The cardiac glycoside sensitive step in the hepatic transport of the bisquaternary ammonium compound, hexafluorenium

EUROPEAN JOURNAL OF PHARMACOLOGY 15 (1971) 245-251 NORTH-HOLLANDPUBLISHINGCOMPANY

THE CARDIAC GLYCOSIDE SENSITIVE STEP IN THE HEPATIC TRANSPORT

OF THE BISQUATERNARY

AMMONIUM COMPOUND, HEXAFLUORENIUM D.K.F. MEIJER, J.W. ARENDS and J.G. WEITERING Department of Pharmacology, State Umverstty of Gronmgen, Bloemsmgel 1, Gromngen, The Netherlands

Received 15 december 1970

Accepted 18 March 1971

D.K.F. MEIJER, J.W. ARENDS and J.G. WEITERING, The cardtac glycostde senstttve step zn the hepattc transport of the btsquaternary ammomum compound, hexafluorentum, European J. Pharmacol 15 ( 1971) 245- 251 When the blsquaternary mtrogen compound hexafluoremum (Hfl) was perfused through isolated rat lwer, It was rapidly cleared from the perfuslon medmm and accumulated m the hver, it was excreted into bile against large concentrat]on grad]ents. Chromatography indicated that blotransformanon was of minor Importance Hfl was bound to the red cells of the perfuslon medmm, slgnff]cant amounts penetrated into the cell but most of the drug was confined to the cell ghost Uptake of Hfl into the erythrocytes was not influenced by cardiac glycosides Hepanc transport of Hfl could be largely blocked by K-strophantos]de and dlgltoxln A stmflar effect was found m anesthet]zed rats with a bflmry fistula When cardiac glycoside was added to the perfusate before rejection of Hfl, both uptake into the hver and bflmry excretion were affected. However, transport into bile was not influenced when K-strophantoslde was added at a time when most of the rejected Hfl had been taken up by the hver suggesting the cardmc glycoside acts on uptake of the blsquaternary compound into the hepatic ceils. Hexafluorenmm Bfllary excret]on

Cardiac glycosides Hepatic uptake mechanisms

Hepatic transport of b]squaternary ammomum compounds

l. INTRODUCTION Hexafluorenlum (Hfl) (fig l ) is used as an adjuvant during anesthesia because of its ability to potentiate and modify succmylcholine-induced relaxation of skeletal muscle. Cavalhto et al. (1956) found that the relaxant activity of Hfl was potentiated by ineffective lipid soluble compounds. Thas was explained by displacement from lipoptuhc sites of loss and Meyer et al. (1970 a) showed that lipid solubility of the quaternary compounds was remarkably hagh. Excretion studies in rat and m man showed that relatively small amounts were excreted unchanged in urine whale in both species bihary excretion was important in the total elimination of the compound (Meyer et a l , 1971). Little IS known about the mechanism by which bisquater-

2 Br'

Hexaftuorer.um bromide

Fig. 1. Structural formula of hexafluoremum. nary nitrogen compounds are transported from plasma into btle It was suggested (Meyer et at., 1970 a) that hpld solubduty is an important determinant in hepatic transport of curare-like drugs. Bthary excretion of the monoquaternary procamamlde ethobromlde is depressed by Hfl pointing to a common step in the transfer to bde (Meyer et a l , 1970 b). The possible involvement of an active transport

246

D K F.Metler et al , Hepatic transport of hexafluoremum

process m the bdiary excretmn of d-tubocurarme, another blsquaternary ammomum compound was suggested by Cohen et al (1967) and Meyer et al. (1968). We have now studied handling of Hfl by the rat liver m more detad.

2 METHODS 2.1. Determmatton o f lift m plasma, bile, hver and erythrocytes Hfl was determined fluonmetncally after formation of a fluorescent complex with the dye, rose bengal. The method has been described prewously for urine and bale (Meyer et al., 1971) Plasma to 1 ml plasma, 1.0 ml phosphate buffer, 0 5 ml rose bengal solutmn and 7 ml chloroformphenol were added. Hfl was then determined accordmg to the procedure described for bile and urine Liver. Specimens of hver (ca 1 g) were homogenized m a Buhler homogenizer (U.M.) with 14 ml Krebs bicarbonate solution 5 ml of the resulting homogenate was extracted at pH 8.2 (KI-'glycme buffer) with 7 ml ethylene dichloride on a rotary mixer After re-extraction with 0.01 N HC1, Hfl was estimated as described for bde, urine and plasma. Erythrocytes The perfuslon medmm, consisting of defibrInated rat blood and Krebs bicarbonate albumin solution 1 1, was centrifuged m haematocnt tubes for 15 mm at 1500 g The supernatant was removed with a pipette, any fluid that remained was suctloned off. 2.5 ml erythrocytes (packed volume) were homogemzed with 7.5 ml Krebs bicarbonate, the volume of extra-cellular fluid was disregarded 5 ml of this homogenate was assayed for Hfl as described for liver. Recovery of known amounts of Hfl added to red cell suspensions amounted to 9 4 - 1 0 0 percent In some experiments, binding to the cell ghost was studied by haemolyslng blood cells with six times the volume of distilled water. The resulting suspension and the supernatant obtained after centrffugatlon for 30 mln at 1500g were assayed for Hfl as described for liver To determine if the Hfl found In this supernatant could be accounted for by drug washed out from the cell ghosts, washed erythrocytes were homogenlzed, during 2 mm under coohng with ice, in a high speed homogemzer at maximum speed, and centrffued at 31,000 g The supernatant representing the cell water was plpetted off and assayed for Hfl

2.2. Isolated perfused rat hver Lwers were taken from fed male Wlstar rats weighIng 250-350 g. The perfuslon medium consisted of 80 ml defibrmated blood and 80 ml Krebs bicarbonate albumin solutmn. The perfuslon techmque and conditmns were similar to those described earher (Meyer et al., 1970 a). The pH of the perfusmn fluid was 7.35-7.40 durmg the experiment, samples plated on blood agar and incubated at 37°C did not show bacterml growth. In all experiments, a 60 mln period was allowed for stabdlsatlon before any materials were introduced, flow rates at this time were about 21 ml per mm. After mjectmn of the drug, dissolved in saline, samples of perfusate and bale were collected after 10, 30, 60, 90 and 120 ram. Bde productmn was 0.40-0.45 ml m the first 30 mm period and decreased to 0 . 2 5 - 0 30 ml/min in the last. K-strophantoslde and Hfl did not affect bale productmn. After perfusion the hver was blotted to remove excess moisture and weighed. Aliquots of lwer welgtung about 1 g were taken from several lobes for fluonmetrtc determmatmns Concentratmn of Hfl in the hver is expressed as the mean of concentrations found m at least three different lobes.

2.3. Experiments m rats Male Wistar rats were anesthetized with pentobarbltal sodium and artificially respired. Normal body temperature was ma_mtained by a heating lamp. The abdomen was opened, the bile duct cannulated. A cannula was placed in the jugular veto. 30 mm after operation, 1 mg/kg Hfl was administered 1.v. during a 5 mm period. Control experiments m which mean blood pressure was measured from the carotid artery mdlcated that 1 mg/kg Hfl alone or combined with k-strophantoslde, did not seriously affect circulation. Bde was collected immediately after injection of the Hfl for three 2 0 m m periods. K-strophantoside (50og) was administered 1.v. over 3 mm period, 5 mm prior to "the Hfl rejection. After 60 mln urme was sampled from the bladder. 2 4 DetectTon ofmetabohtes Plasma, bile and liver homogenate were extracted twice with ethylene dichloride at pH 8.2 (KI-glycine buffer), known amounts of added Hfl were quantitatively removed from the aqueous phase after two extractions After separating the orgamc phase by

D K F Metier et al, Hepatw transport ofhexafluoremum centrifugation, the ethylene dichloride extracts were evaporated to a small volume (ca. 0.1 ml) at room temperature; 0 . 0 1 - 0 . 0 2 ml were applied to slhcagel plates together with the reference substance The plates were developed with 1 N HCl-Isopropanol (1 1) and then sprayed with Iodoplatmate reagent (Hexachloroplatin Merck), at least 0.5 #g of Hfl was detectable, 2.5. B m d m g to erythrocytes Hfl was Incubated 2 or 4 hr at 37 ° with fresh defibrlnated rat blood mixed I 1 with Krebs-albumin solution (4% bovine albumin). During incubation the mixture was frequently shaken and samples were taken at 30 mln intervals. Plasma and red cells were assayed for Hfl as described.

3. RESULTS 3. l . Btotransformanon lift Samples of plasma, bile and hver were taken one and two hours after addition of Hfl to perfused hvers The samples were extracted with ethylene dichloride and after two extractions, all the substance complexmg with rose bengal were removed from the aqueous phase. Thin-layer chromatography of the extracts revealed only one spot with a R f value identical to that of authentic Hfl FluorImetrlc determination o f Hfl in bile, plasma, liver and blood cells after two hours perfuslon resulted m a recovery of more than 90 percent of the amount added. These findings indicate that if any bIotransformatlon of Hfl occurs, ~t affects such a small proportion of the dose that it need not be considered m interpretation of the results. 3.2. Ltver perfuston expertments Handling of Hfl by the isolated perfused rat liver is shown in fig. 2. After administration o f 1000/ag, the drug was rapidly removed from the orculatmg medium (ty2 = 8 min) indicating extensive accumulation in the perfused hver Clearance o f Hfl calculated from concentration differences m 'portal' and venous blood was extremely high, m each passage through the liver about 67 percent of the drug was removed and ttus clearance factor remained constant during the first 45 rain of the experiment Fig. 3 shows such a perfusIon experiment in wluch special arrangements

247

PLASHA jug/W~.

7,

LIVER

Hexoftuoremum (7)

601%

6.,, 5

28 4%

60 S0

t

40

q

30 2

20

1

10 30

60 time (rain)

90

120

PLASHA '

BILE

jug,

i

200 150 100

30 60 90 time (rn~)

LIVER

BILE g

jug,

Hexoftuoremum (41 +K_Strophontoslde

6-

60.

5

50.

4

40.

3

30.

2

20.

1

10-

120

59%

190% 2OO

150 100 50

m.~.~.m. 30

60

90

120

tlme(n~n}

30 60 90 trne(rnmJ

Fig. 2 Hexafluorenmm (a) disappearance from plasma, content of the hver after 2hr and excretion into bile (m t~g/30 mm period) m isolated perfused rat hver (n = 7) after rejection of hexafluoremum (1000 ug) into 160 ml of perfuslon medmm (120 ml plasma). Values are gwen as the mean + S E.M. (b) Similar experiments m the presence of K-strophantoslde (n = 4) (lower part) were made to approximate 'ideal' mixing of venous and 'arterial' perfusIon fluid. Disappearance o f Hfl in the first 45 mIn agrees very well with a theoretical curve calculated with the equation C t = Co e x p ( - l k t / v ) , loss of perfusate due to sampling (less than 15% m the 45 mln period) IS disregarded This indicates that Hfl is primary distributed over the plasma volume and that under these conditions affinity for the hver t]ssue far exceeds that of the red cells and that the decline in plasma concentration can be attributed to uptake into the perfused hver Two hours after its Introduction, the Hfl was almost completely removed from the perfuslon fluid, at this time about 60 per-

120

248

D,K F Metler et al , Hepattc transport o f hexafluoremum ~g/mt Dose

9 8

Ct=Co

7

hexoftuoremurn = lO00jug

•

_lkt v

V= 120 mt pl.osmo i • 16 5 m t ptosma / m l n

k=067

6 5

'

4 3 2 1

30

60

90

120

time (min)

Fig. 3. Concentration m plasma of hexafluorenmm during a perfuslon of two hours. Experimental points are indicated by e. The broken hne represents the theoretical curve calculated with the equation rod]cared. (Co = concentration at t = 0 calculated from the dose and the plasma volume lla the perfusion medmm, z = flow of plasma through the hver m ml/ mm, k = clearance factor, t is time m mm and V is the dlstrlbutton volume = volume of plasma In the perfuslon medmm) cent of the rejected dose was present m the hver. Assuming bde volume in the rat liver to be about 0.005 ml/g hver wet weight (Barber-Riley, 1965), less than 3 percent of the amount present m the hver could be accounted for by Hfl m bile. The transport from plasma into bile occurred agamst high concentration gradients (bale-plasma concentratmn ratio rose from 100 to more than 3000 during the experiments) indicating that the drug

gamed access to bde through a process other than simple chffusmn and suggesting involvement of an active transport process. 3.3. Effect o f cardiac glycostdes Disappearance from plasma, accumulation m the hver and excretion into bile was greatly reduced by prior administration of K-strophantoslde (2.5 × 10-s M) or dlgltoxln (2.5 X 10-s M) see fig. 2. Ttus concentratlon o f car&ac glycosides did not interfere with bde production. A similar result was found in rats (table 1), 50/ag K-strophantoslde rejected 5 min before Hfl was administered led to a reduction of bfllary excretion m the first 20 mm period, excretmn m urine being increased under these circumstances. The blockang of b o t h accumulation m hver and transport into bde could be due to either mhlbltmn o f entrance of Hfl into the liver cells or mhabltmn o f the blhary excretmn process at canahcular sites, assuming that accumulation m the lwer and excretmn into bde are closely related phenoma Therefore, the effect o f K-strophantoslde on bdmry excretion o f Hfl was studled adding the cardmc glycoside when the major part of the rejected drug had been taken up b y the hver. The mean of 6 experiments, m which K-strophantoslde was rejected 60 mm after admm~stratmn o f Hfl, ~s shown m fig. 4. In these experiments the disappearance of Hfl from plasma was blocked b y K-strophantoslde (m fact, some Hfl appeared to be released from the hver) whereas bdlary excretion m the periods following the mjectmn of the glycoside was not affected compared with the normal experiments (fig. 2). (The differences between the two series of experiments was not significant (Wdcoxon p > 0.20) This result indicates that the cardmc glycoside inhibits the uptake of the bisquaternary corn-

Table 1 The effect of K-strophantoslde on the excretion of hexafluoremum m the rat K-stroph

Bale

(50 tzg)

(#g) 0-20 mm

0~g) 20-40 mm

(~g) 40-60 mm

Urine

-

530±25

17.8±14

10.7±08

+

281±46

182±1.8

124±10

n

(/~g) 0-60 mln 9 2 ± 09 244±43

Bile prod (mg) 0-60 mln

6

1210_+30

6

1270±25

K-strophantoslde was administered 5 mm before Hfl (2 mg/kg) was rejected, values are given as the mean ± S E.M of the number of anmaals md]cated.

D K.FMetler et al, Hepatw transport ofhexafluoremum

~ug/g

~ug/mt 7

jug

Hexoftuorenmm(6) + K_Strophontos,:le

5

t.0. 30

2

20

1

10 30

60

trneln~

90

200

50.

1

,

120

6 5 4 3 2 1

30 t.%

5~6%

6

jug/rat

BILE

LIVER

PLASHA

i

249

150

•

60

120

180

I

2/.0

t l m e (ram.}

30

60

90

120

Fig. 5 Hexafluorenmm (-) a) uptake m red cells, 6 7/~g Hfl/ml perfusmn medium was added, concentration per ml erythrocytes (packed volume) Is mdacated dunng an incubation of 4 hr

brae(mini

Fig. 4. Hexafluoremum d~sappearance from plasma, content of the lwer after 2 hr and excretion into bale m Isolated perfused rat hver after rejection of hexafluorenmm (1000 tag) into the perfuslon medmm. K-strophantoslde was rejected after 60 mm. Values are given as the mean + S.E.M. (n = 6). pound into the hver cells or interferes with binding or uptake in some compartment inside the hver cells, the bdiary excretion process appears to be unaffected. 3.4. Binding of lift to erythrocytes In those perfusion experiments in which K-strophantoslde was added and the plasma concentration of Hfl remained high, large amounts of Hfl were found in the red cells of the medaum. Therefore it was of mterest to study uptake of the drug into erythrocytes and to investigate the influence of cardmc glycosides m more detad. Fig. 5 shows that erythrocytes incubated with Hfl (6.7/ag/ml) steaddy take up the drug, after 4 hr mcubatmn, the concentration per ml of erythrocyte was about equal to the concentration of the surrounding medium. Binding or uptake into blood cells was unaffected by K-strophantoslde and ouabaln added 10 mm before Hfl, even in concentrations ten ttmes as high as those used m the perfuslon expertrnents (2.5 X 10-4). To dlstmgmsh between adsorption to the cells and mere penetration into the lntracellular content, the erythrocytes were haemolysed after 2 hr mcubaUon with Hfl and the drug concentration was determined m samples of suspension and supernatant after centrlfugatlon at 1500 g. It appeared that about 70 percent of the drug was bound to the cell ghost while the rest was found in the supernatant. To exclude influence of dilution and contamination w~th plasma, m some expernnents a

part of the packed volume of erythrocytes was washed twice with saline and homogemzed. After centnfugataon at 31,000 g, Hfl was determined m the supernatant, wtuch represented cell sap. The amount of Hfl found m this fired amounted to 3 0 - 4 0 percent of the total Hfl found in the erythrocytes, which is in good agreement with the results from the haemolyzmg experiments. Concentratmns m cell sap never exceeded those m the plasma.

4. DISCUSSION Hexafluorenmm Is accumulated m perfused rat liver and concentrated in bile m unchanged form. The high concentration gra&ents between bile and plasma (of the order of 1000 or more) tend to categorize Hfl as being actively transported from plasma into bile. The possibility that an actwe mechanism ~s revolved ~s supported by the mtub~tory activity of car&ac glycosides. A s~mflar effect was found on the hepatic transport of the structurally related d-tubocuranne (Meyer et al., 1968) suggesting that Na÷, K*-activated ATPase plays a role m the transport of the bisquatemary ammomum agents. Furthermore, ~t was found that Hfl and d-tubocurarme reduce bdmry excretion of the monoquaternary procamamlde ethobromlde (P.A.E.B.) m the rat (Meyer et al., 1970b). However, unhke transfer of curarehke drugs, cardiac glycosides m the concentratmns used did not interfere w~th the transport of P.A.E.B. from plasma into bile, re&catmg that transport of mono- and blsquaternary agents differs m at least one step. The present study attempts to locahze this cardaac-

250

D K F Mezler et al , Hepattc transport o f hexafluoremum

glycoside sensltwe step When K-strophantoslde was injected before Hfl admlmstratlon, both uptake into the hver and excretion Into bile were much reduced. However, ff the cardiac glycoside was gwen when the greatest part of Hfl had already been cleared from the perfuslon medmm and stored m the liver, further uptake was blocked but bdmry excretion was not affected. This result indicates that K-strophantoside probably does not influence transport of Hfl into the bile canahcuh but interferes with uptake from blood into the liver cells or with intracellular binding A similar effect was found with the cardmc glycoside dlgitoxm (2 5 X l0 -s M) and ouabam. Inhibition by ouabam occurred only at much higher doses (5 X 10-4 M) and was of short duration normal excretion was restored 30 mm after administration. This might be due to a rapid removal of ouabain from the perfuslon medium and the hver, caused by an extremely fast elimination into bile such as that found by Kupferberg et al. (1968) in the rat m vlvo. The cardiac glycosides did not interfere with binding and uptake of Hfl into erythrocytes nor was there uptake against a concentration gradient These results show that the drug penetrated the blood cells by diffusion, probably because of its ~elatlvely high lipid solubility This penetrahon into erythrocytes is slow when compared with uptake into the liver The slow decrease in plasma concentration observed in perfusion experiments after addition of K-strophantoslde (fig 2b) could be partly due to this uptake in red cells and to passive diffusion and binding to hepatic hssue, which ~s suggested by a concentration of 16/ag/g hver wet weight under these condlhons (see fig. 2b) Our results on uptake of Hfl into red cells conflict with the conclusion of Foldes et al (1960) that Hfl dM not penetrate into erythrocytes because It failed to inhibit acetylchohnesterase in intact cells but did so in haemolysed cells In contrast, the same authors found that Hfl inhibited acetylchohnesterase m intact red cells washed and resuspended m sahne, and attributed this to a permeablhty change in the red cell membrane. However, our experiments clearly show uptake of Hfl from plasma into the red cells The lack of inhibitory activity of Hfl in heparimzed whole blood found by Foldes et al could be due to the presence of heparm which can bind blsquaternary ammomum compounds (Cheymol et al., 1955) How do our results fit in with present ideas on the

transport of substances into the liver cell and into the bdiary tract9 Substances which are excreted in bile must pass at least two barriers, the membrane between the extracellular and lntracellular space and the canahcular membrane between the intracellular contents and bile Very little seems to be known about the passage of organic compounds after primary uptake through the liver parenchymal cell which could be composed of various compartments It is generally assumed that the outer cell membrane of liver cells forms a restrictive bamer for many substances (Hems et al., 1968). The outer membrane seems to be of a lipid nature because at O°C compounds of widely different hpld solubditles penetrate the liver Ussue at rates related to their hpophlhc character (Kurz, 1961). Lipid solubdIty may be important in the transfer of bisquaternary ammonium compounds (Meyer et al., 1970a) and of fernoxammes (Meyer-Brunot, 1968) from plasma into bale, whether at the site of entry into or passage through cells or at the canicular barrier. Either site may also favour compounds with special hydrophoblc properties (see Schanker, 1968). 'Concentratwe' uptake depending on the free concentration of drug is conceivable In the case of an active effort of hver cells or assuming that high binding to liver cell components and/or excretion of the compounds into bde maintain large concentration gradients of the organic ion between extra- and lntracellular fired. Procalnamide ethobromlde (PAEB), d-tubocurarme, amino-acids, cardiac glycosides, steroids and various dyes attain concentrations in hepatic tissue greatly exceeding that m plasma. In most cases, it is not clear whether ttus accumulation is due to uphdl transport from plasma into lntracellular fired, downhill transport followed by subsequent bmdmg to cell components or actually reflects concentrative transfer into the bile canahcuh. However, while being permeable to molecules of various s~zes and charges, the liver cell membranes can regulate the rate of passage of inorganic cations and maIntam high concentration gradients of Na + and K ÷ (Elshove et al., 1963) resultmg m a membrane potential of about 50 mV m the rat (Blederman, 1968). Other than passive diffusion phenomena must therefore be considered. Possible mechanisms, like faohrated diffusion, countertransport, pnmary and secondary active transport have been extensively rewewed

D K F Meuer et al, Hepatw transport ofhexafluorentum

(Stem, 1967). Sxmllar carrier mechamsms could be located m the liver plasma membranes facing the smusold. Cardmc glycosides affect secondary actwe transport of glucose, amino acids and certam aromatlc amines indirectly by inhibiting Na + , K +, Mg2 + activated ATPase. Ttus results in a loss of K ÷ and a gain of mtraceUular Na ÷ (Stem, 1967, Tissan et a l , 1969). Isolated rat liver plasma-membranes contain tugh actwlty of Na *, K*, Mg2÷ activated ATPase (Emmelot et al., 1966, Bakker et al., 1968) which can be inhibited by cardmc glycosides, erythrophleum alkaloids, Ca 2÷, btle acids, SH reagents. The concentrations of K-strophantoslde we used (2.5 × 10-s M) affect Na ÷ and K ÷ fluxes m rat hver shces (Van Elshove et al., 1963). It ~s temptmg to suggest that such changes m extracellular and mtracelltdar Ion content or direct interference with the energy supply to the transporting system might account for the mlubltlOn of uptake of blsquaternary agents by K-strophantoslde

ACKNOWLEDGEMENT We thank Malhnckrodt Chemical Works (St Lores, USA) for the gift of hexafluorenmm (Mylaxen0~).

REFERENCES Bakkeren, J.A.J.M. and S.L. Bontmg, 1968, Studies on (Na*K~) actwated ATPase. Properties of (Na*-K*) aetwated ATPase m rat hver, Bmchlm. Bmphys. Aeta 150, 460. Barber-Riley, G., 1965, Measurement of the capacity of the bdmry three, m The Bflmry System (BlackweU, Oxford), pp. 89-97 Bledermann, M., 1968, Das verhalten der Membranpotentmle von Leberzellen der Ratte w~thrend und nach Gef'assunterbmdungen, Acta Biol. Med. Germ. 21,827 Cavalhto, C.J., J.G. Arrowood and T.B. O'Dell, 1956, Influence of anesthesm on the neuromuscular blocking actw~ty of Mylaxen ®, Anesthesiology 17, 547. Cheymol, J.F., F. Bourfllet and C. Levassort, 1955, Action

251

antl-curanm6tlque de l'h6parme et d'h6peranoldes de synth~se chez le lapin, J. Physiol. (Pans) 47, 132. Cohen, E.N., B.H. Wmstow and D. Smith, 1967, The metabolism and ehmmatlon of d-tubocurarme -all, Anesthesiology 28, 309. Elshove, A. and G.D.V. van Rossum, 1963, Net movements of sodmm and potassmm and their relation to resplrat~on m shces of rat lwer mcubated m vitro, J. Physiol. 168, 531. Emmelot, P. and C.J. Bos, 1966, Studies on plasma membranes III. Mg~-ATPase (Na÷, K÷, Mg2+)-ATPase and 5' Nucleotldase actwlty of plasma membranes isolated from rat lwer, Bloehem Blophys Acta 120,369 Foldes, F.E., R.E. Molloy, E.K. Zslgmond and J.A. Zwartz, 1960, Hexafluorenmm, its antlcholmesterase and neuromuscular actwlty, J Pharmacol Exptl Therap 129,400 Hems, R., M. Stubbs and H A. Krebs, 1968, Restricted permeabihty of rat laver for glutamate and succmate, Blochem. J. 107,807. Kupferberg, H.J. and L.S. Schanker, 1968, Bthary secretion of ouabam -3H and its uptake by hvershces m the rat, Am. J. Physlol 214, 1048. Kurz, H., 1961, L~ptd solubility as an Important factor for penetration of drugs into the hver, Blochem. Pharmacol. 8, 20-21. Meljer, D.K.F. and A.H.J. Scaf, 1968, lnhlbltmn of transport of d-tubocuranne from blood to bile by K-strophantoslde m the isolated perfused rat ltver, European J. Pharmacol. 4, 343. Meljer, D.K.F., E.S. Bos and K.J. van der Laan, 1970b, Hepatic transport of mono- and blsquaternary ammonmm compounds, European J. Pharmacol. 11,371 Meller, D.K.F. and J.G. Weltering, 1970a, Curareqlke agents Relation between hpld solubility and transport into bile m perfused rat laver, European J. Pharmacol. 10, 283. Meljer, D.KF, GA Vermeer and G Kwant, 1971, The excretion of hexafluorenmm (Mylaxen(~) m man and m the rat, European J. Pharmacol. 14, 276. Meuer-Brunot, H.G. and H. Keberle, 1968, Blhary excretmn of femoxammes of varying hpld solub~hty m perfused rat lwer, Am. J. Physiol. 214, 1193. Schanker, L.S., 1968, Secretion of orgamc compounds m bde, m Handbook of Physiology. Ahmentary canal, Sect 6, Vol 5, pp. 2433 Stem, W.D., 1967, The Movement of Molecules Across Cell Membranes (Academic Press, New York, London) Tlssan, A.H., P.S. Schonhofer, D.F. Bogdanskl and B.B. Brodle, 1969, Mechanism of blogenlc amine transport II, Molec Pharmacol. 5,593.