Comparison of the effects of a bufodienolide and ouabain on neuronal and smooth muscle preparations

Neuroscience Research, 10 (1991) 235-244 © 1991 Elsevier Scientific Publishers Ireland, Ltd. 0168-0102/91/$03.50

235

NEURES 00431

Research Reports

Comparison of the effects of a bufodienolide and ouabain on neuronal and smooth muscle preparations F. Oberfrank, E.S. Vizi, P.F. Baker 1, S. Samuelov and D. Lichtstein Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest (Hungary) Laboratory of the Marine Biological Association, Plymouth and Department of Physiology, King's College, London. (U. K.) and Department of Physiology, Hebrew University- Hadassah Medical School, Jerusalem (Israel) (Received 21 June 1990; Revised version received 8 October 1990; Accepted 15 October 1990)

Key words: Chemical neurotransmission; Na÷,K+-ATPase inhibition; Endogenous inhibitors; Bufodienolide; Noradrenaline release; Blood vessel

SUMMARY

The effect of a bufodienohde (monohydroxy-14,15-epoxy-20,22-dienolideglycoside) purified from toad skin was compared with that of ouabain on 3H-noradrenaline release and on the tension of rabbit pulmonary arterial strips. This compound exerted an ouabaln-like activity. The neuronal effects of this bufodienolide derivative on squid axon were also studied and compared with those of ouabain. Both compounds enhanced the resting and stimulation-evoked (2 Hz, 360 shocks) release of 3H-noradrenaline. Moreover, in the presence of either this bufodienolide or ouabain, the tension of the rabbit artery increased gradually, and the contraction evoked by electrical stimulation was potentiated. Both compounds enhanced, in a prazosin-sensitive way, smooth muscle responses to noradrenaline and to electrical stimulation. In higher concentrations, they contracted smooth muscle cells of pulmonary artery, an action which was insensitive to prazosin. The bufodienolide was about 8 times more active in inhibition of 22Na efflux than was ouabain, but did not affect Ca efflux, which is not sensitive to ouabain. It is therefore concluded that compounds with an inhibitory effect on Na +,K +-ATPase are able to affect chemical neurotransmission of blood vessels in such a way that in lower concentrations they potentiate the release of noradrenaline, and in higher concentrations they contract directly the smooth muscle. These findings indicate that such compounds if they are present in the circulation might be involved in the physiological regulation of blood pressure or in the genesis of hypertension.

INTRODUCTION

The enzymatic and transport properties of Na÷,K+-activated ATPase (ATP phosphohydrolase, EC 3.6.1.3) in nerve and in muscle cells have been extensively studied and found to be important for maintaining membrane potentials and a variety of intracellular activities 32,35,36. In addition, a large body of evidence has accumulated supporting the t Deceased.

Correspondence." E. Sylvester Vizi, Institute of Experimental Medicine, Hungarian Academy of Sciences. Budapest, P.O.B. 67, H-1450-Hungary.

236 possibility that membrane ATPase has a substantial role in the regulation of the release of transmitters in both the peripheral and the central nervous system 24,~0.42.44.45. This has been shown for acetylcholine 2~ and noradrenaline (NA) 4°'42. Whilst inhibition of N a . K '-ATPase results in an increase in transmitter release, stimulation of the enzyme has the opposite effect 43,,u.~. It has even been suggested ~ that neuronal N a - , K ' - A T P a s e is involved in the release of transmitters. Cardiac glycosides such as ouabain or digoxin bind specifically to the catalytic subunit of N a ' , K * - A T P a s e and inhibit enzymatic activity in all tissues 36. The presence of the specific binding site for cardiac glycosides and the wide variety of pharmacological effects induced by these compounds prompted many laboratories to investigate the possibility of the presence of an ouabain-like compound (OLC) in animal tissue. Recently, the existence of an endogenous OLC in animal tissues has been demonstrated (for review, see Refs. 9 and 14). Although the chemical structure of the mammalian OLC has not yet been elucidated, the nature of this compound present in the toad skin and plasma was recently described 21.22. The levels of an OLC have been shown to be increased in hypertensive patients 9,16.29.31 implicating the involvement of this substance in the generation of this pathological state. It seemed of interest to study the effect of a bufodienolide derivative recently prepared and purified from toad skin 21.22 and that of ouabain on chemical neurotransmission in the rabbit pulmonary artery. At this transmission site the transmitter is known to be NA 37 and therefore the release of 3H-NA and the contraction in response to electrical stimulation and administration of this bufodienolide could be easily and simultaneously studied. In addition, the neuronal effect on ouabain-sensitive Na + efflux was also studied and compared with that of ouabain in the squid giant axon of Loligo forbesi. MATERIALS AND METHODS

Isolated rabbit pulmonary arterial strip The experiments were carried out on strips of rabbit main pulmonary artery 37 Rabbits of either sex weighing 2-3 kg were stunned by a blow to the head and bled to death. The main pulmonary artery was dissected and helical strips w e r e s e t up in an organ bath containing Krebs solution. The composition of Krebs solution used was (raM): NaCI, 113; KCI, 4.6; CaC12, 2.5; KH2PO 4, 1.2; Mg2SO4, 1.2; N a H C O 3, 25.0; and glucose, 11.5. The endothelium was retained to preserve near-physiological conditions. Measurement of vascular smooth muscle contractions For isometrical recording of mechanical responses, the vascular strips were mounted in a 5-ml organ bath and connected to an Eltron bioforcemeter (Budapest, Hungary) under a resting tension of 1.5 g. Electrical field stimulation The preparation was stimulated by means of a stimulator (Eltron, Budapest, Hungary) through two platinum electrodes, one at the top and the other at the bottom of the organ bath (field stimulation). Rectangular impulses of I ms duration at a frequency of 2 Hz, 60 V (supramaximal), total 360 shocks in 3 rain, were applied in order to mimic the efferent neuronal activity. The stimulation was controlled by an oscilloscope. Release of labelled noradrenaline The release of 3H-NA was measured as described previously 46. Briefly, a strip of artery prepared from rabbit was loaded with l-7,8-3H.-noradrenaline (490 klkL/ml, 3.555 G B q / m m o l specific activity, Amersham, U.K.) for 45 min at 3 7 ° C in Krebs solution

237 aerated with a 95% 02 + 5% CO 2 mixture containing 0.3 mM ascorbic acid and 30 #M Na 2-EDTA. After 45 min loading, paired arteries were transferred to an organ bath of 5 ml capacity and washed by changing of bath content every 10 min for 120 min, with Krebs solution aerated with a 95% 02 + 5% CO 2 mixture containing 0.3 mM ascorbic acid, 30 #M Na2-EDTA and 2.7 #M prednisolone at 37 o C. Subsequently, 3 rain fractions were collected by changing the organ bath content by aspiration of the solution. The content of radioactivity was also measured. For assay of radioactivity the tissues were blotted on filter paper, weighed and homogenized in 1 ml ice-cold 10% trichloroacetic acid. The homogenates were kept at 4 ° C for 45 min and then centrifuged at 1500 x g for 10 rain. An aliquot of the supernatant (0.1 ml) was processed as described below for the samples. The total radioactivity in the spontaneous outflow of tritiated compounds (3H-NA and its 3H-metabolites) from the tissue into the perfusion fluid was monitored by counting 1 ml samples of the Krebs solution which had been in contact with the tissue. Samples were counted in 7 ml of a mixture of 6 g of 1,4-bis(5-phenyl-2-oxazolyl) benzene (POPOP), 2 g of 2,5-diphenyloxazole (PPO), 2000 ml of toluene and 1000 ml of Triton X-100. The radioactivity of the samples was determined in a liquid scintillation counter (LKB), and the counts were converted to absolute activity by means of the external standard method. The release of tritium was expressed as a fractional rate (10 or 3 m i n 1), i.e. as a percentage of the amount of radioactivity in the tissue at the time when the release was measured. For calculating fractional release, a computing program was made on a computer. The tritiated NA was separated from the other tritiated metabolites and compounds by HPLC combined with radiochemical detection• Evidence was obtained that 37 ± 4% (n = 4) of the total radioactivity released in response to stimulation (supramaximal field stimulation, 2 Hz, 360 shocks) was 3H-NA. Under resting condition 12 ± 1% of the radioactivity released was tritiated NA (n-4; unpublished results) 27

Squid giant axon Squid (Loligo forbesi) giant axons with a diameter of 600-1000 # m were isolated and cleaned by the usual methods 3. The method of injection of 22Na was similar to that described by Caldwell et al. 5, except that a flowing superfusate (1.5 m l / m i n ) was used • 22 and the samples were collected at intervals of 2 man. Na was counted by a gamma-counter. Artificial sea water (ASW) of the following composition was used (mM): NaCI, 400; MgCI2, 100; KCI, 10; CaCI 2, 10; N a H C O 3 2.5. The pH was 7.8. When Li-ASW was used, NaC1 and KC1 were omitted and 400 mM LiC1 was added to the solution. When Ca2--free solution was used, CaCI2 was omitted.

Preparation of bufodienolide derivative Bufodienolide (monohydroxy-14,15-epoxy-20,22-dienolide glycoside) was extracted from toad (Bufo viridis) skin and plasma by methanol and purified using 3 sequential separations on HPLC 21. The material used in this study showed 1 peak on the last HPLC separation as an indication of homogeneity.

Drugs Drugs used were: disodium EDTA (Na2-EDTA, Aldrich, Europe), ascorbic acid (EGA), prednisolone (Organon), ouabain (BDH), Prazosin (Pfizer) and LiCI (Fischer Scientific Co., USA).

Statistical analysis Means 5: SEM are given. Significance of differences was calculated by paired t-test and Student's t-test; the d-test was used when the F-test proved significant differences in the variances of the two groups (n = number of experiments)•

238 RESULTS

Site of action in blood vessel preparation To determine the site of action of both this bufodienolide and ouabain, their effect on electrically evoked NA release was investigated on isolated arteries preloaded with 3H-NA and the contractions of the circular smooth muscle were concomitantly recorded (Fig. 1). Bufodienolide (28.6 ~M) markedly increased the 3H-NA release from the pulmonary arteries and contracted the smooth muscle. The peak release of 3H-NA developed 80 min after application of bufodienolide. Later, in spite of the presence of • • 3 bufodtenohde, H-NA release ~radually decreased. Similarly, exposure to ouabain (10 ~M) caused a gradual increase in H-NA release. The effect reached a maximum during the period of 60-80 min, and then gradually declined to a resting level (Fig. 2). The contraction of isolated pulmonary artery in response to supramaximal electrical field stimulation (60 V, 2 Hz, 360 shocks) was significantly potentiated by ouabain and bufodienolide (Table I). Both effects were dose-dependent. Prazosin (0.1 ~tM), a selective a]-adrenoceptor antagonist, even in the presence of this bufodienolide (2.86 /~M) completely prevented the contraction produced either by field stimulation or by administration of I-NA (0.1 ~tM; data not shown). Bufodienolide administered in a higher con-

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Fig. 1. Effect of bufodienohde derivative purified from toad skin (OLC, 28.6 ~M) on tension and tritium release from rabbit pulmonary arterial strip preloaded with ~H-noradrenaline. Concomitant recording of contraction and 3H-release. Upper tracing: Isometricrecordingof tension of rabbit pulmonaryartery. Force of contraction (g) is indicated. Sl a n d S2 = electrical s t i m u l a t i o n (supr.m~ximal, 2 Hz, 360 shocks). L o w e r t r a c i n g : F r a c t i o n a l release of r a d i o a c t i v i t y (%). ($1 = 7010 BCL/g.10 min). C o l l e c t i o n t i m e - 1 0 min.

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Fig. 2. Effect of ouabain (10 # M ) on tension and tritium release from rabbit pulmonary arterial strip preloaded with 3H-noradrenaline. Concomitant recording of contraction and 3H-release. Upper tracing: Isometric recording of tension of rabbit pulmonary artery. Force of contraction (g) is indicated. S l and S2 = electrical stimulation (supramaximal, 2 Hz, 360 shocks). Lower tracing: Fractional release of radioactivity (%). (S l = 6850 Bq/g.10 min). Collection time = 10 rain. TABLE 1 T H E E F F E C T O F B U F O D I E N O L I D E D E R I V A T I V E P U R I F I E D F R O M T O A D SKIN A N D O U A B A I N ON T H E C O N C E N T R A T I O N O F RABBIT M A I N P U L M O N A R Y A R T E R I A L STRIP E V O K E D BY ELECTRICAL STIMULATION Concentration ( # M )

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0.98:1:0.04 1.07±0.11 1.36±0.13 2.34

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Significance: * P < 0.05, * * P < 0.01. The results are expressed as the mean of the ratios of the contractions evoked by electrical stimulation (60 V, 2 Hz, 1 ms, 360 shocks) in the presence ($2) and absence (S 0 of O L C or ouabain added in cumulative doses. Contractions were recorded isometrically. Contraction in response to electrical stimulation (S 1) was 1082 + 57 mg (n = 48). In the control experiments (no drug) the preparation was stimulated 6 times; the S~/S I ratios were between 1.03+0.08 and 1.07±0.04 (n = 5-8).

240 TABLE I1 E F F E C T OF B U F O D I E N O L I D E D E R I V A T I V E P U R I F I E D F R O M T O A D SKIN ON T H E R E L E A S E ()F 3H-NA FROM RABBIT P U L M O N A R Y A R T E R Y Effect on 3H-NA R E L E A S E ( $ 2 / S 1 ratio)

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0.72+0.11 0.52 _*0.16 0.53 +0.06 1.14:t:0.06 * 2.33+0.46 * 21.36+4.78 *

1.18+0.22 1.09 + 0.19 1.51 +0.20 2.76+0.32 * " 3.58:1:0.16 * * * 3.69:1:2.15

Significance: * P < 0.05, * * P < 0.01, * * * P < 0.001 a Ratio of the fractional release values of the fractions before stimulation, in the presence and absence of bufodienolide. b Ratio of the fractional release values ( S - R ) of the stimulated fractions in the presence and absence of bufodienolide. Collection period = 3 rain. Bufodienolide derivative was added to the organ bath between S I and $2, 36 min prior to the second stimulation. Mean 5: SEM of 4 experiments.

centration (28.6 ~M), however, caused constriction of the rabbit arterial smooth muscle strip, and this response was not inhibited by administration of 0.1 pM prazosin. Effects on noradrenaline release

Therefore, in our experiments the effect of this bufodienolide on 3H-NA release was studied and compared with those of ouabain. After 3H-NA loading the radioactivity of the tissue at the end of the experiment was 728344 -t- 91474 Bq/g (n = 12). The resting release was 1686 + 296 Bq/g.3 min (n = 12). The release of tritium collected in 3 min following the stimulation increased significantly (fractional release= 1.03 + 0.20%, e . , ~ e Ouabain

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Abscissa: ouabain or OLC concentration (M). Each determination was made after 10 min pretreatment in the o u a b a i n / O L C solution. The bathing solution was normal ASW throughout. Temperature - 1 9 ° C.

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Loligo forbesi. The axon was superfused with 10-K(Na) (normal) and 0-K(Na) artificial sea water (ASW) or 10-K-Ca(Li) and 10-K-0-Ca(Li) ASW as indicated, and the superfusate collected at 2-rain intervals. Bufodienolid• and ouabain were applied during the periods indicated. Axon diameter = 600 # m. Temperature = 19 o C. P < 0.01, n = 12). T h e resting a n d electrically-evoked release of t r i t i u m i n c r e a s e d g r a d u ally in p a r a l l e l with the elevation of b u f o d i e n o l i d e d o s e in the i n c u b a t i o n fluid ( T a b l e II). O n a d m i n i s t r a t i o n of 50 # M b u f o d i e n o l i d e the resting release i n c r e a s e d d r a m a t i c a l l y .

Effects on 22Na efflux from giant axons of Loligo forbesi F u r t h e r clear-cut evidence that b u f o d i e n o l i d e has an effect on n e u r o n s c o m e s from o b s e r v a t i o n o f its effect on 22 N a efflux from the g i a n t a x o n s of Loligo forbesi. F i g u r e 3 shows a d o s e - d e p e n d e n t i n h i b i t i o n b y o u a b a i n a n d b u f o d i e n o l i d e o f N a ÷ efflux from squid axons i m m e r s e d in n o r m a l artificial sea w a t e r ( A S W ) . T h e curves show that the c o n c e n t r a t i o n n e e d e d to p r o d u c e 50% i n h i b i t i o n is 0.1/~M for o u a b a i n a n d 12 n M for the b u f o d i e n o l i d e . T h e m a x i m u m i n h i b i t o r y effect was o b t a i n e d b y 0.2 # M b u f o d i e n o l i d e (Fig. 4). A t this b u f o d i e n o l i d e c o n c e n t r a t i o n , a d d i t i o n of o u a b a i n c a u s e d no further i n h i b i t i o n of N a ÷ efflux. In the a b s e n c e of o u a b a i n , r e m o v a l of e x t e r n a l C a :+ reversibly r e d u c e d N a ÷ efflux from the axon i m m e r s e d in N a + - f r e e L i - A S W to the s a m e extent ( a b o u t 15%) as it d i d in the presence of 0.1 m M b u f o d i e n o l i d e . T h e s e findings i n d i c a t e that Ca2+-sensitive a n d o u a b a i n - i n s e n s i t i v e N a ÷ efflux was not affected b y b u f o d i e n o lide. DISCUSSION M a n y y e a r s ago S z e n t - G y 6 r g y i suggested that there is an e n d o g e n o u s digitalis: " . . . the digitalis glycosides are no drugs at all. T h e y are s u b s t i t u t e s for a missing screw in our

242 machinery, which had a role in one of the most basic physiological regulations" ~s. In recent years evidence has been presented for the presence of an ouabain-like compound in animal tissues. Such compounds were extracted and partially purified from mammalian brain 2"1°'17-1", heart s, plasma 6.7.13.16.2q and c s F 15"2° and from toad skin ~121 and plasma ~2.2~. None of these mammalian factors has yet been obtained in a pure form. Recently we have purified a bufodienolide derivative from toad skin 21.22 and showed that the same compound is present in the toad plasma. Based on N M R and mass spectroscopy, the structure of this endogenous ouabain-like compound was suggested to be monohydroxy-14,15-epoxy-20,22-dienolide glycoside (resibufogenin). It is tempting to hypothesize that the mammalian OLC is also a steroidal compound resembling bufodienolide derivative purified from the toad skin and plasma. This compound inhibits Na +,K +-ATPase activity and produces a positive inotropic effect on frog atrium 4.21.22.~.~4 In our experiments both bufodienolide derivative and ouabain enhanced dose-dependently the resting and evoked release of radioactivity from rabbit pulmonary artery previously loaded with 3H-NA. The bufodienolide derivative purified from toad skin and plasma contracted smooth muscle of the isolated rabbit pulmonary artery in a dosedependent manner. Previously, it has been demonstrated that the responses of this preparation to field stimulation are due to the release of NA from the nerve terminals and its effect on at-adrenoceptors of the smooth muscle cells 37.46. The bufodienolide derivative enhanced ~H-NA release and potentiated the effect of electrical stimulation, but only slightly enhanced the effect of exogenous NA added to the organ bath. Ouabain was shown to release NA from pulmonary arteries sg.~ and other noradrenergic axon terminals 25 and to augment the constrictor response of blood vessels to sympathetic nerve stimulation 41. These data suggest that both bufodienolide derivative and ouabain possess a presynaptic effect and that they are able to potentiate NA release from axon terminals. The finding that prazosin inhibits contractions potentiated by a low dose (14.3/xM) of bufodienolide, yet failed to affect those produced by a high dose (28.6/~M) of bufodienolide indicates that its effect was due to the potentiated release of NA. The contraction of blood vessels produced by ouabain was antagonized by a~-adrenoceptor antagonists, which suggests a possible involvement of activation of postjunctional a-adrenoceptors by NA released 1,23,26. Nevertheless, it seems very likely that bufodienolide and ouabain in high doses have a direct effect on smooth muscle. The exact mechanism by which this bufodienolide affects chemical neurotransmission in the pulmonary artery seems to be rather clear. It may act directly on axon terminals releasing NA a n d / o r by an unidentified substance which contracts smooth muscle. The primary site of action of a compound with an inhibitory effect on Na+,K 4-ATPase seems to be on the axon terminals. Our findings also indicate that bufodienolide and ouabain in higher concentrations have a direct effect on the smooth muscle. Since evidence has been obtained '~ that Na*,K+-ATPase is involved in transmitter release and bufodienolide inhibited the pump activity 33, these effects may be mediated via partial or complete inhibition of membrane ATPase. Further direct evidence of a neuronal effect of bufodienolide on neurones was obtained from studies of 22Na efflux from squid axon and compared to those of ouabain. The effect of bufodienolide was very similar to that of ouabain: it inhibited the 22Na efflux and failed to affect ouabain-insensitive Ca: + efflux. Our data suggest that bufodienolides cause noradrenaline release in blood vessels directly, but it cannot be excluded that they may contract vascular smooth muscle by other mechanisms of action, e.g. through endothelium-dependent factors 48 The basic conclusion of the present study is that any compound with an inhibitory

243 effect on Na+,K+-ATPase may control the activity of the sodium pump and hence may function as a physiological modulator of chemical neurotransmission in arteries. REFERENCES 1 Aarhus, L.L., Shepherd, J.T., Tyce, G.M., Verbeuren, T.J. and Vanhoutte, P.M., Contractions of canine vascular smooth muscle cells caused by ouabain are due to release of norepinephrine from adrenergic nerve endings, Circ. Res., 52 (1983) 501-507. 2 Akagawa, K., Hava, N. and Tsukada, Y., Partial purification and properties of the inhibitors of Na+,K +ATPase and ouabain binding in bovine central nervous system, J. Neurochem., 42 (1984) 775-780. 3 Baker, P.F., Blaustein, M.P., Keynes, R.D., Manil, J., Shaw, T.I. and Steinhardt, R.A., The ouabain-sensitive fluxes of sodium and potassium in squid giant axons, J. Physiol. (Lond), 200 (1969) 459-496. 4 Brownlee, A.A., Lee, G. and Mills, 1.H., Marked inhibition of N a / K ATPase by a bufodienolide but weak natriuretic activity in the rat, J. Physiol. (Lond.), (1987) 108P. 5 Caidwell, P.C., Hodgkin, A.L., Keynes, R.D. and Shaw, T.I., The effects of injecting 'energy-rich' phosphate compounds on the active transport of ions in the giant axons of Loligo, J. Physiol. (Lond), 152 (1960) 561-590. 6 Cloix, J.-F., Miller, F.D., Pernollet, M.G., Devynck, M.A. and Meyer, P., Purification d'un inhibiteur endog/me de la sodium-potassium ATPase, C.R. Acad. Sci. (Paris), 296, (1983) 213-216. 7 Dasgupta, A., Kiang-Teck, Y., Malik, S., Sandu, P., Abroad, S. and Kenny, M., Two novel endogenous digoxin-like immunoreactive substances isolated from human plasma ultrafiltrate, Biochem. Biophys. Res. Commun., 148 (1987) 623-628. 8 De Pover, P., Castaneda, A., Hernandoz, G. and Godfraind, T., Water versus acid extraction of digitalis-like factor from guinea pig heart, Biochent Pharmacol., 31 (1982) 267-271. 9 De Wardener, H.E. and Clarkson, E.M., Concept of natriuretic hormone, Physiol. Rev., 65 (1985) 658-745. 10 Fishman, M., Endogenous digitalis like activity in mammalian brain, Proc. Natl. Acad Sci. USA 76 (1979) 4661-4663. 11 Flier, J.S., Edwards, M.W., Daly, J.J. and Meyers, C.W., Widespread occurrence in frogs and toads of skin compounds interacting with ouabain site of Na +,K +-ATPase, Science, 208 (1980) 503-505. 12 Flier, J.S., Maratos-Flier, E., Pallotta, J.A. and Mclsaac, D., Endogenous digitalis like activity in the plasma of the toad Bufo marinus, Nature, 279 (1979) 341-343. 13 Gruber, K.A., Whitaker, J.M. and Buckalew, V.M., Endogenous digitalis-like substance in plasma of volume-expanded dogs, Nature, 287 (1980) 743-745. 14 Haber, E. and Haupert, G.T., The search for hypothalamic Na+,K*-ATPase inhibitor, Hypertension, 9 (1987) 315-324. 15 Halperin, J., Schaeffer, R., Galvez, L. and Malave, S., Ouabain-like activity in human cerebrospinal fluid (CSF), Proc. Natl. Acad. Sci. USA, 80 (1983) 6101-6104. 16 Hamlyn, J.M., Levinson, P.D., Ringel, R., Levin, P.A., Hamilton, B.P., Blaustein, M.P. and Kowarski, A.A., Relationships among endogenous digitalis-like factors in essential hypertension, Fed Proc., 44 (1985) 2782-2788. 17 Haupert, Jr., G.T. and Sancho, J.M., Sodium transport inhibitor from bovine hypothalamus, Proc. Natl. Acad. Sci. USA, 76 (1979) 4685-4690. 18 Leppaluoto, J., Vakkuri, O., Arjamaa, O. and Ruskoaho, H., Rat hypothalamic Na,K-ATPase inhibitory activity is heterogenous and mostly of small-molecular-weight size, J. Physiol. (Lond.), 390 (1987) 97P. 19 Lichtstein, D. and Samuelov, S., Endogenous ouabain like activity in rat brain. Biochem. Biophys. Res. Commun., 96 (1980) 1518-1523. 20 Lichtstein, D., Minc, D., Bourrit, A., Deutsch, J., Karlish, S.J.D., Belmaker, H., Rimon, R. and Palo, J., Evidence for the presence of ouabain-like compound in human cerebrospinal fluid, Brain Res., 325 (1985) 13-19. 21 Lichtstein, D., Kachalsky, S. and Deutsch, J., Identification of a ouabain-like compound in toad skin and plasma as a bufodienolide derivative, Life Sci., 38 (1986) 1261-1270. 22 Lichtstein, D., Samuelov, S., Deutsch, J., Xu, H., Lutz, R.A., Chernick, S.S. and Chernick, S.S., The ouabain receptor in animal tissues and its endogenous ligand, Klin. Wochenschr., 65 (Suppl. VIII) (1987) 40-48. 23 Lorenz, R.R., Powis, D.A., Vanhoutte, P.M. and Shepherd, J.T., The effects of acetylstrophanthidin and ouabain on the sympathetic adrenergic neuroeffector junction in canine vascular smooth muscle, Circ. Res., 47 (1980) 845-854. 24 Meyer, E.M. and Cooper, J.R., Correlations between Na +,K + ATPase activity and acetylcholine release in rat cortical synaptosomes, J. Neurochem., 36 (1981) 467-475.

244 25 Nakazato. Y., Ohga, A. and Onoda, Y., The effect of ouabain on noradrenaline output from peripher~d adrenergic neurones of isolated guinea-pig vas deferens, J. Physiol. (Lond.), 278 (1978) 45-54. 26 Oberfrank, F., Bern, Th.S., Lichtstein, D., Samuelov, S. and Vizi. E.S.. Effect of Na-K-ATPase inhibition on pre- and postsynaptic site of signal transmission in rabbit pulmonary artery. Neuroscience, 22 ~1987) $69g. 27 Oberfrank, F., H~rsing, Jr., L.G., l,ichtstein. D. and Vizi, E.S., Ouabain-like compound releases noradrenaline from blood vessel. (Submitted for publication.) 28 Paton, W.D.M., Vizi, E.S. and Zar, M.A., The mechanism of acetylcholine release from parasympatheuc ne~'es, J. Physiol. fLorid.), 215 (1971) 819-848. 29 Poston, L., Sewell, R.B., Wilkinson, S.P., Richardson. P.S., Williams, R., Clarkson, E.M., MacGregor, G.A. and De Wardener, H.E., Evidence for a circulating sodium transport inhibitor in essential hypertension, Br. Med. J.. 232 (1981) 847-849. 30 Powis, D.A.. Cardiac glycosides and autonomic neurotransmission, J. Autonom. Pharmacol. 3 (1983) 127 -154. 31 Sagnella, G.A.. Jones, J.C., Shore, A.C., Markandu, N.D. and MacGregor, G.A., Evidence for increased levels of a circulating ouabain like factor in essential hypertension, Hypertension, 8, (1986) 433-437. 32 Schwartz, A., Lindenmayer, G.R. and Allen, J.C., The sodium-potassium adenosine triphosphatase. Pharmacological, physiological and biochemical aspects. Pharmacol. Rev., 27 (1975) 3-134. 33 Serf6zb, Z.P., Oberfrank, F. and Vizi, E.S., Effect of ouabain-like compound and bufalin on Na,K-activated ATPase and p-nitrophenylphosphatase activity of synaptic membrane preparation of rat brain. (Submitted for publication.) 34 Shimoni, Y.. Gotsman, M., Deutsch, J., Kachalsky, S. and Lichtstein, D., Endogenous ouabain like compound increases heart muscle contractility, Nature 307 (1984) 369-371. 35 Skou, J.C., Enzymatic basis for active transport of Na 4 and K ÷ across the cell membrane, PhysioL Ret:., 45 (1965) 596-617. 36 Stahl, W.I,., The Na,K-ATPase of nervous tissue. Neurochem. Int., 8 (1986) 449-476. 37 Starke, K., Montel, H., Gayk, W. and Merker, R., Comparison of the effect of clonidine on pre- and postsynaptic adrenoceptors in the rabbit pulmonary artery, Naunyn-Schmiedeberg~ Arch., 285 (1974) 133-138. 38 Szent-Gyitrgyi, A., Chemical Physiolog)' of Contraction in Body and Heart Muscle. Academic Press, New York (1953). 39 'l't~rt~k, T.L., Salamon, Z., Nguyen, T.T. and Magyar, K., Spontaneous (3H)noradrenaline release from the main pulmonary artery of the rabbit induced by sodium-pump inhibition, Quart. J. Exp. Physiol., 69 (1984) 841-865. 40 Tt~rt~k, T.L. and Magyar, K., Ouabain-evoked (3H)noradrenaline release from the rabbit pulmonary artery, in calcium-free solution, Quart. J. Exp. Physiol., 71 (1986) 105-114. 41 Vanhoutte, P.M. and Lorenz, R.R., Na*,K*-ATPase inhibitors and the adrenergic neuroeffector interaction in the blood vessel, J. Cardiovasc. Pharmacol., 6 (1984) $88-$94. 42 Vizi, E.S., Stimulation by inhibition of (Na+-K*-Mg *÷ )-activated ATPase of acetylcholine release in cortical slices from rat brain, J. Physiol. (Lond.), 226 (1972) 95-117. 43 Vizi, E.S., Termination of transmitter release by stimulation of sodium potassium activated ATPase, .L PhvsmL (Lond), 267 (1977) 261-280. 44 Vizi, E.S., Na +-K +-activated adenosinetriphosphatase as a trigger in transmitter release, Neuroscience, 3 (1978) 367-384. 45 Vizi, E.S. and Vyskocil, F., Changes in total and quantal release of acetylcholine in the mou~ diaphragm during activation and inhibition of membrane ATPase, J. Physiol. (Lond), 286 (1979) 1-14. 46 Vizi, E.S., TiSr~Sk, T. and Magyar, K., Effect of potassium on the release of (3H)noradrenaline from rabbit and human pulmonary artery, J. Neurochem., 42 (1984) 670-676. 47 Vizi, E.S., Oberfrank, F., Bern, Th.S. and Lichtstein, D., Noradrenaline releasing effect of an ouabain-like compound on pulmonary artery, Neuropharmacology, 26 (1987) 1541-1544. 48 Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, M., Mitsui, Y., Yazaki. Y., Goto, K. and Masaki, T., A novel potent vasoconstrictor peptide produced by vascular endothelial cells, Nature, 332 (1988) 411-415.