Ascorbic acid enhances the formation of prostaglandin E1 in washed human platelets and prostacyclin in rat aortic rings

Prostaglandins

Leukotrienes

ASCORBIC ACID ENHANCES WASHED HUMAN PLATELETS

and Medicine

18:

227-233,

1985

THE FORMATION OF PROSTAGLANDIN El AND PROSTACYCLIN IN RAT AORTIC RINGS

IN

K.C. Srivastava, Department of Environmental Medicine, Institute of Community Health, Odense University, J.B. Winsl@ws Vej 19, DK-5000 Odense C, Denmark. ABSTRACT Effects of ascorbic acid at physiologically achieved concentrations were examined on the metabolism of exogenous dihomo-‘1 -1inolenic acid and arachidonic acid (AA) in washed human platelets, and of AA in rat aortic rings. In the presence of ascorbic acid an increased formation of PGFI, , PCEl and PCE2 was observed in platelets. Also this vitamin induced an increased production of, prostacyclin (measured as its stable metabolite 6-keto-PGF1, ) from exogenously provided substrate in aortic rings. In addition, from endogenous stores of AA in aortic rings ascorbic acid induced an increased generation of prostacyclin as revealed by inhibition of ADP-induced platelet aggregation. INTRODUCTION The biochemical function of ascorbic acid is not well understood. Experimental evidence suggests that this vitamin has a specific role in collagen synthesis failure of which results in scurvy with typical symptoms such as hemorrhages, loosening of the teeth, poor wound healing, and the easy fracturability of the bones. In recent years evidence has been accumulating that this vitamin plays a role in the metabolism of prostaglandin precursors (1,2). In this paper results are presented describing a selective role of this vitamin in the metabolism of dihomoY-linolenic acid (DHGLA) and arachidonic acid in human platelets, and of arachidonic acid in aortic rings of the rat, wherein it induced an increased formation of - both substances working as protective agents in the PGE 1 and prostacyclin development of cardiovascular disease. MATERIALS

AND METHODS

Labelled dihomo-Y -1inolenic acid and arachidonic acids were purchased respectively from The Radiochemical Centre, Amersham, England, and New England Nuclear. Standard prostaglandins of ‘1’ series were obtained from the Upjohn Company, Kalamazoo, Michigan, and those of ‘2’ series from ON0 Pharmaceutical Company, Osaka, Japan. The animals used were male albino SPS Wistar rats.

227

Platelet

incubation Venous blood from healthy donors who had not taken any medication for at least 10 days was collected into acid citrate-dextrose, and platelet suspensions were prepared by the method of Schmidt et al. (3). Platelet suspensions (200 ul containing 108 platelets) were first incubated with ascorbic acid (570 uM) for 5 min at OOC, followed by 5 min at 370C. The suspensions were cooled to OoC followed by addition of labelled prostaglandin precursors (DHCLA 0.30 uCi; AA 0.12 uCi). The suspensions were incubated for 10 min at 370C. The reaction was terminated by cooling the tubes on ice and adding I ml saline-HCI mixture to give a pH 3.0. Extraction of the incubation mixture was done in ethyl acetate twice (3 ml + 2 ml), and the solvent evaporated under vacuum. The residue thus obtained was resolved by TLC on silica gel G plates prepared in our laboratory by developing in a solvent system consisting of chloroform-methanol-acetic acid (90:8:6, v/v). The PG spots were revealed by iodine, marked, scraped off, and finally counted in a liquid scintillation counter. Aorta

incubation experiments Rats were killed by a blow on the head and exanguination. The thorax was opened and aorta removed. It was perfused in cold saline, and extra tissue was removed from it. The aorta was gently blotted on a piece of filter paper and stored at -7OOC until used in the incubation experiment. Aortae were collected from control rats used in some other experiment in our laboratory. They were cut into equal rings, which were cut open longitudinally (average area ca. 51 mm2) and placed in incubation tubes. From each rat, three pieces, each 1 cm, were used. Of these, one piece served as control and the other two as experimental (treated with two different ascorbic acid concentrations). To each tube were added 300 ul cold phosphate buffer (60 mM, pH 7.4 containing 2 mM EDTA) and the contents were incubated at 370C for 10 min. The incubate was rejected. The tubes were kept on ice, and to each tube were added 300 ul phosphate buffer and 5 ul ascorbic acid solution (0.57 and 1.0 mM); to controls were added 5 ul buffer. The tubes were incubated for 15 min at 37OC. Then they were cooled on ice for 5 min followed by addition of labelled AA (13 uM) and incubated for 30 min at 37OC. The reaction was mixture and cooling on ice. Arachidonic terminated by adding 1 ml saline-HCOOH acid (AA) metabolites were extracted in ethyl acetate (3 ml + 2 ml) and the solvent evaporated to dryness in vacuum. The residue was dissolved in 200 ul chloroformmethanol-ethyl acetate (2:l:l v/v) mixture; 25 ul were used for the resolution of 6keto-PGF1 a from other AA metabolites by TLC using the solvent ethyl acetateacetic acid-iso-octane-water (55:10:25:100 v/v, upper organic phase was used after 2-5 min of mixing). The spot due to 6-keto-PGFl, was revealed by iodine, marked, scraped off and finally counted in a liquid scintillation counter. Aggregation experiments I The effect of ascorbic acid on platelet aggregation induced by ADP, epinephrine, collagen and arachidonate was examined. Aggregation studies were carried out using human platelet-rich plasma (PRP). II In another set of aggregation experiments, the effect of ascorbic acid on the ability of rat aortic rings to produce prostacyclin judged by the latters inhibiting effect on ADP-induced platelet aggregation was examined. Prior to the aggregation step, incubation of aortic pieces was done as described below. TO the control and experimental plastic tubes, each containing a piece of fresh aorta (1 cm, opened longitudinally, area ca. 60 mm*) were added 800 ul cold tris-HCI (50 mM, pH 8.5) and incubated for 15 min at 370C. The experimental tube contained ascorbic acid (1.0 mM) in the incubation 228

medium. The incubates from both the tubes were carefully pipetted out and rejected. This step was necessary to minimize the effect of manipulation/sample handling on prostacyclin production. The tubes were kept in ice-cold water. Then to one tube at a time, beginning with the control, were added 600 ul tris-HCl and incubated at 370C for 7 min. From the incubation tube the entire incubate was transferred to another tube. Immediately I5 ul from this were transferred to the aggregation tube, the contents mixed for 30 set by stirring followed by addition of a threshold concentration of ADP which produced an irreversible aggregation in the PRP sample used for this purpose. The rest of the incubate was rejected. The incubation tube was kept in ice-cold water. To the experimental tube was added an equal volume of tris-HCl containing ascorbic acid (1.0 mM) followed by an incubation and aggregation procedure as described for the control. The rest of aortaincubate was rejected. Incubation and aggregation steps were repeated first with the controland then with the experimental aorta piece. Incubates were rejected. used in aggregation of The same aorta pieces were incubated and incubate PRP from another blood donor. The sequence was as follows: control, experimental, experimental, control. This experiment was performed using aorta pieces from three rats. RESULTS In the presence of ascorbic acid washed human platelets produced increased amounts of PCEl and PGFla (respectively 37 and 48% over control values) from labelled dihomo-Y-linolenic acid. Under similar incubation conditions platelets produced increased amounts of PGF2a, PGE2 and TxB2 (resp. 13, 33 and 10% over control values, a significant increase only in the case of PGE2)(Table 1). Table 2 presents data on the effect of ascorbic acid on the formation of ) in rat aortic rings from exogenous prostacyclin (measured as 6-keto-PGFb arachidonate. A significantly increased formation was observed at 0.57 and 1.0 mM ascorbic acid concentrations. Ascorbic did not show any effect on ADP-, epinephrine-, collagenand arachidonate-induced platelet aggregation. Figure 1 shows the effect of ascorbic acid on the production of prostacyclin by aortic rings of the rat. Prostacyclin formation was assessed by the ability of aorta incubates to inhibit ADP-induced platelet aggregation. As is obvious platelet aggregation was inhibited more by incubates containing ascorbic acid as compared to control without ascorbic acid.

DISCUSSION Some years ago it was demonstrated that platelet prostaglandin synthetase metabolized P&precursors - DHCLA and AA, differently in the presence of ascorbic acid (11, and ethyl alcohol (4) -in vitro at physiologically achievable concentration levels. In the presence of the two substances, platelets produced significantly more PCEl, PGFlo and TxBI compared to controls, whereas no such effect was observed on the metabolism of exogenous AA by platelets. Recently it was shown that ascorbic acid induced an increased formation of prostacyclin in aortic rings from several species, such as rat, guinea pig and rabbit (5). Further it was demonstrated that ascorbic acid prevented the decrease of endothelial production of PG12 in rabbits during experimental atherosclerosis, and delayed the development of atherosclerotic lesions (6). These observations have added a new dimension to our understanding of the many diversified effects of ascorbic acid. In 229

Table 1. Effect of ascorbic washed human platelets

acid

PCPl C. Control +Ascorbic

Control +Ascorbic

acid (n=4)

69X+3 16 1036+140*

on the metabolism

PGEf

of labelled

PCP2 u

DHGLA

TxB2

and AA !n

_--_

PGE2

8175216 1120+349*

3515136 399+ 62

acid (n=6)

633452810 700452586

7975322 10625446”

The values (DPM, Mean + SD) are l/X of the total amount produced by IO8 platelets in the incubation medium. *p < 0.05 (Student’s t-test for paired data)

230

Table 2. Effect arachidonate

of ascorbic

acid on the formation

of 6-keto-PGFl,

pmola/aorta Control + Ascorbic + Ascorbic * p < 0.01

area of aorta

labelled

min

21.3 + 2.9 32.9 + 2.2* 37.2 + 3.8”

acid (0.57 mM) acid (1.0 mM) average

piece/30

from

piece = 50.8 mm2

a Mean + SD (n=5)

With fresh aortic rings (i.e., not stored frozen) an increased production of 6-ketoPGFlu and PGE2 (respectively by 48 and 39%, p < 0.01) was observed in the in tris-HCl (50 presence of ascorbic acid (1.0 mM). The aortic rings were incubated mM, pH 8.5). Amounts (pmol, Mean + SD, n = 4)(control/experimental) of 6-ketoPGFl u: 50 + 23174 + 21, and of PGE2: 115 + 34/160 + 44 were produced per aorta piece (56 mm2) during 30 min incubation. Statistics: Student’s t-test for paired data.

x Addition

of an

Figure 1. Effect of ascorbic acid on the platelet aggregation induced by ADP apparently due to an increased formation of prostacyclin from rat aortic rings. A. Aggregation curves with the PRP from blood of a donor. 1. ADP (1.5 uM); 2. ADP (2.0 uM) - other aggregation curves were obtained with 2.0 uM ADP; 3. control, treated with aorta incubate without ascorbic acid; 4. experimental, treated with aorta incubate with ascorbic acid; 5. control; 6. experimental. B. Aggregation curves with the PRP from blood of another donor. 1. ADP (2.0 uM) 2. control; 3. experimental; 4. experimentah 5. control. Incubates from the same two aorta pieces were used with A and B (see under Methods: Aggregation experiment II). The numbers of the aggregation curves represent the sequence in which aggregation was performed. 231

situations where there is an increased risk of thromboembolic disease (ageing, smoking, estrogen therapy, pregnancy, infection, trauma, surgery, soft water consumption and winter), all are associated with low blood ascorbic acid level (7). Our data confirm the results of earlier studies in regard to increased platelet formation of PGEl and PGFl, from DHGLA in the presence of ascorbic acid. However, we did not observe as great an increase as reported earlier (I). This could be because of our radiolabelled DHGLA solution, though stored under nitrogen at -3OoC, was more than two years old. In contrast to the earlier report (1) we observed an increased formation of the AA metobolites (PGF2,, PGE2 and TxB2) in platelets, though this was significant (PcO.05) only in the case of PGE2. The formation of PGs is controlled at several steps of the AA cascade: as for example, release of AA by the activation of a phospholipase or lipase, conversion of the released AA into PC-endoperoxides by cyclooxygenase, and finally generation of several metabolites both enzymatically and non-enzymatically from the pivotal PC-endoperoxides. The first step of the cascade is of great significance in the production of AA metabolites both under physiological and disease situations. However, desirable effects of drugs and other chemicals that may enter the organism may not necessarily be mediated through the release of AA from lipid stores: instead they may influence one or more of the subsequent steps. As far as the effect of ascorbic acid on the increased formation of PGI2 in aortic rings is concerned, it might exercise its influence at two points on the cascade. Firstly, it might be protecting the cyclooxygenase from the deleterious effects of high concentrations of PGG2 (a hydroperoxide) by converting it into PGH2 with the help of peroxidase and, secondly it might protect PG12 synthetase by scavanging hydroxyl radicals which are produced from hydroperoxides. 15hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) was first demonstrated to specifically inhibit PG12 synthetase, and later hydroperoxides of other fatty acids were shown to possess a similar effect. It has been shown that inactivation of PG12 synthetase by 15-HPETE involves an hydroxyl radical or a species of similar reactivity (8). The inhibition of PC12 synthetase by a variety of hydroperoxides seems to suggest that a common reactive species is involved in this process (9). The strong anti-oxidation property of ascorbic acid, would support these ideas (Fig. 2). A similar argument has been put forward for the increased PG12 formation in blood vessels in the presence of dipyridamole (10). Moreover, it has been shown that the inhibitory effect of 15-HPETE on vascular PG12 synthesis was completely abolished by ascorbic acid (5). Further, an increased formation of PGE2 from added AA in the aortic rings in the presence of ascorbic acid suggests that it protects the cyclooxygenase enzyme. lor

AA

cycle oxygenase

hif” cont.

b P;G2

e?y

ascorbic acid

PGH2

yyyt!hetase

b PGI2

ascorbic acid,,

Figure 2. Proposed mechanism for the prolongation rings treated with ascorbic acid. AA; arachidonic prostaglandin-endoperoxides possessing respectively droxyl group at C-15.

232

of

PC12 formation by aortic acid; PGG2 and PGH2 are a hydroperoxy (OOH) or hy-

On the basis of the arguments given above, one would expect an increased production of PCs and TX in platelets in the presence of ascorbic acid from both DHGLA and AA. Significantly increased formation of ‘1’ series PCs has indeed been reported without any such increase of ‘2’ series PCs (1). Inspite of our observation on the increased formation of ‘2’ series PCs in platelets in the presence of ascorbic acid, though significant only in the case of PGE2, the over all desirable effects of ascorbic acid on the metabolism of DHGLA and AA warrant a systematic study with humans. ACKNOWLEDGEMENTS The author wishes to thank the personnel of the Blood Bank, Odense University Hospital for the procurement of blood samples. Mrs. Ruth 8. Alexandersen provided technical assistance. Mrs. Inge Bogelund typed the manuscript. REFERENCES 1.

Manku, M.S., Oka, M., Horrobin, D.F. Differential regulation of the formation of prostaglandins and related substances from arachidonic acid and from dihomogammalinolenic acid. II. Effects of vitamin C. Prostaglandins and Medicine 3: 129, 1979.

2.

Van Dorp, Biochemical

3.

Schmidt, K.G., Rasmussen, J.W. Preparation of platelet suspensions from whole blood in buffer. Description of a method which gives a large platelet yield. Scandinavian Journal of Haematology 23: 88, 1979.

4.

Manku, M.S., Oka, M., Horrobin, D.F. Differential regulation of the formation of prostaglandins and the related substances from arachidonic acid and from dihomogammalinolenic acid. I. Effects of ethanol. Prostaglandins and Medicine 3: 119, 1979.

5.

Beetens, J.R., Herman, A.G. Vitamin C increases the formation of prostacyclin by aortic rings from various species and neutralizes the inhibitory effect of 15-hydroperoxy-arachidonic acid. British Journal of Pharmacology 80: 249, 1983.

6.

Beetens, J.R., Coene, M.-C., Verheyen, A., Zonnekeyn, L., Herman, A.G. Influence of vitamin C on the metabolism of arachidonic acid and the development of aortic lesions during experimental atherosclerosis in rabbits. Biomedica Biochimica Acta 43: 273, 1984.

7.

Clemetson, C.A.B. Some thoughts on the epidemiology of cardiocascular disease. Role of ascorbic acid. Medical Hypotheses 5: 825, 1979.

8.

Weiss, S.J., Turk, J., Needleman, P. A mechanism for the hydroperoxidemediated inactivation of prostacyclin synthetase. Blood 53: 1191, 1979.

9.

Salmon, J.A., Smith, D.R., Flower, R.J., Moncada, S., Vane, J.R. Further studies on the enzymatic conversion of prostaglandin endoperoxide into prostacyclin by porcine aorta microsomes. Biochimica Biophysics Acta 523: 250, 1978.

10.

Deckmyn, H., Gresele, prostacyclin production 1984.

D.A. Aspects Pharmacology

of the biosynthesis 3: 71, 1967.

of prostaglandins.

Progress

in

P., Arnout, J., Todisco, A., Vermylen, J. Prolonging by nafazatrom or dipyridamole. The Lancet, ii: 410,

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