EFFECT OF METABOLIC POISO S AND DIGOXIN 0 THE ADRENERGIC NEURONEBLOCKING ACTIO OF GUANETHIDINE

SAROJA JAYKAR * AND O.D. GULATI

Department of Pharmacology, Medical College, Baroda

Summary: The adrenergic neurone blocking action of guanethidine in isolated rabbit ileum prepa-ration (Finkleman) was neither prevented nor reversed by either iodoacetate or dinitrophenol.However iodoacetate and dinitrophenol together partia lIy prevented but did not reverse theneurone blocking action of guanethidine. Digoxin totally prevented but did not reverse theneurone blocking action of guanethidine.

Key Words: Guanethidine Iodoacetate and dinitrophenolAdrenergic neurone blockade

Digoxin

Gulati and Jaykar (7) studied the uptake of guanethidine indirectly by testing the abilityof various procedures to prevent or reverse adrenergic neurone blockade in theperiarterially stimulated isolated rabbit ileum preparation described by Finkleman (4).Theyshowed that adrenergic neurone blockade was prevented but not reversed hy equilibrationwithguanethidine at low temperature (l0°C), in the absence of sodium or in the presence oftetrodotoxin or noradrenaline. It was concluded that the neuronal mechanism concerned withthe uptake of noradrenaline could take up guanethidine and that this uptake of guanethidinewasenergy and sodium dependent. This paper reports the effects of metabolic inhibitors andof digoxin since it is known that digitalis glycosides inhibit the sodium-potassium ATP-ase (12)

METERJALS A D METHODS

Details of the method for setting up Finkleman preparation are described in the earlierpaper(7). The relaxant responses of the rabbit ileum to mesenteric nerve stimulation showedvariationsfrom preparation to preparation. Therefore, two preparations were set up simulta-neously. After eliciting control responses to different frequencies one preparation was treatedwiththe test drugs for a specific period of time. Both the preparations were now exposed toguanethidine (3.3 x 10_6 M) for 10 min. The preparations were washed several times with Me-Ewan'ssolution (the solution used for bathing) and the responses to nerve stimulation wererecorded. The effects of the test drug on responses to nerve stimulation were studied by settingupa third preparation from the same ileum and exposing it to the test drug for a specified time.Guanethidine (3.8 x 10-6 M) produced substantial to complete block of responses of the ileum toperiarterial sympathetic nerve stimulation at frequencies ranging from 2 sec to 60 sec. Responses

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·Presentaddress: 3245, East Jafferson Avenue, Detroit, Michigan, U.S.A.

76 Jaykar and Gulati January I'Ind. J. Physiol. Phar

Volume 16Number 1

to noradrenaline (0.59 x lO-8_0.59 x 10-7M) were unaffected. The blocking action lasted ~more than 3 hr.

RESULTS

Iodoacetate: Exposure of the tissue to iodoacetate (2.7 x 1O-6M) for 30 min reduceditone and pendular movements which did not recover completely even after several washes ~over 2 hr. Responses to nerve stimulation were partially blocked (Fig. 1). Responses to norsdrenaline (0.59 x 10-8-0.59 x lO-7M) were not blocked.

In control preparations guanethidine produced complete adrenergic neurone blockadeall the frequencies. Iodoacetate (2.7 x 10-6M) kept in the bath for 20 min before exposing ttissue to guanethidine did not prevent adrenergic neurone blockade due to guanethidine (Fig.!In two experiments exposure of the preparation to iodoacetate (2.7 x 10-6M) for 30 min aftguanethidine had completely blocked responses to nerve stimulation, did. not reverse the adre.nergic neurone blocking action of guanethidine.

Dinitrophenol: Exposure of preparations to dinitrophenol (0.5 x lO-3M) for 30 roilreduced the tone and abolished .the pendular movements. Responses to nerve stimulatiwere abolished. Responses to exogenous noradrenaline (0.59 x 10-8-0.59 x lO-7M) were naffected. Soon after a few washes, the tone and the pendular movements and responses t

nerve timulation were restored to normal (Fig. 1).

10.@

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o~0.. 60

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In control preparatiall the frequencies. Expofore adding guanethidine fdine (Fig. I). Dinitropheiadrenergic neurone blockii

Iodoecetate and dim(2.7 x 1O-6M) and dinitropments were abolished andponses to exogenous noradral washes the tone and J

nerve stimulation continue

In control preparatiacetate (2.7 x 10-6M) andexposing the tissue to guaof guanethidine (Fig. 1). (nol (6.5 x 10-3M) for 30 nuthidine (2 experiments).

Digoxin: Fig. 2 SUI

Following exposuretone and pendular movemeiThe action of digoxin laste(0.59 x 10-8-0.59 x 1O-7M

In control preparaticstimulation. In test prepanfollowed by further exposuraction of guanethidine was

In the control experiidine it was treated with digrdifferent frequencies of nervresponses to different frequesignificantly (p > 0.05) diffenot reverse the adrenergic b

C0 CD CV CV CV2 5 10 20 j 2 i 5"0'20' i I , i I

2' 5'10'20'i i i i j j I I I

2 6 10 20 2 5 10 20 2 ~ 102'0I I .J I I ! I

CONTROL GUA IDA lOA + GUA ONP ONP+ GUA lOA + QNP

Fig. I: R'!'pJ:lD of Finklernan preparation to periarterial nerve stimulation at different frequencies, epressed as % of controls. From left to right the first panel shows control and second, third, fifthseventh panels show responses after exposure of the preparations to guanethidine (3.3xlO-G M) for 10iodoacetate (2.7xlO-G M) for 30 min, dinitrophenol (0.5xlO-3 M) for 30 min and iodoacetate (2.7xlO...•and dinitrophenol (0.5xlO-3 M) for 30 min respectively. Responses shown in the fourth, sixth and ei,panels were obtained by exposing the preparations to iodoacetate (2.7xlO-6 M) for 20 min, dinitroph(0.5xlO-3 M) for 20 min and iodoacetate (2.7x10-8 M) and dinitrophenol (0.5xlO-3 M) for 20 minpectively followed by further exposure to guanethidine (3.3.xlO-s M) for 10 - min. Vertical lines indistandard errors. Encircled figures above each panel indicate the number of observations.

Interference with eneithe isolated tissues (3,6,13).

January 1972Ind. J. Physiol. Pharrnac,

I. The blocking action lasted for

2.7 x ID-GM)for 30 min reduced itslelyeven after several washes for)cked(Fig. 1). Responses to nora-

te adrenergic neurone blockade atuh for 20 min before exposing theckade due to guanethidine (Fig. 1).e (2.7 x lO-6M) for 30 min afterdation, did not reverse the adre-

phenol (0.5 x ID-3M) for 30 min. Responses to nerve stimulationi9x 10-8-0.59 x ID-7M) were notdular movements and responses to

o CV CV~ ~ i ill---J 2 5 10 20 S, 10 20

iD" DNP+ GUA lOA + ONP

mulation at different frequencies, ex-M'S control and second, third, fifth andto guanethidine(3.3x10-G M) for 10 min,)for30min and iodoacetate (2.7x10-6 M)sshownin the fourth, sixth and eighth(2.7xI0-aM) for 20 min, dinitrophenolitrophenol (0.5xlO-3 M) for 20 min res-M) for 10 -min. Vertical lines indicatenumberof observations.

Adrenergic Neurone Blocking Action of Guanethidine 77

In control preparations guanethidine totally blocked responses to nerve stimulation atallthe frequencies: Exposure of the preparation to dinitrophenol (0.5 x IQ-3M) for 20 min be-foreadding guanethidine failed to prevent the adrenergic neurone blocking action of guanethi-dine(Fig. 1). Dinitrophenol (0.5 x IO-3M) kept for 30 min in the bath failed to reverse theadrenergic neurone blocking action of guanethidine (2 experiments).

Iodoecetate and dinitrophenol: When the prepatations were exposed to dicoacetate .(2.7 x ID-GM) and dinitrophenol (0.5 x 10-3M) for 20 min they were relaxed, the pendular move-mentswere abolished and responses to nerve stimulation were totally blocked (Fig. 1). Res-ponsesto exogenous noradrenaline (0.59 x IQ-8-0.59 x IQ-7M) were not affected. After seve-ralwashes the tone and pendular movements were only partially restored, but responses tonervestimulation continued to remain blocked for over 2 hr.

In control preparations guanethidine produced total adrenergic neurone blockade. Icdo-acetate (2.7 x IQ-6M) and dinitrophenol (0.5 x IO-3M) placed in the bath for 20 min beforeexposing the tissue to guanethidine partially prevented the adrenergic neurone blocking actionofguanethidine (Fig. 1). Combined treatment with iodoacetate (2.7 x IQ-GM) and dinitrophe-nol (6.5 x IQ-3M) for 30 min failed to reverse the adrenergic neurone blocking action of guane-thidine (2 experiments).

Digoxin: Fig. 2 summarises the data.

Following exposure of the tissue to digoxin (3.0 x IQ-GM) for 20 min and washing, thetone and pendular movements were reduced and responses to nerve stimulation were blocked.The action of digoxin lasted for more than 2 hr. Responses to exogenous noradrenaline(0.59x IQ-8-0.59 x IQ-7M) were not affected.

In control preparations guanethidine substantially blocked responses to periarterial nervestimulation. In test preparations digoxin (3.0 x IO-GM) was placed in the bath for 10 minfollowed by further exposure of the tissue to guanethidine. The adrenergic neurone blockingaction of guanethidine was totally prevented.

In the control experiments described above, where the tissue was exposed to guanethi-dineit was treated with digoxin (3.0 x IQ-GM for 212, min) after eliciting a panel of responses todifferent frequencies of nerve stimulation. The tissue was now washed five to six times and theresponses to different frequencies were re-elicited. Responses to nerve stimulation were notsignificantly (p > 0.05) different from the control guanethidine responses. Thus digoxin couldnot reverse the adrenergic blocking action of guanethidine.

DISCUSSION

Interference with energy metabolism reduces the uptake and retention of noradrenaline inthe isolated tissues (3,6,13). The uptake of noradrenaline by the guinea pig isolated left atrium

78 Jaykar and Gulati

'4U

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80

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January 19Ind, J. Physiol. Pharm

CD

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CONTROL

Fig. 2 : Responses of Finkleman preparations to periarterial nerve stimulation at different frequencies, expresas % of controls. From left to right the first panel shows control responses; second and third panels sho'responses after exposure of the preparations to guanethidine (3.3xlD-6 M) for 10 rnin and digoxi(3.xlO-6 M) for 20 min respectively; responses shown in the fourth panel were obtained after exposing I:

preparations to digoxin (3.0xl0-6 M) for 10 min followed by exposure to gunethidine (3.3.xlo-6 M) for 11

min in the pressence of digoxin and several wash-outs; after eliciting responses shown in the second panthe preparations were treated with digoxin (3.0xl0-G M) for 20 min, washed and the responses re-elicit(fifth panel). Vertical lines indicate standard errors. Encircled figures above each panel indicate t

number .of observations.

is not exclusively dependent on carbohydrate metabolism, and the energy required for the uptake!can also be provided by aerobic oxidation of noncarbohydrate endogenous substrates (13). I1

the present study the metabolic inhibitors, iodoacetate or dinitrophenol individually did not pre·vent the adrenergic neurone blockade due to guanethidine but together were able to prevent illpartially. This agrees with the reported metabolic requirements for the uptake of the noradenaline (13) and a- methyl-noradrenaline (8,9). The failure of iodoacetate and dinitrophenol t,

completely prevent the adrenergic neurone blocking action of guanethidine may partly be due to.

its passive diffusion into the adrenergic neurone. Alternatively the persistent adrenergic neurone blocking action of iodoacetate may have masked the preventing action of metabolic inhib~1'tion on adrenergic neurone blockade by guanethidine. When iodoacetate and dinitropheneither separately or in combination were added to the bat h after exposure of the tissuet,

Volume 16Number 1

guanethidine, the adremay, therefore, be jusone is in part energy-

Digoxin prevendemonstrated that ouaslices. Ouabain exertsmem brane rather than,that cuabain can prodjspleen of the cat. QUImembrane (11). Thesodium-potassium ATnaline (2,5,10) and su~the preventing action6the sodium-potassiumconcentration is blocketory role.

It is a pleasuret

and dinitrophenol by PBaroda; and of noradrework was supported byNew Delhi.

1. Carlsson, A., N.A. Hil\amine granulla of the a

2. Crane, R.K. ProceediiH. Amsterdam, Elsevie

3. Dengler, H.J., LA. Miby isolated brain and o.

4. Finklernan, B. On the5. Gerrahan, EJ. and l.M.6. Green, R.D. and J.W.

uterus of the rat. J. Ph7. Gulati, O.D. and S. Jay

Pharmac., 42: 352-3'8. Hamberger, B. Reserpif

Scand., 71, Suppl. 295:9. Ha.nberger, B. and T.

treatment, histochemic

January 1972Ind, J. Physiol. Pharmac.

2 5 10 20I 2 5 10 20 I

DIG+ GUA GUA + DIG

tion at different frequencies, expressedesponses;second and third panels show(3.3xlQ-eM) for 10 min and digoxinpanel were obtained after exposing the

sureto gunethidine (3.3.xlQ-6 M) for 10g responsesshown in the second panel,in, washed and the responses re-elicitedfiguresabove each panel indicate the

the energy required for the uptakeendogenous substrates (13). In

ophenol individually did not pre-together were able to prevent its for the uptake of the noradre-iodoacetate and dinitrophenol touanethidine may partly be due tothe persistent adrenergic neur-

nting action of metabolic inhibi-iodoacetate and dinitrophenol

th after exposure of the tissue to

Adrenergic Neurone Blocking Action of Guanethidine 79Volume16umber1

guanethidine,the adrenergic neurone blocking action of guanethidine was not affected at all. Itmay,therefore, be justifiably concluded that the uptake of guanethidine by the adrenergic neur-oneis in part energy-dependent.

Digoxin prevented the neurone blocking action of guanethidine. Dengler et at (3) firstdemonstratedthat ouabain could block the accumulation of noradrenaline by brain and heartslices. Ouabain exerts this effect by inhibiting the transport of noradrenaline across the cellmembranerather than by acting on storage granules (1). Kirpekar and Wakade (10) showedtbatcuabain can produce marked interference with the uptake of noradrenaline by the perfusedspleenof the cat. Ouabain blocks the transport of 5-HT into platelets by an action on the cellmembrane(11). The amine transport system is blocked indirectly through the inhibition ofsodium-potassium A TP-ase (12). An ion-coupled transport such as that described for noradre-naline(2,5,10) and suggested for guanethidine (7) finds further support from the present data ontbepreventing action of digoxin of the adrenergic neurone blocking action of guanethidine sincethesodium-potassium dependent ATP-ase which is crucial in determining extracellular sodiumconcentration is blocked by digitalis glycosides (12). It appears that sodium ions play an obliga-toryrole.

ACKNOWLEDGEMENTS

It is a pleasure to acknowledge the gifts of guanethidine by Ciba, Basle; of iodoacetateanddinitrophenol by Prof. C.V. Ramakrishnan, Department of Biochemistry, Faculty of Science,Baroda; and of noradrenaline by Mr. A.V. Mody of Unichem Laboratories, Bombay. Thisworkwas supported by a research grant from the Council of Scientific and Industrial Research,NewDelhi.

REFERENCES

I. Carisson, A., .A. Hillarp and B. Waldeck. Analysis of the Mg.-ATP dependent storage mechanism in theamine granulla of the adrenal medulla. Acta Physiol. Scand., 59, Suppl. 215 : 1-38, 1963.

2. Crane, R.K. Proceedings of XV Annual Symposium on Peptides of the Biological Fluids. ed. by Peeters,H. Amsterdam, Elsevier Publishing Co., p. 227-235, 1967.

J. Dengler, H.J., LA. Micbaelson, H.E. Spiegel and E.O. Titus. The uptake of labelled norepinephrineby isolated brain and other tissues of the cat. Intern. J. Neuropharmac., 1: 23-38, 1962.

4. Finkleman, B. On the nature of inhibition in the intestine. J. Physiol. (Lond.), 70: 145-157, 1930.5. Gerrahan, F.J. and I.M. Glynn. The stoicheometry of sodium pump. J.Physiol. (Lond.), 192 : 217-235, 1967_6. Green, R.D. and J.W. Miller. Evidence for the active transport of epinephrine and norepinephrine by the

uterus of the rat. J. Pharmac. Exp. Ther., 152 : 42-50, 1966.7. Gulati, O.D. and S. Jaykar. Factors affecting the action of guanethidine on adrenergic neurones. Br. J.

Pharmac., 42: 352-363, 1971.8. Harnberger, B. Reserpine-resistant uptake of catecholamines in isolated tissues in the rat. Acta Physiol.

Seand., 71, Suppl. 295: 1-58, 1967.9. Ha.nberger, B. and T. Malmfors. Uptake and Release of a-methyl-noradrenaline in vitro after reserpine pre-

treatment, histochemical study, Acta-Physiol. Seand., 70 : 312-418, 1967.

80 Jaykar and Gulati

10. Kirpekar, S.M. and A.R. Wakade. Factors influencing noradrenaline uptake by the perfused spleenthe cat. J. Physiol. (Land.)., 194: 609-626, 1968.

11. PJetscher, A., W.P. Burkard, J.R. Tranger and K.F. Gey. Two sites of 5-hydroxytryptamine uptakeblood platelets. Life Sci. Oxford., 6: 273-280, 1967.

12. Tissari, A.H., P.S. Schonhoffer, D.F. Bogdanski and B.B. Brodie. Mechanism of biogenic amine transprll. Relationship between sodium and mechanism of ouabain. Blockade of the accumulation of serotoand norepinephrine by synaptosemes. Mol. Pharmac., 5 : 593-604, 1969.

13. Wakade, A.R. and R.F. Furchgott, Metabolic requirements for the uptake and storage of norepinepby the isolated left atrium of the guinea pig. J. Pharmac. Exp. Ther., 163 : 123-135, 1968.

SHORT COMMUNiCA

ANTICONVULSA T

Department of PediatrilPostgraduate inslil

Summary: Anticonvulsaidecussata (Shankhpushpiserved as the reference .sodium were found to prwere 62 mgjlOO g, 7.6 n

Key Words: Canscon

-Shankhpushpi' isSamhita (1), an ancient Adescribed under the name'to Filliozat (3) the Vedic pscompiled in 1st or 2nd cent

The name 'Shankhppushpi' is Clitoria tenatea Ldes Linn. in Uttar Pradesh 2

arisen due to a lack of corn

Canscora decussatabeen made on this planCanscora decussate.

Crude powder of Cata fine powder form.

The extraction of thedark green, sticky, thick ~powder was obtained. Forprior to feeding the rats.