Changes in erythrocyte sodium, sodium transport and 3H ouabain binding capacity during digoxin administration in the pig

Life Sciences, Vol. 32, pp. 747-754 Printed in the U.S.A.

Pergamon Press

CHANGES IN ERYTHROCYTE SODIUM, SODIUM TRANSPORT AND 3H OUABAIN BINDING CAPACITY DURING DIGOXIN ADMINISTRATION IN THE PIG 3. Whittaker, M. Hawkins, R. Swaminathan

Department of Chemical Pathology, University of Leeds, Leeds LSI 3EX (Received in final form November 2, 1982) Summary Time course3of the changes i n e r y t h r o c y t e sodium c o n t e n t , sodium t r a n s p o r t , H ouabain b i n d i n g c a p a c i t y and Na+,K+-ATPase a c t i v i t y were measured f o r 14 weeks, in 6 young pigs t r e a t e d w i t h d i g o x i n and i n 6 c o n t r o l p i g s . A f t e r one week o f treatment the e r y t h r o e y t e sodium content increased from 5.4 mmol/kg c e l l s to 6.9 mmol/kg c e l l s and the e f f l u x r a t e constant o f sodium decreased. With prolonged treatment the e r y t h r o e y t e sodium content returned to normal and the H ouabain b i n d i n g c a p a c i t y increased by week 5. The plasma d i g o x i n c o n c e n t r a t i o n decreased from 1.1 ng/ml at week 5 t o 0.6 ng/ml at week 8 probably due t o the d e c l i n e i n dose (pg/kg) o f d i g o x i n w i t h age. The e f f l u x r a t e constant o f sodium and Na+,K+-ATPase a c t i v i t y were h i g h e r towards the end o f t r e a t m e n t . I t i s concluded t h a t w i t h prolonged a d m i n i s t r a t i o n of d i g o x i n t h e r e i s an increase in e r y t h r o c y t e sodium pump u n i t s . Digoxin has been used s u c c e s s f u l l y i n the treatment o f h e a r t disease f o r s e v e r a l decades. I t has been suggested t h a t the membrane bound enzyme Na+,K +ATPase i s the r e c e p t o r f o r d i g o x i n in the h e a r t ( I ) . The i n h i b i t o r y e f f e c t o f d i g o x i n on Na+,K+-ATPase could be demonstrated i n many t i s s u e s i n c l u d i n g erythrocytes. Aronson et a l (2) attempted to use i n h i b i t i o n o f e r y t h r o c y t e sodium pump as a b i o l o g i c a l index of d i g o x i n t r e a t m e n t . Such an in d e x was considered because plasma d i g o x i n l e v e l s are not always h e l p f u l i n the diagnosis o f d i g o x i n t o x i c i t y (3). However, when e r y t h r o c y t e sodium was examined i n d i g o x i n t r e a t e d p a t i e n t s , i t was observed t h a t i n p a t i e n t s t r e a t e d w i t h d i g o x i n f o r a s h o r t time (2 weeks) the e r y t h r o c y t e sodium was high but i n p a t i e n t s " t r e a t e d w i t h d i g o x i n f o r l o n g e r periods (>2 months) the e r y t h r o c y t e sodium was not e l e v a t e d ( 4 , 5 , 6 , 7 ) . 3Ford e t a l (4) also showed t h a t i n c hr onic d i g o x i n treatment the number o f H d i g o x i n b i n d i n g s i t e s was not low and we have shown an e l e v a t e d Na+,K+-ATPase a c t i v i t y i n e r y t h r o c y t e s from c h r o n i c a l l y t r e a t e d p a t i e n t s (6). However, i n a l l these s t u d i e s l o n g i t u d i n a l data was not a v a i l a b l e . We have examined the time course o f the e f f e c t s o f d i g o x i n adminis t r a t i o n on e r y t h r o c y t e s i n p i g s . Pig was chosen as the e x p e r i m e n t a l animal as the p i g e r y t h r o e y t e sodium content l i k e i n humans and u n l i k e i n the dog i s low (8). M a t e r i a l s and Methods Twelve young pigs (8 weeks of age and weighing between 13-17 kg) from the same litter were allocated to two groups so as to match weight and sex. Animals were fed individually twice a day. One group was given digoxin 0.5 mg (approx. 0.035 mg/kg) daily for 3 days and then given a maintenance dose 0024-2305/83/070747-08503.00/0 Copyright (c) 1983 Pergamon Press Ltd.

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o f d i g o x i n f o r 14 weeks. The maintenance dose was 0.25 mg i n i t i a l l y which was increased to 0.375 mg d a i l y from week i i . The d i g o x i n t a b l e t s were put i n w i t h a small amount o f feed in the morning and i t was made sure t h a t the t a b l e t s were eaten before the r e s t o f the food was given. As very l i t t l e is known about d i g i t a l i s i n g doses i n pigs the above regime was i n s t i t u t e d so as to o b t a i n a reasonable drug c o n c e n t r a t i o n i n plasma. Blood samples were taken from a l l animals before treatment and a f t e r a week o f t r e a t m e n t . T h e r e a f t e r 3 animals from each group were bled on a l t e r n a t i v e weeks. The pigs were t r a n q u i l i z e d w i t h an i n t r a m u s c u l a r i n j e c t i o n o f e t o r p h i n e and acepromazine (Large animal Immobilion, R i c k e t t and Coleman L t d . , H u l l ) (0.01 mg/kg), before t a k i n g the sample by j u g u l a r vein puncture. 10-15 ml o f blood were taken i n h e p a r i n i s e d s y r i n g e s , t r a n s f e r r e d t o l i t h i u m heparin tubes f o r the measurement o f e ~ y th r o e y t e sodium c o n t e n t , sodium t r a n s p o r t , e r y t h r o c y t e Na+,K+-ATPase, ~H ouabain b i n d i n g eapaeity and plasma d i g o x i n l e v e l s and e l e c t r o l y t e s . Sodium t r a n s p o r t was not measured during weeks 2-10. A l l blood samples were taken in the morning before the d a i l y dose of digoxin. •

W

The methods used to measure the erythroeyte sodium content ([N~ pR ) and . . . . o os . . . ,,~C the ouabaln-sensztzve e f f l u x rate of sodlum ( M ~ ) and ouabaln sensltzve e f f l u x rate constant of sodlum ( K~a) have been prevlously described (6,9). •

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.

.

Erythroc~te sodium content The sodium content of erythrocytes was calculated from the concentration of sodium in haemolysates prepared from a known weight of cells which had been packed by eentrifugation at 15,000 g for 30 minutes at 4°C. The measurements

of sodium were mad~ on either unwashed erythroeytes ( ~

HBC) or wasbed

erythroeytes ( [ N ~ B C ) . Erythrocytes were washed in magnesium chloride (285 mosm/kg) to remove trapped plasma. Equal volumes of ice cold magnesium chloride was added to erythrocyte and the suspension was centrifuged at 1500 g for 5 minutes and the supernatant was discarded. This procedure was repeated twice before centrifugation at 15,O00 g. 0nly true or washed erythrocytes

sodium content ([N4~B c) values were given in the results section. Sodium transport iO~OS

The o u a b a i n - s e n s i t i v e e f f h x r a t e o f sodium ~ ~Na; mmol h -1) was measured from the increase i n e r y t h r o c y t e sodium content during i n c u b a t i o n o f whole blood w i t h ouabain. Three 3 ml a l i q u o t s o f h e p a r i n i s e d blood was p i p e t t e d i n t o 5 ml tubes. To one o f these 20 p l o f 20% e t h a n o l was added and to others 20 ~1 o f ouabain i n 20% e t h a n o l was added t o give a f i n a l c o n c e n t r a t i o n o f 10 -4 m o l / l which was s u f f i c i e n t t o maximally i n h i b i t the sodium pump. The tubes were incubated at 37°C f o r 2 hours. At the end of 2 hours the sodium content o f e r y t h r o c y t e s i n a l l the tubes ~eR~.determioed ( [ N a ] ~ C ) . T h e ouabain s e n s i t i v e e f f l u x r a t e o f sodium (~M~; was ealcuza~eo Trom the d i f f e r e n c e in e r y t h r o c y t e sodium content in-~he presence and absence o f ouabain. In the absence o f ouabain the sodium content o f c e l l s was maintained during incubation. The ouabain s e n s i t i v e e f f l u x r a t e constant o f sodium, °K°S Na' was c a l c u l a t e d from the e q u a t i o n : OMOS Na OKOS Na RBC -

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where ( [ N ~ B C ) i s the sodium content of washed erythroeytes sampled before the addition of ouabain. 3H ouabain b i n d i n 9 c a p a c i t y Erythrocytes were washed in potassium free isotonic buffer (pH 7.4) twice and a suspension of erythrocytes (15-25% packed cell vohme) was prepared in th~s buffer. A portion (0.5 ml) of this suspension was incubated with 20 pl of H ouabain (specific activity 15 Ci/mmol, final concentration 10 -7 mol/1) and 20 pl of 20% ethanol/saline or 20 pl of ~H2ouabain and 20 pl of unlabelled ouabain dissolved in 20% ethanol/saline (10- mol/1), for 2 hours at 37°C. Another aliquot of the cell suspension was used for cell counting. After incubation three I00 ul aliquots were taken from each tube and the cells were washed four times in 2 ml of ice-cold isotonic magnesium chloride. A~ter the final wash 200 ul of 10% perchloric acid were added to extract the H ouabain. The extraction was repeated twice and the radioactivity in the extract counted in a liquid scintillation counter. Quench correction was applied and the amount of H ouabain bound calculated. 3H ouabain binding capacity (c.p.m.)

=

total counts - non specific counts cell counts

In p r e l i m i n a r y experiments i t was observed t h a t the maximal b i n d i n g was o b t a i n e d w i t h t h i s technique (Whittaker and Swaminathan, unpublished r e s u l t s ) . Na+~K+-ATPase A c t i v i t y The Na+,K+-ATPase a c t i v i t y i n f r e e z e thawed haemolysates was measured by the r e l e a s e o f phosphate from ATP i n the absence and presence o f ouabain (6).

a kit

Plasma d i g o x i n c o n c e n t r a t i o n was assayed by a radioimmunoassay using w i t h I I 2 5 - t y r o s o y 1 - d i g o x i n (Beckton-Dickinson & Co. New York).

The group data are given as means + SEM and the groups were compared using p a i r e d and unpaired t t e s t s as a p p r o p r i a t e . A P value o f 0.05 or less was eonsidered s i g n i f i c a n t . Results Figure 1A shows the plasma d i g o x i n l e v e l s : the mean plasma l e v e l s was 1.2 ng/ml at week I and remained above I ng/ml t i l l week 5. From week 6 plasma d i g o x i n l e v e l decreased to 0.6 ng/ml at 8 weeks and 0.4 ng/ml at 11 weeks. When the maintenance dose o f d i g o x i n was increased, the plasma l e v e l s tended to increase but returned to 0.5 ng/ml at 14 weeks. The erythrocyte sodium changes are shown in Figure lB. The erythrocyte sodium in the control group of animals was 5.2 mmol/kg cells at the start of the experiment and was 4.5 mmol/kg at 4 weeks, and was 5.3 mmol/kg at 14 weeks. In the digoxin treated group the erythrocyte sodium increased i n i t i a l l y . After a week of treatment the sodium content of erythrocytes increased to 6.9 mmol/kg cells from 5.4 mmol/kg cells (P
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compared t o 5.3 + 0.28 mmol/kg c e l l s in the c o n t r o l s .

Figure

1

A 1.2

plasma digoxin 0.8 level

(ng m1-1 ) 0.4

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RBC (mmol kg ceils

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ouabain binding 16 capacity

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Changes in plasma digoxin concentration (A), sodium content (B) and erythrocyte ouabain binding capacity (C) in control ( ) and digoxin (.... ) groups of pigs. Vertical bars are SEM Significantly different from either corresponding control group (x) or from all values at week 0 ((a)).

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Changes in ouabain b i n d i n g c a p a c i t y are shown i n Figure61C. Erythrocyte ouabain binding capacity remained at a mean of 11.4 c.p.m~/10 cells throughout the study period of 14 weeks. In the di@oxin group the ouabain binding capacity increased from 12.9 + ~ 7 e.p.m./lO ~ cells before treatment to a maximum of 19.4 + 2.3 c.p.m./~0 ~ cells. The increase was seen after 5 weeks of digoxin administration when the erythroeyte sodium had returned to normal (Figure i). The increase in ouabain binding capacity was significantly different from the pre-treatment value and from the control groups as indicated in Figure le. IO~OS~

Changes in ouabain-sensitive efflux rate constant of sodium ~ ~Na p and e f f l u x r a t e o f sodium ( ° M ~ ) i n c o n t r o l and diQoxin groups are shown i n Table 1. The e f f l u x r a t e constant o f sodium ( ° K ~ ) a f t e r one week o f treatment was 18% lower compared t o the p r e - t r e a t m e n t . This was s i g n i f i c a n t l y d i f f e r e n t from a l l values a t week 0 (P < 0 . 0 1 ) . The e f f l u x r a t e constant o f

TABLE I •

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The Erythrocyte Ouabain-Sensitive Efflux Rate Constant of Sodlum ( K~ ) and o. os) . .sa Ouabain-Sensitive Efflux Rate of Sodium ( MNa in Control and Dlgoxln Treated Pigs• oKNa os (h-l) Week 0 1 Ii 12 13 14

Control group 0.140+0.0027 0.128~0.0125 0.160~0.0063 0.140~0.0153 0.144~0.0065 0.149~0.0066

°M.~S s a (n~nol h-lkg -I)

Digoxin group (6) (5) (3) (3) (5) (3)

0.124+0.0046 0.I02~0.0110 0.184~0.0063 0.168~0.0130 0.167~0.0068 0.193~0.0149

(5) (6)* (3)** (3)* (6)~* (4)

Control group

Digoxin group

0.71+0.025 0.63~0.065 0.78~0.021 0.71~0.079 0.72~0.039 0.84~0.034

0•67+0.040 (5) 0.71~0.070 (6) 0.82~0.084 (3) 0.82~0.084 (3) 0.81~0.063 (6) 0.85~0.045 (4)

(6) (5) (3) (3) (5) (3)

Results are Mean + SEM * or X P < 0.01 ** P < 0.001 * Signigicantly different from all values at week 0. X Significantly different from corresponding control group. Figures in parentheses indicate the number of animals bled.

sodium measured on weeks I I , 12, 13 and 14 were higher than the pre-treatment or corresponding control values. The e f f l u x rate constant was on average 24% higher than the mean control levels. The ouabaln sensitive e f f l u x rate of sodium (°M~) in the digoxin group tended to be higher than the corresponding-galues in the control group. The a c t i v i t y of Na+,K+-ATPase was measured before treatment and at weeks 7, 8, 9 and 12 of treatment in the dicoxin group. The a c t i v i t y of Na+,K+-ATPase was 6.35 ± 0.17 pmol Pi, h-1 g Hb-1 before treatment and at weeks 7, 8, 9 and 12 i t was 10.2 + 0.87, 11.9 + 2.07, 11.1 + 1.91 and 12.4 + 0.92 pmol Pi, h-1 g Hb- I r~spectively. --These were sTgnificantly higher than ~he pretreatment values (P <0.01).

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Discussion During the early stages of digoxin administration erythrocyte sodium increased and the efflux rate constant of sodium decreased (Figure iB and Table i). We have shown this previously in man (6). Loes et al (7) showed that the erythrocyte sodium content was higher during early stages of digoxin administration in children. Ford et al (i) in addition showed a decrease in erythrocyte 86Rb uptake and in vitro 3H digoxin binding during early stages of digoxin treatment. In this study we have shown a decrease in sodium transport and an increase in erythrocyte sodium content. However, a decrease in ouabain binding capacity could not be demonstrated. This could be due to dissociation of digoxin during the measurement of ouabain binding capacity. However, this is unlikely since Ford et al (ii) have shown that dissociation constants for digoxin and ouabain were similar (9.1 x 10 -9 M and 3.5 x 10 -9 M respectively). Furthermore, in 2 experiments the time course of ouabain was studied. The ouabain binding would have increased with time if there was dissociation of digoxin during incubation. This was not observed. A poor precision of the ouabain binding method could also explain the lack of decrease in ouabain binding capacity. However, the coefficient of variation of the method was 5% compared to an expected change of 20% (calculated from changes in efflux rate constant of sodium). In patients treated with digoxin the maximum effect of digoxin was seen around 4 days(4). As ouabain binding capacity was measured weekly, we may have missed the expected decrease. During continued digoxin administration the erythrocyte sodium decreased to normal values (Figure 1B). In patients treated for more than 2 months with digoxin, erythrocyte sodium was normal (4. 5, 6, 7). In a few patients studied longitudinally, the erythrocyte 86Rb uptake was found to fluctuate after a week or two (4). In this longitudinal study we show that with prolonged digoxin administration there is return of the erythrocyte sodium content to normal at a time when the digoxin level was still above 1 ng/ml on week 4 and 5. Erythrocyte 3H digoxin binding in patients treated with digoxin for more than 2 months was found to be the same as in control subjects (4). Digoxin can inhibit the Na+,K+-ATPase system (12, 13) and the inhibition is possibly responsible for its inotropic effect (i). We suggest that during initial stages of digoxin administration digoxln occupies some of the binding sites in the erythrocyte causing a decrease in efflux rate constant of sodium (Table l)oand an increase in erythrocyte sodium content (Figure iB). With continued treatment, the number of binding sites (sodium pump units) on the erythropyte membrane increases, resulting in the restoration of erythrocyte sodium content. This must happen in the new cells formed during treatment as several weeks elapse before changes are seen. The time taken to bring the erythroeyte sodium back to normal in the pigs was 4 - 5 weeks and in man it was about 5 - 8 months (6). This difference is probably due to the fact that the pigs were young and growing and the rate of new erythrocyte production would have been high. The normalisation of erythrocyte sodium content and increase in ouabain binding observed here could be due to failure of the digoxin levels to be maintained (Figure IA). However, as can be seen clearly the changes were seen before the digoxin levels started to decrease. The fall in plasma digoxin concentration (Figure IA) is probably due to the decline in the dose (ug/kg) of digoxin with age.

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These adaptive changes in sodium pump units (Na+,K+-ATPase o r glycoside binding s i t e s ) have been shown in v i t r o during exposure to ouabain or in hypokalaemia where there i s a decrease in sodium pump a c t i v i t y and an increase in sodium content. In HeLa c e l l s grown in tissue culture exposed to a low concentration of ouabain, an increase in ouabain binding sit e s was demonstrated (14, 15). This increase in ouabain binding sit e s was shown to be dependent on protein synthesis and i t restored cation transport to normal (15). In patients with hypokalaemia, Rubython et al (16) showed that ouabain binding capacity was higher in patients whose erythrocyte sodium content was normal. Erdmann & Krawietz (17) have reported s i m i l a r results. An increase in Na+,K+-ATPase was shown in the cardiac muscle of guinea pigs treated chronically with d i g i t o x i n or a potassium d e f i c i e n t d i e t (18). Our results confirm t h i s with chronic digoxin administration there is an adaptive increase in binding s i t e s . However, others have f a i l e d to show t h i s adaptive increase in the myoeardiumof dogs (19, 20) and the kidney of guinea pigs (21). However, why the ouabain binding capacity should increase higher than normal has not been fully discussed. If the adaptive response is to bring the erythrocyte sodium content to normal, the the number of 'free' binding sites (not occupied by digoxin) should return to normal. However, we have observed here that the ouabain binding capacity is higher. This could again be explained by dissociation of digoxin during the procedure of measuring ouabain binding capacity so that the total number of binding sites was higher. However, we have argued earlier that this is unlikely, and furthermore this would not explain the increase in efflux rate constant of sodium in this study (Table I) and that observed previously in man (6) in a crosssectional study. In the measurement of efflux rate constant of sodium, whole blood was incubated and therefore it is unlikely that there would have been significant dissociation of digoxin. These observations therefore suggest that the number of binding sites not occupied by digoxin has increased. Cumberbatch & Morgan (22) reported that in human erythrocytes, the ouabain /o~os~ sensitive efflux rate constant of sodium ~ ~Na J and membrane permeability io~os~ are related. In the steady state, the efflux rate of sodium ~ nNa j must be lo~os~ equal to the net influx of sodium, so that ~ mNa J is a measure of membrane O~OS permeability (22). In the pigs there was a good c o r r e l a t i o n between ~Na o os . p < and Mu~ as in man ( r = 0.788, 0.01, n = i i ) , so that an increase in oKN~ os would ..u . . be accompanied by an increase in membranepermeability. Therefore an ~ increase in KNa or ouabaln binding to normal would not return the erythrocyte sodium to normal as permeability would also increase. Only by i n c r e a s i n g the pump u n i t s (which would be shown by a high O~OS ~Na and ouabain b i n d i n g c a p a c i t y ) t o h i g h e r than normal could the e r y t h r o c y t e sodium be brought t o normal. The expected increase in membrane p e r m e a b i l i t y i s shown by a h i g h e r e f f l u x r a t e o f sodium (Table 1 and Cumberbatch e t a l ( 6 ) ) . We suggest t h a t i n c h r o n i c d i g o x i n a d m i n i s t r a t i o n , there i s ah attempt to reduce the i n t r a c e l l u l a r sodium content by producing more pump u n i t s . However, as the pump u n i t s increase the p e r m e a b i l i t y would a l s o increase and the f i n a l r e s u l t i n order t o normalise i n t r a c e l l u l a r sodium content i s an increase in the pump u n i t s h i g h e r than normal. •

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I t i s concluded that administration of digoxin resulted in an increase in erythrocyte sodium i n i t i a l l y which returned to normal and t h i s was accompanied by a higher ouabain binding capacity. Acknowledgements We would like to thank Mr. A. Calder and Dr. A. Teller of the Department of Animal Physiology and Nutrition for their help in the study, and Professor D.B. Morgan for helpful criticism and advice. This study was supported by a grant from the British Heart Foundation (80/66).

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References 1. 2. 3. 4. 5. 6. 7. 8. 9. I0. ii. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

T. AKERA and T.M. BRODY, Pharmac. Rev. 29, 187-220 (1978) 3.K. ARONSON, D.C. CRAHAEM-SMITH, K.F. HALLIS, A.A. HIBBLE and F. WIGLEY, Br. 3. C l i n . Pharm. 4, 213-221 (1977) 3.A. INGELFINCER and P. GOLDMAN, New Eng. J. Med. 294, 866-870 (1976) A.R. FORD, J.K. ARONSON, D.C. GRAHAEM-SMITH and J.C. CARVER, Br. 3. C l i n . Pharm. 8, 135-1#2 (1979) D.B. MORGAN, M. CUMBERBATCH, S. COHN, D. SCOTT, T. CUNASUNTHARAM, C. DAVIDSON and C. CHAPMAN, Br. J. C l i n . Pharm. 10, 127-133 (1980) M CUMBERBATCH, K. ZARIAN, C. DAVIDSON, D.B. MORGAN, and R. SWAMINATHAN Br. J. C l i n . Pharm. 11, 565-570 (1981) M.W. LOES, S. SINGH, 3.E. LOCK and B.L. MIRKIN, New Eng. 3. Med. 299, 501-504 (1978) 3.3. KANEKO, C l i n i c a l Biochemistry of Domestic Animals 3rd E d i t i o n P.120, Academic Press, New York (1980) M. CUMBERBATCHand D.B. MORGAN, C l i n . Chim. Acta 89, 221-230 (1978) A.R. FORD, 3.K. ARONSON, D.C. CRAHAEME-SMITHand J.O. CARVER. Br. 3. C l i n . Pharm. 8, 125-134 (1979) A.R. FORD, J.K. ARONSON, D.C. CRAHAEM-SMITH and 3.A. ROSE, Br. 3. C l i n . Pharm. 8, 115-124 (1979) I.M. GLENN, Pharmac. Rev. 16, 381-407 (1964) K.GIBSON and P. HARRIS, C a r d i o v a s c u l a r Res. ~, 367-370 (1968) L.3. BOARDMAN, J.F. LAMB and D. McMALL, J. P h y s i o l . (Lond). 225, 619-635 (1972) C.L. VAUCHAN and 3.S. COOK, Proc. Nat. Aead. Sci. USA 69, 2627-2631 (1972) E.J. RUBYTHON, M. CUMBERBATCHand D.B. MORGAN. Clin. Sci. 61, 43P (1981) E. ERDMANN and W. KRAWlETZ, Aeta B i o l . Med. Germ. 36, 879-883 (1977) V. BLUSCHKE, R. BONN and K. CREEF, Eur. 3. Pharmac. 37, 189-191 (1976) D.D. KU, T. AKERA, J.M. BRODY and L.C. WEAVER, Naunyn-Schmiedberg's Arch. Pharmcol. 301, 39-47 (1977) B.R. NECHAY, R.E. JACKSON, M.C. ZIECLER, S.L. NELDON and 3.D. THOMPSON. C i r . Res. 49, 655-660 (1981) R. BONN and--K. CREEF. Arch. I n t . Pharm. Ther. 233, 53-64 (1978) M. CUMBERBATCHand D.B. MORGAN, Clin. Sci. 60, 555-564 (1981)