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The effect of some diuretics on the electrogenic component of the sodium pump in mammalian non-myelinated nerve fibres
EUROPEAN JOURNALOF PHARMACOLOGY10 (1970) 249-254. NORTH-HOLLANDPUBLISHINGCOMPANY
T H E E F F E C T O F SOME D I U R E T I C S ON T H E E L E C T R O G E N I C C O M P O N E N T O F T H E S O D I U M PUMP IN M A M M A L I A N N O N - M Y E L I N A T E D N E R V E F I B R E S A. DEN HERTOG and R. RAS Department of Pharmacology, University of Groningen, Groningen, The Netherlands
Accepted 29 December 1969
Received 15 August 1969
A. DEN HERTOG and R. RAS, The effect of some diuretics on the electrogenic component of the sodium pump in mammalian non-myelinated nerve fibers, European J. Pharmacol. 10 (1970) 249-254. A study has been made of the effect of some diuretics on the electrogenic component of the sodium pump in non-myelinated fibres of the desheated rabbit vagus nerve. It was found that mercurials inhibit this sodium pump; however, it seems unlikely that this action can account for their diuretic action. Ethacrynic acid and amiloride increased the sodium permeability of the nerve fibre membrane; the importance of this permeability change with respect to the diuretic action is discussed. Chlorothiazide and triamterene appeared to be ineffective on the nerve fibre membrane. Diuretics
C fibres
1. INTRODUCTION The metabolic processes in nerve fibres during recovery after a period of activity are accompanied by membrane potential changes (hyperpolarization), which reflect an increased rate of sodium extrusion (Ritchie and Straub, 1957; Connelly, 1959; Holmes, 1962; Rang and Ritchie, 1968b). This post-tetanic hyperpolarization is abolished by interference with the mechanism of active sodium extrusion by applying metabolic inhibitors. Ouabain, an inhibitor of the Na-K sensitive adenosine triphosphate (ATPase), affected the post-tetanic hyperpolarization in the same way as metabolic inhibitors such as cyanide and deoxy-D-glucose (Ritchie and Straub, 1957; Holmes, 1962; Den Hertog and Ritchie, 1969; Den Hertog, Greengard and Ritchie, 1969). This hyperpolarization representing the sodium pump activity is thought to be related to the Na-K activated membrane ATPase representing the energy-consuming step in active ion transport (Bonting, 1962; Skou,
Electrogenic sodium pump
1965; Rang and Ritchie, 1968b; Den Hertog and Ritchie, 1969). This ouabain-sensitive sodium pump can be activated by potassium and other cations (Rang and Ritchie, 1968b). In the absence of potassium, the sodium pump is switched off as soon as the potassium that is extruded during activity by the nerve fibres has been recaptured, or has diffused away from the peri-axonal space (Rang and Ritchie, 1968b; Den Hertog and Ritchie, 1969). Active sodium transport begins again after addition of potassium; this phenomenon is called the potassium-activated response (PAR) and can be recorded as a hyperpolarization. Such a response is shown in the left-hand part of fig. 1; the addition of potassium is indicated by the arrow about 5 min after the period of activity of the nerve (upward deflection) and the following posttetanic hyperpolarization (downward deflection). The fibres will gain a constant amount of sodium ions during a tetanus and thus the elicited potassiumactivated response will be independent of changes in
250
A.Den Hertog, R.Ras, Diuretics and the electrogenic Na pump in C-fibres
the action potential, if the drug has been added after the tetanus. The action of drugs on the ouabainsensitive sodium pump can be studied by recording the PAR; a membrane permeability change can be distinguished from an action on the pump by analyzing the amplitude and time constant of the PAR. The effect of the diuretics triamterene, amiloride, chlorothiazide, ethacrynic acid, mersalyl and the non-diuretic mercurial PCMB (p-chloromercuribenzoate) on the membrane permeability and the inhibiting action on the ouabain-sensitive sodium pump in mammalian non-myelinated nerve fibers has been studied. A comparison of the action of these diuretics on the Na-K transport in nerve fibres and the kidney has been made. The possibility that the underlying mechanism of the Na-K exchange in the distal tubules, which can be inhibited by triamterene and amiloride (Bear, 1966), is identical with the active Na-K transport in nerve fibres has been investigated. Furthermore, the relation of the ATPase inhibiting action of the mercurials in the kidney to the activity of the sodium pump in nerve fibres during exposure to these drugs is of interest. The action of represensatives from other groups of diuretics, such as ethacrynic acid and chlorothiazide, is also discussed.
2. METHODS Rabbits were killed by the injection of air into an ear vein. The cervical vagi were removed, desheathed under a dissecting microscope and mounted in a sucrose-gap apparatus for measuring membrane potential changes (Stampfli, 1954; Straub, 1957; Armett and Ritchie, 1960). The experiments were carried out at room temperature (20-24°C). The composition of the chloridefree Locke solution used was (mM): sodium isethionate, 154; K2SO4, 2.8; CaSO4, 5.0; tris (hydroxy methyl) aminomethane brought to pH 7.2 with sulphuric acid, 2.5; D-glucose, 5.0. The drugs were added as sulfates and brought to pH 7.2 if necessary. The nerve was stimulated between in-dwelling platinum electrodes in the sucrose-gap apparatus. Square wave pulses of 0.5 msec duration and of an intensity sufficient for supra-maximal stimulation were delivered by a stimulator (Grass, $8). Responses of the nerve were obtained to a 5 sec period of stimulation at a
frequency of 30 stimuli per sec. The resulting membrane potential changes across the sucrose bridge were recorded through calomel electrodes via a cathode-follower; the cathode-follower was connected with a servo-driven potentiometric pen recorder (Varian 2000) and a storagescope (Tektronic 564) to reproduce respectively the rather slow and fast membrane potential changes. The potassium-activated response that was recorded as a downward deflection (hyperpolarization) was elicited by 2 mM potassium added 5 min after the nerve was stimulated (indicated with an arrow in fig. 1) unless otherwise stated. This potassium-activated response (PAR) declines exponentially with time as long as the coupling ratio of the pump does not change during recovery (Rang and Ritchie, 1968b). Such a PAR is determined by the maximal amplitude V m and the time constant. The time constant can be calculated from the slope of the straight line in the log V-time characteristic (right-hand part of fig. 1). The time constant reflects the ability of the pump to extrude an amount of sodium in a fixed period; the amplitude V m is related to the maximum amount of sodium transported electrogenically per unit time and with the membrane permeability. Therefore, chloride-free bathinsolutions were used because the resulting decrease in membrane conductance greatly increases the amplitude of the PAR and thus facilitates its measurement.
3. RESULTS The drugs are not grouped according to the kind of diuretic action assumed but to the effects on the nerve fibre membrane. 3.1. M e r s a l y l a n d P C M B
Not only mersalyl (100/aM) but also parachloromercuribenzoate (PCMB; 25-1 O0/aM) affected the amplitude and the time constant of the PAR. The diuretic mersalyt (100/aM) diminished the amplitude 30% whereas the decrease of the time constant was about 20%. The same concentration of the non-diuretic mercurial PCMB (100/aM) diminished both the amplitude and the time constant of the PAR up to 50%. A record of an experiment with PCMB and the relation between the amplitude plotted logarithmical-
A.Den Hertog, R.Ras, Diuretics and the electrogenic Na pump in C-filters
251
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TIME Fig. 1. The effect of p-chloromercuribenzoate (PCMB) on the potassium-activated response of the non-myelinated fibres of a rabbit desheathed vagus nerve. The upward deflection at the beginning of each record (left-hand part of the figure) results from the 5 sec series of action potentials elicited at a rate of 30 per sec. A downward deflection represents a hyperpolarization of the nerve. The potassium-activated responses are obtained 5 min after stimulation by adding 2 mM potassium to the Locke solution (arrow). During the second record the drug was added just after stimulation of the nerve (broken arrow). In the right-hand part of the figure, the amplitudes of the potassium activated responses in the absence ( o - - - o ) and the presence ( o - - - o ) of PCMB have been plotted logarithmically against time.
ly a n d t h e t i m e is s h o w n in fig. 1; t h e a d d i t i o n o f t h e d r u g is i n d i c a t e d w i t h a b r o k e n arrow. T h e straight line w i t h t h e steeper slope ( r i g h t - h a n d p a r t o f fig. 1) t h a t was c a l c u l a t e d f r o m the P A R d u r i n g PCMB i n d i c a t e s a smaller t i m e c o n s t a n t t h a n t h a t o f t h e
c o n t r o l r e s p o n s e ; a smaller t i m e c o n s t a n t m e a n s a h i g h e r overall p u m p i n g rate o f sodium. T h e n u m e r i c a l d a t a o f m e r s a l y l a n d PCMB are given in table 1; a review o f t h e c h a n g e d p a r a m e t e r s o f t h e P A R in t h e p r e s e n c e o f t h e m e r c u r i a l s is given in fig. 3. I t can b e
Table 1 The effects of a number of diuretics and diuretic-like agents on the electrogenic component of the sodium pump. The initial value of the amplitude of the K-activated response (2 mM) before exposure to the drug was 4.8 + 2.5 mV (means + S.E.) and the initial value of the time constant was 1.88 + 0.56 min (means + S.E.).
Drug
Amplitude ratio of K-activated response Conc. after and before exposure to the drug (~M) (means + S.E.)
Time constant ratio of K-activated response after and before exposure to the drug (means -+ S.E.)
Number of experiments
Mersalyl
100
0.68 + 0.07
0.78 + 0.06
6
PCMP
25 100
0.85 +0.05 0.58 +0.06
0.81 +0.04 0.61 +0.11
2 5
300 1000
0.84 + 0.07 0.65 +0.12
0.93 -+ 0.02 0.98 +0.09
2 4
20 50
0.71 + 0.09 0.47 +0.15
0.90 + 0.04 0.97 +0.05
2 2
Chlorothiazide
250 500
1.11 + 0.02 0.93 + 0.02
1.00 + 0.07 0.94 + 0.09
2 2
Triamterene
100
1.06 + 0.08
1.00 + 0.01
4
Amiloride Ethacrynic acid
252
A.Den Hertog, R.Ras, Diuretics and the electrogenic Na pump in C-fibres
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Fig. 2. The effect of amiloride on the potassium-activated response of the non-myelinated fibres of a rabbit desheathed vagus nerve. The upward deflection at the beginning of each record (left-hand part of the figure) results from the 5 sec series of action potentials elicited at a rate of 30 per sec. A downward deflection represents a hyperpolarization of the nerve. The potassium activated responses were obtained 5 rain after stimulation by adding 2 mM potassium to the Locke solution (arrow). During the second record the drug was added just after stimulation of the nerve (broken arrow). In the right-hand part of the figure, the amplitude of the potassium activated respoose in the absence ( e - - - e ) and the presence ( o - - o) of amiloride have been plotted logarithmically against time. noticed than no changed was observed in the compound action noticed than no change was observed in the compound action the drugs. 3.2. Amiloride and ethacrynic acid These diuretics were tested in the same manner as the mercurials. A 5 min exposure to amiloride (0.3 and 1.0 raM) and ethacrynic acid (20 and 50/aM) diminished the maximal amplitude of the PAR 1 0 - 4 0 % and 2 0 - 6 0 % respectively whereas the time constant remained constant (fig. 3; table 1). Thus, ethacrynic acid appeared to be about 10 times more potent than amiloride (table 1). That the time constant was not affected by these drugs is demonstrated in fig. 2; the left-hand part of the figure shows a record of the posttetanic hyperpolarization and the PAR in the absence and presence of amiloride and in the right-hand part the amplitude is plotted logarithmically against-time. The straight lines (fig. 2), representing an exponential relationship between the PAR amplitude and the time, are parallel in the control situation and in the presence of amiloride; this means that these drugs did not affect the rate o f sodium extrusion. The resting potential and action did not change in amplitude during exposure to the drugs.
3.3. Chlorothiazide and triamterene High concentrations of chlorothiazide (250 and 500/~M) and triamterene (saturated in Locke solution) did not affect the PAR, the resting potential or the action potential. Thus these compounds do not affect the sodium pump the permeability of the membrane of non-myelinated nerve fibres.
TIME CONSTANT
AMPLITUDE
[]
%
o t. u_ u_ ta 50
MersoLyL
PCM B
15DpM
100NM
Am,lor,Oe
1 mM
Etho{ry.,c o:,d
50rim
Chloroth,oi,~, Tt,amtar.ne
0 5 mM
~ O1 mM
DRUG
Fig. 3. Percentage change, as mean + standard error, of the maximal amplitude (white column) and the time constant (dark column) of the potassium-activated response of the non-myelinated fibres of a rabbit desheathed vagus nerve during exposure to drugs (as indicated on the horizontal axis) related to controls.
A.Den Hertog, R.Ras, Diuretics and the electrogenic Na pump in C-filters
4. DISCUSSION 4.1. Mersalyl and PCMP
The results of these experiments with mercurials differ from those obtained with metabolic inhibitors such as deoxy-D-glucose and cyanide. The sodium debt after a period of activity as paid off more slowly during the PAR (Den Hertog and Ritchie, 1969; Den Hertog, Greengard and Ritchie, 1969) in the presence of these metabolic inhibitors (deoxy-D-glucose and cyanide). This is reflected in an increased time constant and a lower maximal amplitude Vm of the PAR whereas the area of the response remains constant. However, it was observed that the PAR amplitude decreased in the presence of the mercurials but that the time constant decreased up to 40% resulting in a diminished area of the response. Furthermore, a change in the resting potential and action was not observed; thus, a permeability increase of the membrane is unlikely to account for the decreased amplitude. The same effect on the potassium response as with the mercurials was found with ouabain added in a low concentration (Den Hertog and Ritchie, 1969) although it is generally accepted that ouabain inhibits the sodium pump quite specifically. The assumption was made to explain the ouabain effect on the electrogenic component of the sodium pump, namely that pumping is inhibited completely in a number of fibres in a random manner with respect to their diameter. This produces a progressive decrease in the amplitude of the PAR and thus a decrease in the maximal amplitude and a smaller time constant. Thus, these results indicate an inhibitory action of the mercurials on the potassium sensitive sodium pump in nerve fibres as found with ouabain. The ability of mercurials to inhibit not only the sodium- and potassium-sensitive renal membrane ATPase but also the total ATPase activity (Nechay et al., 1967) was considered to be related to the inhibition of the active reabsorption of sodium and thus with their diuretic action (Taylor, 1963; Jones et al., 1965). A positive correlation between potentiation of the mercurial diuresis and the Na- and K-sensitive ATPase by acidosis (Nechay et al., 1967) was suggested. However, it can be concluded from the comparable findings of Nechay (1967), and the resuits of our experiments, that not only diuretic
253
(mersalyl) but also non-diuretic (PCMB) mercurials inhibit the Na-K activated ATPase. If there is a comparable distribution of mersalyl and PCMB in the kidney then it is unlikely that the inhibition of the Na- and K-sensitive ATPase can completely account for the diuretic action of mersalyl. 4.2. Ethacrynic acid and amiloride
An action similar to that of these diuretics on the PAR, that is a diminished amplitude and a constant time constant, was also seen with tetramethyl ammonium (Den Hertog and Ritchie, 1969). A constant pumping rate and a decreased amplitude of the PAR suggest an increased membrane permeability; a higher sodium permeability can account for the observed increase in permeability rather than a change in the potassium permeability, for no change in the resting potential was recorded. This action seems to contrast with the inhibitory action of this diuretic on membrane ATPase described elsewhere (Duggan and Noll, 1965; Baer, 1966); a 50% reduction of the Na- and K-activated ATPase was found by Nechay (1967) with a dose of 0.1 mM ethacrynic acid. The amplitude of the PAR was reduced to 50% of its control value in our experiments due to an increase in membrane permeability with a dose of 50/aM ethacrynic acid. This means that no effect of ethacrynic acid on the sodiumpump could be detected because the change in membrane permeability was dominant (fig. 3, table 1). The diuretic action of this drug, localized mainly in the loop of Henle (Early and Friedler, 1964), can be explained by an increase in permeability in the tubules. Assuming a constant reabsorption rate of sodium, such a permeability change would result in an increased net excretion of sodium in that part of the tubules where a concentration gradient exist between inside and outside; this is mainly found in the loop of Henle. In our experiments, amiloride had a similar action on the non-myelinated nerve fibres as ethacrynic acid. However, it is unlikely that the permeability change found in nerve fibers is responsible for the diuretic action of both ethacrynic acid and amiloride because of the quite different nature of the diuretic response (Baer, 1966). The dose of amiloride on a weight base has to be about 15 times higher than ethacrynic acid to a cause a comparable permeability change, whereas
254
A.Den Hertog, R.Ras, Diuretics and the electrogenic Na pump in C-fibres
the dose o f amiloride is 10 times lower than with ethacrynic acid for a diuretic action. Thus, in contrast w i t h ethacrynic acid, the sodium permeability increase caused b y amiloride is unlikely to account for the diuretic action. The conclusion that amiloride does n o t effect the sodium p u m p in nerve fibres is c o n f i r m e d by the insensitivity o f the Na-K activated ATPase in rabbit liver to this substance (Baer, 1966). 4.3. C h l o r o t h i a z i d e a n d t r i a m t e r e n e A l t h o u g h b o t h chlorothiazide and triamterene (Baba et al, 1964; Wiebelhaus et al., 1965; Early, 1967) inhibit sodium reabsorption in the distal tubules, neither the electrogenic c o m p o n e n t o f the ouabain-sensitive sodium p u m p , the permeability, the resting potential nor the action p o t e n t i a l o f the nerve fibre m e m b r a n e , were affected.
ACKNOWLEDGEMENTS Amiloride, ethacrynic acid and chlorothiazide were kindly supplied by Merck Sharp and Dohme. Triamterene was kindly supplied by R.I.T. Nederland N.V.
REFERENCES Baer, J.E., 1966, Recent developments in the pharmacology of salturetic-diuretic agents, in: Salt and water balance, vol. 8 (Pergamon press, London) p. 317. Baba, W.I., G.R, Tudhope and G.M. Wilson, 1964, Site and mechanism of action of the diuretic triamterene, Clin. Sci. 27, 181. Bonting, S.L., L.L. Caravaggio and N.M. Hawkins, 1962, Studies in sodium-potassium-activated adenosinetripbosphatase. IV. Correlation with cation transport sensitive to cardiac glycosides, Arch. Biochem. Biophys. 98,413. Connelly, C.M., 1959, Recovery processes and recovery in nerve, Rev. Mod. Phys. 31,474. Den Hertog, A. and J.M. Ritchie, 1969, A comparison on the effect of temperature, metabolic inhibitors, and of oua-
bain on the electrogenic component of the sodium pump in mammalian non-myelinated nerve fibres, J. Physiol. (London) 1969, 204,523. Den Hertog, A. and J.M. Ritchie, 1969, The effect of some quaternary ammonium compounds and local anesthetics on the electrogenic component of the sodium pump in mammalian non-myelinated nerve fibres, European J. Pharmacol. 6, 138. Den Hertog, A., P. Greengard and J.M. Ritchie, 1969, On the metabolic basis of nervous activity, J. Physiol. (London) 204,511. Duggan, D.E. and R.M. Moll, 1965, Effects of ethacrynic acid and cardiac glycosides upon a membrane adenosinetriphosphatase of renal cortex, Arch. Biochem. Biophys. 109,388 Early, L.E. and R.M. Friedler, 1964, Renal tubular effects of ethacrynic acid, J. Clin. Invest. 43, 1495. Early, L.E., 1967, Diuretics, New England J. Med. 276, 17, 966. Holmes, O., 1962, Effects of pH, changes in potassium concentration and metabolic inhibitors on the afterpotentials of mammalian non-medulated nerve fibers, Arch. Intern. Physiol. 70, 211. Jones, V.D., J. Lockett and E.J. Landon, 1965, Cellular action of mercurial diuretics. J. Pharmacol. Exptl. Therap. 147, 23. Nechay, B.R., R.F. Palmer, D.A. Chiney and V.A. Posey, 1967, The problem of Na¢+ K ÷ adenosine triphosphatase as the receptor for diuretic action of mercurials and ethacrynic acid. J. Pharmacol. Exptl. Therap. 157,599. Rang, H.P. and J.M. Ritchie, 1968, On the electrogenic sodium pump in mammalian non-myelinated nerve fibres and its activation by various external cations, J. Physiol. (London) 196, 183. Ritchie, J.M. and R.W. Straub, 1957, The hyperpolarization which follows activity in mammalian non-meduUated fibres, J. Physiol. (London) 136, 80. Skou, J.C., 1962, Preparation from mammalian brain and kidney of the enzyme system involved in active transport of Na ÷ and K ÷, Biochem. Biophys. Acta 58,314. Taylor, C.B., 1963, The effect of mercurial diuretics on adenosine triphospbatase of rabbit kidney in vitro, Biochem. Pharmacol. 12, 539. Wiebelhaus, V.P., J. Weinstock, A.R. Mass, F.T. Brennau, G. Sosnowki and T. Larsen, 1965, The diuretic and natrureric activity of triamterene and several related pteridines in the rat, J. Pharmacol. Exptl. Therap. 149,397.
T H E E F F E C T O F SOME D I U R E T I C S ON T H E E L E C T R O G E N I C C O M P O N E N T O F T H E S O D I U M PUMP IN M A M M A L I A N N O N - M Y E L I N A T E D N E R V E F I B R E S A. DEN HERTOG and R. RAS Department of Pharmacology, University of Groningen, Groningen, The Netherlands
Accepted 29 December 1969
Received 15 August 1969
A. DEN HERTOG and R. RAS, The effect of some diuretics on the electrogenic component of the sodium pump in mammalian non-myelinated nerve fibers, European J. Pharmacol. 10 (1970) 249-254. A study has been made of the effect of some diuretics on the electrogenic component of the sodium pump in non-myelinated fibres of the desheated rabbit vagus nerve. It was found that mercurials inhibit this sodium pump; however, it seems unlikely that this action can account for their diuretic action. Ethacrynic acid and amiloride increased the sodium permeability of the nerve fibre membrane; the importance of this permeability change with respect to the diuretic action is discussed. Chlorothiazide and triamterene appeared to be ineffective on the nerve fibre membrane. Diuretics
C fibres
1. INTRODUCTION The metabolic processes in nerve fibres during recovery after a period of activity are accompanied by membrane potential changes (hyperpolarization), which reflect an increased rate of sodium extrusion (Ritchie and Straub, 1957; Connelly, 1959; Holmes, 1962; Rang and Ritchie, 1968b). This post-tetanic hyperpolarization is abolished by interference with the mechanism of active sodium extrusion by applying metabolic inhibitors. Ouabain, an inhibitor of the Na-K sensitive adenosine triphosphate (ATPase), affected the post-tetanic hyperpolarization in the same way as metabolic inhibitors such as cyanide and deoxy-D-glucose (Ritchie and Straub, 1957; Holmes, 1962; Den Hertog and Ritchie, 1969; Den Hertog, Greengard and Ritchie, 1969). This hyperpolarization representing the sodium pump activity is thought to be related to the Na-K activated membrane ATPase representing the energy-consuming step in active ion transport (Bonting, 1962; Skou,
Electrogenic sodium pump
1965; Rang and Ritchie, 1968b; Den Hertog and Ritchie, 1969). This ouabain-sensitive sodium pump can be activated by potassium and other cations (Rang and Ritchie, 1968b). In the absence of potassium, the sodium pump is switched off as soon as the potassium that is extruded during activity by the nerve fibres has been recaptured, or has diffused away from the peri-axonal space (Rang and Ritchie, 1968b; Den Hertog and Ritchie, 1969). Active sodium transport begins again after addition of potassium; this phenomenon is called the potassium-activated response (PAR) and can be recorded as a hyperpolarization. Such a response is shown in the left-hand part of fig. 1; the addition of potassium is indicated by the arrow about 5 min after the period of activity of the nerve (upward deflection) and the following posttetanic hyperpolarization (downward deflection). The fibres will gain a constant amount of sodium ions during a tetanus and thus the elicited potassiumactivated response will be independent of changes in
250
A.Den Hertog, R.Ras, Diuretics and the electrogenic Na pump in C-fibres
the action potential, if the drug has been added after the tetanus. The action of drugs on the ouabainsensitive sodium pump can be studied by recording the PAR; a membrane permeability change can be distinguished from an action on the pump by analyzing the amplitude and time constant of the PAR. The effect of the diuretics triamterene, amiloride, chlorothiazide, ethacrynic acid, mersalyl and the non-diuretic mercurial PCMB (p-chloromercuribenzoate) on the membrane permeability and the inhibiting action on the ouabain-sensitive sodium pump in mammalian non-myelinated nerve fibers has been studied. A comparison of the action of these diuretics on the Na-K transport in nerve fibres and the kidney has been made. The possibility that the underlying mechanism of the Na-K exchange in the distal tubules, which can be inhibited by triamterene and amiloride (Bear, 1966), is identical with the active Na-K transport in nerve fibres has been investigated. Furthermore, the relation of the ATPase inhibiting action of the mercurials in the kidney to the activity of the sodium pump in nerve fibres during exposure to these drugs is of interest. The action of represensatives from other groups of diuretics, such as ethacrynic acid and chlorothiazide, is also discussed.
2. METHODS Rabbits were killed by the injection of air into an ear vein. The cervical vagi were removed, desheathed under a dissecting microscope and mounted in a sucrose-gap apparatus for measuring membrane potential changes (Stampfli, 1954; Straub, 1957; Armett and Ritchie, 1960). The experiments were carried out at room temperature (20-24°C). The composition of the chloridefree Locke solution used was (mM): sodium isethionate, 154; K2SO4, 2.8; CaSO4, 5.0; tris (hydroxy methyl) aminomethane brought to pH 7.2 with sulphuric acid, 2.5; D-glucose, 5.0. The drugs were added as sulfates and brought to pH 7.2 if necessary. The nerve was stimulated between in-dwelling platinum electrodes in the sucrose-gap apparatus. Square wave pulses of 0.5 msec duration and of an intensity sufficient for supra-maximal stimulation were delivered by a stimulator (Grass, $8). Responses of the nerve were obtained to a 5 sec period of stimulation at a
frequency of 30 stimuli per sec. The resulting membrane potential changes across the sucrose bridge were recorded through calomel electrodes via a cathode-follower; the cathode-follower was connected with a servo-driven potentiometric pen recorder (Varian 2000) and a storagescope (Tektronic 564) to reproduce respectively the rather slow and fast membrane potential changes. The potassium-activated response that was recorded as a downward deflection (hyperpolarization) was elicited by 2 mM potassium added 5 min after the nerve was stimulated (indicated with an arrow in fig. 1) unless otherwise stated. This potassium-activated response (PAR) declines exponentially with time as long as the coupling ratio of the pump does not change during recovery (Rang and Ritchie, 1968b). Such a PAR is determined by the maximal amplitude V m and the time constant. The time constant can be calculated from the slope of the straight line in the log V-time characteristic (right-hand part of fig. 1). The time constant reflects the ability of the pump to extrude an amount of sodium in a fixed period; the amplitude V m is related to the maximum amount of sodium transported electrogenically per unit time and with the membrane permeability. Therefore, chloride-free bathinsolutions were used because the resulting decrease in membrane conductance greatly increases the amplitude of the PAR and thus facilitates its measurement.
3. RESULTS The drugs are not grouped according to the kind of diuretic action assumed but to the effects on the nerve fibre membrane. 3.1. M e r s a l y l a n d P C M B
Not only mersalyl (100/aM) but also parachloromercuribenzoate (PCMB; 25-1 O0/aM) affected the amplitude and the time constant of the PAR. The diuretic mersalyt (100/aM) diminished the amplitude 30% whereas the decrease of the time constant was about 20%. The same concentration of the non-diuretic mercurial PCMB (100/aM) diminished both the amplitude and the time constant of the PAR up to 50%. A record of an experiment with PCMB and the relation between the amplitude plotted logarithmical-
A.Den Hertog, R.Ras, Diuretics and the electrogenic Na pump in C-filters
251
O
mV tu co
10
n u~ LtJ
o:I 3
10 mV
5(7
D i,--
1
I
10min. i
i
i
i
2
t.
6
8 rain.
TIME Fig. 1. The effect of p-chloromercuribenzoate (PCMB) on the potassium-activated response of the non-myelinated fibres of a rabbit desheathed vagus nerve. The upward deflection at the beginning of each record (left-hand part of the figure) results from the 5 sec series of action potentials elicited at a rate of 30 per sec. A downward deflection represents a hyperpolarization of the nerve. The potassium-activated responses are obtained 5 min after stimulation by adding 2 mM potassium to the Locke solution (arrow). During the second record the drug was added just after stimulation of the nerve (broken arrow). In the right-hand part of the figure, the amplitudes of the potassium activated responses in the absence ( o - - - o ) and the presence ( o - - - o ) of PCMB have been plotted logarithmically against time.
ly a n d t h e t i m e is s h o w n in fig. 1; t h e a d d i t i o n o f t h e d r u g is i n d i c a t e d w i t h a b r o k e n arrow. T h e straight line w i t h t h e steeper slope ( r i g h t - h a n d p a r t o f fig. 1) t h a t was c a l c u l a t e d f r o m the P A R d u r i n g PCMB i n d i c a t e s a smaller t i m e c o n s t a n t t h a n t h a t o f t h e
c o n t r o l r e s p o n s e ; a smaller t i m e c o n s t a n t m e a n s a h i g h e r overall p u m p i n g rate o f sodium. T h e n u m e r i c a l d a t a o f m e r s a l y l a n d PCMB are given in table 1; a review o f t h e c h a n g e d p a r a m e t e r s o f t h e P A R in t h e p r e s e n c e o f t h e m e r c u r i a l s is given in fig. 3. I t can b e
Table 1 The effects of a number of diuretics and diuretic-like agents on the electrogenic component of the sodium pump. The initial value of the amplitude of the K-activated response (2 mM) before exposure to the drug was 4.8 + 2.5 mV (means + S.E.) and the initial value of the time constant was 1.88 + 0.56 min (means + S.E.).
Drug
Amplitude ratio of K-activated response Conc. after and before exposure to the drug (~M) (means + S.E.)
Time constant ratio of K-activated response after and before exposure to the drug (means -+ S.E.)
Number of experiments
Mersalyl
100
0.68 + 0.07
0.78 + 0.06
6
PCMP
25 100
0.85 +0.05 0.58 +0.06
0.81 +0.04 0.61 +0.11
2 5
300 1000
0.84 + 0.07 0.65 +0.12
0.93 -+ 0.02 0.98 +0.09
2 4
20 50
0.71 + 0.09 0.47 +0.15
0.90 + 0.04 0.97 +0.05
2 2
Chlorothiazide
250 500
1.11 + 0.02 0.93 + 0.02
1.00 + 0.07 0.94 + 0.09
2 2
Triamterene
100
1.06 + 0.08
1.00 + 0.01
4
Amiloride Ethacrynic acid
252
A.Den Hertog, R.Ras, Diuretics and the electrogenic Na pump in C-fibres
mV b
tl
w 03
10
g 3c t
10mV taJ C3 D
I
(3_
!
I
10rain. 0
A
i
i
i
2
4
6
8 rnin.
TIME
Fig. 2. The effect of amiloride on the potassium-activated response of the non-myelinated fibres of a rabbit desheathed vagus nerve. The upward deflection at the beginning of each record (left-hand part of the figure) results from the 5 sec series of action potentials elicited at a rate of 30 per sec. A downward deflection represents a hyperpolarization of the nerve. The potassium activated responses were obtained 5 rain after stimulation by adding 2 mM potassium to the Locke solution (arrow). During the second record the drug was added just after stimulation of the nerve (broken arrow). In the right-hand part of the figure, the amplitude of the potassium activated respoose in the absence ( e - - - e ) and the presence ( o - - o) of amiloride have been plotted logarithmically against time. noticed than no changed was observed in the compound action noticed than no change was observed in the compound action the drugs. 3.2. Amiloride and ethacrynic acid These diuretics were tested in the same manner as the mercurials. A 5 min exposure to amiloride (0.3 and 1.0 raM) and ethacrynic acid (20 and 50/aM) diminished the maximal amplitude of the PAR 1 0 - 4 0 % and 2 0 - 6 0 % respectively whereas the time constant remained constant (fig. 3; table 1). Thus, ethacrynic acid appeared to be about 10 times more potent than amiloride (table 1). That the time constant was not affected by these drugs is demonstrated in fig. 2; the left-hand part of the figure shows a record of the posttetanic hyperpolarization and the PAR in the absence and presence of amiloride and in the right-hand part the amplitude is plotted logarithmically against-time. The straight lines (fig. 2), representing an exponential relationship between the PAR amplitude and the time, are parallel in the control situation and in the presence of amiloride; this means that these drugs did not affect the rate o f sodium extrusion. The resting potential and action did not change in amplitude during exposure to the drugs.
3.3. Chlorothiazide and triamterene High concentrations of chlorothiazide (250 and 500/~M) and triamterene (saturated in Locke solution) did not affect the PAR, the resting potential or the action potential. Thus these compounds do not affect the sodium pump the permeability of the membrane of non-myelinated nerve fibres.
TIME CONSTANT
AMPLITUDE
[]
%
o t. u_ u_ ta 50
MersoLyL
PCM B
15DpM
100NM
Am,lor,Oe
1 mM
Etho{ry.,c o:,d
50rim
Chloroth,oi,~, Tt,amtar.ne
0 5 mM
~ O1 mM
DRUG
Fig. 3. Percentage change, as mean + standard error, of the maximal amplitude (white column) and the time constant (dark column) of the potassium-activated response of the non-myelinated fibres of a rabbit desheathed vagus nerve during exposure to drugs (as indicated on the horizontal axis) related to controls.
A.Den Hertog, R.Ras, Diuretics and the electrogenic Na pump in C-filters
4. DISCUSSION 4.1. Mersalyl and PCMP
The results of these experiments with mercurials differ from those obtained with metabolic inhibitors such as deoxy-D-glucose and cyanide. The sodium debt after a period of activity as paid off more slowly during the PAR (Den Hertog and Ritchie, 1969; Den Hertog, Greengard and Ritchie, 1969) in the presence of these metabolic inhibitors (deoxy-D-glucose and cyanide). This is reflected in an increased time constant and a lower maximal amplitude Vm of the PAR whereas the area of the response remains constant. However, it was observed that the PAR amplitude decreased in the presence of the mercurials but that the time constant decreased up to 40% resulting in a diminished area of the response. Furthermore, a change in the resting potential and action was not observed; thus, a permeability increase of the membrane is unlikely to account for the decreased amplitude. The same effect on the potassium response as with the mercurials was found with ouabain added in a low concentration (Den Hertog and Ritchie, 1969) although it is generally accepted that ouabain inhibits the sodium pump quite specifically. The assumption was made to explain the ouabain effect on the electrogenic component of the sodium pump, namely that pumping is inhibited completely in a number of fibres in a random manner with respect to their diameter. This produces a progressive decrease in the amplitude of the PAR and thus a decrease in the maximal amplitude and a smaller time constant. Thus, these results indicate an inhibitory action of the mercurials on the potassium sensitive sodium pump in nerve fibres as found with ouabain. The ability of mercurials to inhibit not only the sodium- and potassium-sensitive renal membrane ATPase but also the total ATPase activity (Nechay et al., 1967) was considered to be related to the inhibition of the active reabsorption of sodium and thus with their diuretic action (Taylor, 1963; Jones et al., 1965). A positive correlation between potentiation of the mercurial diuresis and the Na- and K-sensitive ATPase by acidosis (Nechay et al., 1967) was suggested. However, it can be concluded from the comparable findings of Nechay (1967), and the resuits of our experiments, that not only diuretic
253
(mersalyl) but also non-diuretic (PCMB) mercurials inhibit the Na-K activated ATPase. If there is a comparable distribution of mersalyl and PCMB in the kidney then it is unlikely that the inhibition of the Na- and K-sensitive ATPase can completely account for the diuretic action of mersalyl. 4.2. Ethacrynic acid and amiloride
An action similar to that of these diuretics on the PAR, that is a diminished amplitude and a constant time constant, was also seen with tetramethyl ammonium (Den Hertog and Ritchie, 1969). A constant pumping rate and a decreased amplitude of the PAR suggest an increased membrane permeability; a higher sodium permeability can account for the observed increase in permeability rather than a change in the potassium permeability, for no change in the resting potential was recorded. This action seems to contrast with the inhibitory action of this diuretic on membrane ATPase described elsewhere (Duggan and Noll, 1965; Baer, 1966); a 50% reduction of the Na- and K-activated ATPase was found by Nechay (1967) with a dose of 0.1 mM ethacrynic acid. The amplitude of the PAR was reduced to 50% of its control value in our experiments due to an increase in membrane permeability with a dose of 50/aM ethacrynic acid. This means that no effect of ethacrynic acid on the sodiumpump could be detected because the change in membrane permeability was dominant (fig. 3, table 1). The diuretic action of this drug, localized mainly in the loop of Henle (Early and Friedler, 1964), can be explained by an increase in permeability in the tubules. Assuming a constant reabsorption rate of sodium, such a permeability change would result in an increased net excretion of sodium in that part of the tubules where a concentration gradient exist between inside and outside; this is mainly found in the loop of Henle. In our experiments, amiloride had a similar action on the non-myelinated nerve fibres as ethacrynic acid. However, it is unlikely that the permeability change found in nerve fibers is responsible for the diuretic action of both ethacrynic acid and amiloride because of the quite different nature of the diuretic response (Baer, 1966). The dose of amiloride on a weight base has to be about 15 times higher than ethacrynic acid to a cause a comparable permeability change, whereas
254
A.Den Hertog, R.Ras, Diuretics and the electrogenic Na pump in C-fibres
the dose o f amiloride is 10 times lower than with ethacrynic acid for a diuretic action. Thus, in contrast w i t h ethacrynic acid, the sodium permeability increase caused b y amiloride is unlikely to account for the diuretic action. The conclusion that amiloride does n o t effect the sodium p u m p in nerve fibres is c o n f i r m e d by the insensitivity o f the Na-K activated ATPase in rabbit liver to this substance (Baer, 1966). 4.3. C h l o r o t h i a z i d e a n d t r i a m t e r e n e A l t h o u g h b o t h chlorothiazide and triamterene (Baba et al, 1964; Wiebelhaus et al., 1965; Early, 1967) inhibit sodium reabsorption in the distal tubules, neither the electrogenic c o m p o n e n t o f the ouabain-sensitive sodium p u m p , the permeability, the resting potential nor the action p o t e n t i a l o f the nerve fibre m e m b r a n e , were affected.
ACKNOWLEDGEMENTS Amiloride, ethacrynic acid and chlorothiazide were kindly supplied by Merck Sharp and Dohme. Triamterene was kindly supplied by R.I.T. Nederland N.V.
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bain on the electrogenic component of the sodium pump in mammalian non-myelinated nerve fibres, J. Physiol. (London) 1969, 204,523. Den Hertog, A. and J.M. Ritchie, 1969, The effect of some quaternary ammonium compounds and local anesthetics on the electrogenic component of the sodium pump in mammalian non-myelinated nerve fibres, European J. Pharmacol. 6, 138. Den Hertog, A., P. Greengard and J.M. Ritchie, 1969, On the metabolic basis of nervous activity, J. Physiol. (London) 204,511. Duggan, D.E. and R.M. Moll, 1965, Effects of ethacrynic acid and cardiac glycosides upon a membrane adenosinetriphosphatase of renal cortex, Arch. Biochem. Biophys. 109,388 Early, L.E. and R.M. Friedler, 1964, Renal tubular effects of ethacrynic acid, J. Clin. Invest. 43, 1495. Early, L.E., 1967, Diuretics, New England J. Med. 276, 17, 966. Holmes, O., 1962, Effects of pH, changes in potassium concentration and metabolic inhibitors on the afterpotentials of mammalian non-medulated nerve fibers, Arch. Intern. Physiol. 70, 211. Jones, V.D., J. Lockett and E.J. Landon, 1965, Cellular action of mercurial diuretics. J. Pharmacol. Exptl. Therap. 147, 23. Nechay, B.R., R.F. Palmer, D.A. Chiney and V.A. Posey, 1967, The problem of Na¢+ K ÷ adenosine triphosphatase as the receptor for diuretic action of mercurials and ethacrynic acid. J. Pharmacol. Exptl. Therap. 157,599. Rang, H.P. and J.M. Ritchie, 1968, On the electrogenic sodium pump in mammalian non-myelinated nerve fibres and its activation by various external cations, J. Physiol. (London) 196, 183. Ritchie, J.M. and R.W. Straub, 1957, The hyperpolarization which follows activity in mammalian non-meduUated fibres, J. Physiol. (London) 136, 80. Skou, J.C., 1962, Preparation from mammalian brain and kidney of the enzyme system involved in active transport of Na ÷ and K ÷, Biochem. Biophys. Acta 58,314. Taylor, C.B., 1963, The effect of mercurial diuretics on adenosine triphospbatase of rabbit kidney in vitro, Biochem. Pharmacol. 12, 539. Wiebelhaus, V.P., J. Weinstock, A.R. Mass, F.T. Brennau, G. Sosnowki and T. Larsen, 1965, The diuretic and natrureric activity of triamterene and several related pteridines in the rat, J. Pharmacol. Exptl. Therap. 149,397.