatographic (TLC) plate, and developed e i t h e r once in solvent system l-chloroform:methanol:acetic acid, (180:10:10)-to separate prostaglandins, or twice in solvent system r l heptane:diethyl ether:acetic acid, (60:40:1)-for separation of arachidonic and hydroxyfatty acids. Radioactive peaks were located using a Packard 7220/21 radiochromatogram scanner, and matched with the authentic prostaglandins and arachidonic acid which were visualized using iodine vapors. Peaks were scraped and quantified by l i q u i d s c i n t i l l a t i o n (Beckman LS 8100). R a d i o a c t i v i t y was converted to picomoles using the specific a c t i v i t y of the arachidonic acid and quench correction. Platelet TxB2 and PGE2 Washed platelets were prepared as above and suspended in Krebs buffer. The p l a t e l e t count was adjusted to I x I08/0.5 ml incubation volume. Platelets were s t i r r e d with collagen, thrombin, ADP, or arachidonic acid at 37°C, the reaction terminated with 1N HCl, and the products extracted with 3 ml diethyl ether. Recovery of TxB2 and PGE2 was greater than 90%. After ether evaporation, the residue was dissolved in TRIS-BSA buffer for RIA of TxB2 and PGE2. Cross r e a c t i v i t y of the TxB2 antiserum was as follows: PGD2, 1.6%; PGE2, 0.021%; PGF2m, 0.26%; arachidonic acid 0.001%. Cross r e a c t i v i t y of the PGE2 antiserum was as follows: PGD2, 0.051%; PGF2~, 0.31%; TxB2, 0.0078%; arachidonic acid, 0.001%. A o r t i c PGI2 Production from Exo~nous Arachidonic Acid A o r t i c PGI2 release was determined by measuring the formation of 6-keto -PGFlm. The procedure was performed as previously described ( I i ) . 1-14C-arachidonic acid (8.8 nmoles) was incubated with 10-15 mg a o r t i c tissue in 0.5 ml TRIS-NaCI-EDTA buffer (pH 8.0) at 37°C. Aliquots were withdrawn at 15,30,60, and 90 minutes and analyzed for 6-keto-PGFlm formation using TLC separation and l i q u i d s c i n t i l l a t i o n ( I I ) . Aortic PGI2 Production from Endogenous Substrate A.

RIA of 6-keto-PGFlm

Aortic t i s s u e was prepared as above and 10-12 mg slices incubated at room temperature in 0.5 ml TRIS-NaCI buffer (without EDTA, pH 8.0). Aliquots were withdrawn at 30, 60, and 90 minutes i n t o TRIS-BSA buffer for RIA. Incubations for dipyridamole studies were identical except that the buffer (I ml) contained dipyridamole dissolved in ethanol ( 2 . 5 ~ I ) , and the incubations were performed at 37°C. Cross r e a c t i v i t y of the 6-keto-PGFlm antiserum was as follows: 0.02%; PGE2, 0.15%; PGF2m, 0.10%; arachidonic acid, 0.005%.

95

PGD2,

B.

PGI2 Bioassay

A o r t i c PGI 2 production was measured by the a b i l i t y of PGI2 to i n h i b i t ADP induced p l a t e l e t aggregation. Washed p l a t e l e t s from a normal control rat were prepared as above and suspended in Krebs b u f f e r (9 p a r t s ) and homologous p l a t e l e t - f r e e plasma (1 p a r t ) . The f i n a l p l a t e l e t count was adjusted to Ix10~/0.5 ml incubation volume, and p l a t e l e t aggregation was performed as described e a r l i e r (12). Aortas were prepared as above and incubated at 25°C i n TRIS-NaCI b u f f e r (pH 9,0). Aliquots of the a o r t i c supernate were added t o the p l a t e l e t suspension at 37% and incubated f o r 30 seconds p r i o r to a d d i t i o n of 1.2-2.4 ~M ADP. A standard curve was prepared by p l o t t i n g the maximal slopes of aggregation using authentic PGI 2 standards, The PGI 2 in the samples was measured by comparing the maximal slopes of the samples to those of the standards. S t a t i s t i c a l Analysis Significance of differences between means was determined using Student's o n e - t a i l e d t t e s t . Data is presented as mean ± SEM, and numbers in parentheses represent the number of animals in each group. RESULTS Diabetic Model Diabetic r a t s , as compared to age-matched c o n t r o l s , f a i l e d to gain weight and demonstrated hyperglycemia and hypoinsulinemia. Plasma glucose (mM) and i n s u l i n (ng/ml) were 8.8 ± 0,2 and 15.8 ± 3.3, r e s p e c t i v e l y , f o r c o n t r o l s , and 32.6 ± 3.0 and 1.2 ± 0.6, r e s p e c t i v e l y , f o r d i a b e t i c s . Oxygenation of 14-C-Arachidonic Acid in Washed P l a t e l e t s As seen in Table 1, conversion of exogenous arachidonic acid i n t o HETE (I 2-hydroxyei cosatetraenoi c a c i d ) , HHT (I 2-hydroxyheptadecatri enoi c aci d), and TxB2 by the d i a b e t i c p l a t e l e t s was not d i f f e r e n t from control pl atel ets. PGE2 formation was s i g n i f i c a n t l y higher (p
HETE 20.8±3.1 20.1±3.1

96

HHT 24.8±0.4 25.1±1.8

PGE2 2.4±0.2 3.8±0.8

TxB2 29.7±0.6 30.0±1.4

TxA2 and PGE? Generation by Washed Platelets

Figures 1 (a) and 1 (b) show the time dependent production of TxB2 and PGE2 induced by thrombin. These data c l e a r l y show t h a t p l a t e l e t s from d i a b e t i c rats generate increased amounts of TxB2 and PGE2 compared to control p l a t e l e t s . In the next experiment, TxB2 and PGE2 synthesis was measured a f t e r the a d d i t i o n of arachidonic acid to the p l a t e l e t suspension. There was a s i g n i f i c a n t increase in the amount of PGE2 produced from the d i a b e t i c p l a t e l e t s (diabetic = 50 ± 5; and control = 28 ± 3 pmoles/lO 8 p l a t e l e t s , p<0.0025). This difference, however, was not reflected in TxB2 synthesis (diabetic = 250 ± 14, and control = 282 ± 18 pmoles/ 108 p l a t e l e t s ) . 90 p
C = Control

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97

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98

Release of PGI2 From Aorta Figure 2 depicts release of PGl2-1ike a c t i v i t y normal and diabetic rats measured at d i f f e r e n t from diabetic rats released s i g n i f i c a n t l y less controls. Differences were s i g n i f i c a n t at all in Fig. 2.

from the thoracic aorta of times with bioassay. Aortas PGI2 compared to aortas from times of incubation as shown

In the next experiment, using d i f f e r e n t groups of control and diabetic rats, we measured the formation of 6-keto-PGFla by RIA using thoracic aortas.

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99

Figure 3 shows the decreased 6-keto-PGFl~ formation from the aortas of d i a b e t i c r a t s . S i m i l a r r e s u l t s were obtained with abdominal aortas. To ascertain the d i f f e r e n c e between control and d i a b e t i c r a t a r t e r i a l PGI 2 production,we incubated the t h o r a c i c aorta w i t h 1-14C - arachidonic acid and measured ~C-6-keto-PGFl~ by TLC. Figure 4 shows a time-dependent decrease of 6-keto-PGFl~ synthesis from added arachidonic acid i n the aortas of d i a b e t i c r a t s . S i m i l a r r e s u l t s were obtained from the incubation of abdominal aorta.

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Stimulation of Control and Diabetic PGI? Production by Dipyridamole Dipyridamole (IO0~M) stimulated control PGI2 production, as measured by 6-keto-PGFl~ RIA, from a mean of 62 + 5 to 85.5 + 4.5 picomoles/mg t i s s u e , a 38% s t i m u l a t i o n (p<0.0025). Diabet--ic a o r t i c PGI 2 production increased from a mean of 31.5 ± 2.5 to 54.5 + 3.0 picomoles/mg t i s s u e with the a d d i t i o n of IO0~M dipyridamole, a 73% s t i m u l a t i o n (p
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60 80 IO0 TIME (rain) Fig. 4. Time dependent synthesis of prostacyclin estimated by TLC in control (C) and diabetic (D) r a t thoracic (T) aorta. DISCUSSION The present study shows s i g n i f i c a n t changes in TxB2 and PGE2 formation from endogenous substrates in streptozotocin induced diabetic p l a t e l e t s . TxB2 synthesis in diabetic platelets incubated with arachidonic acid was unchanged. This f i n d i n g is in agreement with Gerrard et al. ( I ) , and points to the a v a i l a b i l i t y of arachidonic acid from p l a t e l e t phospholipids as the aberrent point in diabetic p l a t e l e t arachidonate oxygenation. The observed increment in PGE2 synthesis is consistent with the report of Halushka e_t_t a l . ( 1 3 ) using diabetic human p l a t e l e t s . Either a higher a c t i v i t y of cyclooxygenase or PGE2 isomerase in diabetic p l a t e l e t s is compatible with these data. We found (data not presented) that rat p l a t e l e t TxB2 production from exogenous arachidonic acid is rapid and reaches completion in 2 minutes, whereas PGE2 production proceeds at a slower rate, reaching completion in I0 minutes. Possibly, changes in a v a i l a b i l i t y of endoperoxide in diabetic platelets are not reflected by TxB2 production, but due to the slower nature of PGE2 production, are reflected in PGE2 synthesis. HETE formation induced with 14C-arachidonic acid was the same for control and diabetic p l a t e l e t s . We demonstrated that diabetic rat p l a t e l e t synthesis of TxB2 and PGE2 is more sensitive to thrombin and ADP when compared to control p l a t e l e t s . Enhancement of p l a t e l e t aggregation induced by thrombin and ADP in d i a b e t i c r a t s has been reported by Eldor et al. (2). Gerrard et al. ( I ) however, using diabetic rat platelets pre-labelled with 14C-arachidonic acid, f a i l e d to show s i g n i f i c a n t changes of TxB2 formation.

101

Eldor et a l . (2) showed a decrease in d i a b e t i c rat p l a t e l e t aggregation induced by collagen when the aggregation was performed i n PRP. They found no d i f f e r e n c e in aggregation between d i a b e t i c and control p l a t e l e t s when aggregation was performed w i t h washed p l a t e l e t s . Our r e s u l t s show no s i g n i f i c a n t difference in collagen induced TxB2 or PGE2 production between p l a t e l e t s of d i a b e t i c and control r a t s . The reason f o r the f a i l u r e to f i n d differences i n collagen stimulated TxB2 and PGE2 production in d i a b e t i c p l a t e l e t s is unclear. Decreased PGI 2 production in d i a b e t i c rat aorta has been reported (1,3,4). This study confirms a decrease in PGI2 production by d i a b e t i c r a t aorta both from exogenous and endogenous arachidonic acid. We found t h a t dipyridamole, an a n t i t h r o m b o t i c drug and s t i m u l a t o r of PGI 2 synthesis (14), e s s e n t i a l l y restored the decreased PGI 2 release from d i a b e t i c a o r t i c t i s s u e when incubated with the a o r t i c s l i c e s . Although the mechanism of s t i m u l a t i o n has not been c o n c l u s i v e l y determined, dipyridamole has been shown to i n h i b i t p l a t e l e t aggregation (15) and p l a t e l e t adenosine uptake (16). The s t i m u l a t i o n of PGI 2 production from aortas of d i a b e t i c rats by dipyridamole implies t h a t the t i s s u e contains active PGI 2 synthetase t h a t can be stimulated to release control l e v e l s of PGI 2, but does not suggest that a d d i t i o n of dipyridamole normalizes the t i s s u e enzyme, since the control aortas incubated w i t h dipyridamole produce greater amounts of PGI 2 than the d i a b e t i c aortas incubated with dipyridamole. Owing to the opposing actions so potently displayed by PGI 2 and TxA2 on p l a t e l e t f u n c t i o n , the balance between PGI 2 and TxA2 is probably a more c r i t i c a l parameter than the absolute q u a n t i t i e s of each prostanoid alone. When the molar a o r t i c P G l 2 / p l a t e l e t TxA2 r a t i o is c a l c u l a t e d using thrombin as a TxA2 inducer, the r a t i o in the d i a b e t i c rats is 0.4 whereas f o r normal rats the r a t i o i s 2.0° I f the decreased r a t i o is r e l a t e d to the in vivo s i t u a t i o n , t h i s altered r a t i o might r e f l e c t a p r e d i s p o s i t i o n of the d i a b e t i c rats to a pro-thrombotic s t a t e , and c o n t r i b u t e to the reported development of vascular lesions with appearance of p l a t e l e t s in the vascular wall (17). Important in f u t u r e studies w i l l be the study of agents, such as dipyridamole, t h a t might help normalize the steady state in vivo PGI2/TxA 2 r a t i o . ACKNOWLEDGEMENTS This study was supported in part by Research Grants HL-23439 from the National I n s t i t u t e s of Health and by Grants from the Central Ohio Heart Chapter, Inc. Antibodies were the kind g i f t of Dr. L. Levine. REFERENCES I.

Gerrard JM, S t u a r t MJ, Rao GHR, Steffes MW, Mauer SM, Brown DM, White JG. A l t e r a t i o n in the balance of prostaglandin and thromboxane synthesis in d i a b e t i c r a t s . J Lab C l i n Med 95:950-958, 1980.

2.

Eldor A, Merin S, Bar-On H. The e f f e c t of s t r e p t o z o t o c i n diabetes on p l a t e l e t f u n c t i o n in r a t s . Thromb Res 13:703-714, 1978.

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Carreras LO, Chamone DAF, Klerck P, Vermylen Jo Decreased vascular prostacyclin (PGI2) in diabetic rats. Stimulation of PGI 2 release in normal and diabetic rats by the antithrombotic compound Bay 6575. Thromb Res 19:663-670, 1980.

4.

Harrison HE, Reece AH, Johnson M. Decreased vascular prostacyclin in experimental diabetes. Life Sci 23:351-356, 1978.

5.

Harrison HE, Reece AH, Johnson M. Effect of insulin treatment on prostacyclin in experimental diabetes. Diabetologia 18:65-68, 1980.

.

Nakanishi M, Imamura H, Goto R. Potentiation of the ADP-induced platelet aggregation by collagen and i t s i n h i b i t i o n by a tetrahydrothienopyridine derivative (Y-3642). Biochem Pharmacol 20:2116-2118, 1971.

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Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 193:163-165, 1951.

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Trinder P. Determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor. Ann Clin Biochem 6:24-27, 1969.

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Desbuguois B, Aurbach GD. Use of polyethylene glycol to separate free and antibody-bound peptide hormones in radioimmunoassays. J Clin Endocrinol Metab 33:732-738, 1971.

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Gwebu ET, Trewyn RW, Cornwell DG, Panganamala RV. Vitamin E and the inhibition of platelet lipoxygenase. Res CommChem Pathol Pharmacol 28:361-376, 1980.

II.

PanganamalaRV, Gillespie A, Merola AJ. Assay of prostacyclin synthesis in intact aorta by aqueous sampling. Prostaglandins 21:I-7, 1981.

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Panganamala RV, M i l l e r JS, Gwebu ET, Sharma HM, Cornwell DG. Differential i n h i b i t o r y effects of vitamin E and other antioxidants on prostaglandin synthetase, platelet aggregation and lipoxidase. Prostaglandins 14:261-271, 1977.

13.

Halushka PV, Lurie D, Colwell JA. Increased synthesis of prostaglandin-E-like material by platelets from patients with diabetes mellitus. N Eng J Med 297:1306-1310, 1977.

14.

Blass KE, Block HU, Forster W, Ponicke K. Dipyridamole: A potent stimulator of prostacyclin (PGI2) biosynthesis. Br J Pharmac 68:71-73, 1980.

15.

Ally AI, Manku MS, Horrobin DF, Morgan RO, Karmazin MM, Karmali RA. Dipyridamole: A possible potent i n h i b i t o r of thromboxane A2 synthetase in vascular smooth muscle. Prostaglandins 14:607-609, 1977.

16.

Moncada S, Korbut R. Dipyridamole and other phosphodiesterase inhibitors act as antithrombotic agents by potentiating endogenous prostacyclin. Lancet I:1286-1289, 1978.

17.

Reinila A. Long-term effects of untreated diabetes on the arterial wall in rat: An ultrastructural study. Diabetologia 20:205-212, 1981. 103