J. Physiol. (1977), 270, pp. 383-414 With 9 text-ffJgure Printed in Great Britain
383
THE EFFECT OF CATECHOLAMINES ON NA-K TRANSPORT AND MEMBRANE POTENTIAL IN RAT SOLEUS MUSCLE
BY TORBEN CLAUSEN AND JOHN A. FLATMAN From the Institute of Phy8iology, Univer8ity of Arhu8, Univer8itetaparken, DK-8000 Arhus C, Denmark (Received 29 November 1976) SUMMARY
1. The action of catecholamines on the transport and the distribution of Na and K and the resting membrane potential (EM) has been investigated in soleus muscles isolated from fed rats. 2. In a substrate-free Krebs-Ringer bicarbonate buffer adrenaline (ADR) (6 x 106 M) increased 22Na efflux by 83 %, 42K influx by 34 %, and Em by 10%. Similar effects were exerted by noradrenaline (NA), phenylephrine, salbutamol and isoprenaline. The effects of ADR on Na-K transport and EM were suppressed by ouabain (10-3 M) and propranolol (10-5 M), but not by thymoxamine (10-5 M) or tetracaine (10-4 M). 3. Following 90 min of incubation in the presence of ADR (6 x 106 M), the intracellular K/Na-ratio was increased threefold. NA produced almost the same change, and both catecholamines seem to induce a new steady-state distribution of Na and K which can be maintained for several hours in vitro. 4. The effect of ADR on 22Na efflux and EM could be detected at concentrations down to 6 x IO- and 6 x 10-10 M, respectively, and halfmaximum increase was obtained at around 2 x 10-8 M. NA was at least one order of magnitude less potent. 5. The effect of low concentrations of ADR on 22Na efflux was potentiated by theophylline (2 mM). When added together, dibutyryl-cyclic AMP and theophylline mimicked the action of ADR on 22Na efflux, 42K influx, Na/K content and EM. Ouabain (10-3 M) also suppressed the effect of dibutyryl-cyclic AMP and theophylline on Na-K transport. 6. Following the addition of ouabain (10-3M), EM rapidly dropped from a mean of -71 to -63 mV, and then showed a slow linear fall for up to 4hr. 7. The hyperpolarization induced by ADR was associated with a decrease in membrane conductance, 22Na influx and 42K efflux. The time course and the response to ouabain suggests that all of these effects are secondary to stimulation of the active coupled transport of Na and K.
384 T. CLAUSEN AND J. A. FLATMAN 8. It is concluded that in rat soleus muscle, the active Na-K transport is electrogenic and susceptible to stimulation by catecholamines via beta-adrenoceptors. This effect is mediated by adenyl cyclase activation and may account for the increase in EM and the intracellular K/Na ratio. INTRODUCTION
Several studies have demonstrated that catecholamines influence the transport of sodium and potassium as well as the resting membrane potential in striated muscle cells (for reviews, see Bowman & Nott, 1969; Daniel, Paton, Taylor & Hodgson, 1970). ADR was found to stimulate the accumulation of K in rabbit atria (Waddell, 1961), and the efflux of 24Na from frog atria (Haas & Trautwein, 1963). Dockry, Kernan & Tangney (1966) showed that isoprenaline favours the extrusion of Na and augments the resting membrane potential in rat soleus muscles, and, more recently, it was found that ADR stimulated the efflux of isotopic Na from frog sartorius muscle (Hays, Dwyer, Horowicz & Swift, 1974), and rat diaphragm muscle (Evans & Smith, 1973). In addition, it is evident from studies with avian slow muscle fibres that catecholamines induce hyperpolarization by an ouabain-suppressible mechanism (Somlyo & Somlyo, 1969). In soleus and extensor digitorum longus muscles of the guinea-pig, it was recently demonstrated that stimulation of f8-adrenoceptors with isoprenaline induces an increase in the resting membrane potential (Tashiro 1973). Each of these observations lent some support to the idea that the catecholamines induce a stimulation of an electrogenic active transport of Na and K in muscle cells. In order to reach a more integrated analysis of this possibility, it seemed desirable to obtain information about both active and passive fluxes of Na and K as well as the time course of changes in resting membrane potential and Na-K distribution across the plasma membrane in the same preparation. Furthermore, the possible regulatory role of these hormones in electrolyte homeostasis had to be assessed in experiments with concentrations close to the physiological range. In the present study, these questions were explored using the isolated rat soleus muscle, which in previous reports has already been well characterized with respect to hexose transport (Kohn & Clausen, 1971, 1972), Na-K transport, ouabain-binding and insulin action (Clausen & Kohn, 1977; Clausen & Hansen, 1974; Dahl-Hansen & Clausen, 1973). Part of the data presented in this article have briefly been described in preliminary reports (Clausen & Flatman, 1975; Wang & Clausen, 1976).
CATECHOLAMINES AND ELECTROGENIC Na+ PUMP 385 METHODS All experiments were performed with isolated soleus muscles obtained from fed females or male rats of the Wistar strain. All measurements of the distribution and transport of Na and K were performed using rats weighing from 60 to 70 g, and in order to allow direct comparison, most of the membrane potential recordings were performed using rats of the same size. In some series of experiments, however, where it was important to maintain stable membrane potential values, rats weighing between 150 and 250 g were used. Krebs-Ringer bicarbonate buffer, containing 1-27 mM-Ca and no metabolizable substrate, was used as the standard medium (Cohen, 1951). The buffer was continuously equilibrated with a mixture of 95 % 0° and 5 % C02, and apart from a few instances, all experiments were performed at 30° C in order to reduce the metabolic requirements and thus to ensure sufficient oxygenation of the muscles. The procedures for the preparation and incubation of the muscles have been described earlier (Kohn & Clausen, 1971). The measurements of the uptake and the washout of isotopic Na and K, Na-K contents and extracellular space were performed as described in recent reports (Dahl-Hansen & Clausen, 1973; Clausen & Kohn, 1977). Further details are given in the legends to Figures and Tables.
Mea8urements of membrane potentials The dissection technique was modified so that a small plate of bone was left attached to the proximal tendon and part of the calcaneus to the Achilles tendon. The muscles were quickly transferred to a petri dish containing Krebs-Ringer bicarbonate buffer at room temperature and under a stereomicroscope the small stainless-steel hooks used for fixing the muscles were passed through the two bone plates. No attempt was made to clean connective tissue from the surface of the muscles as this was found to lead to a decline of the membrane potential with time. Pairs ofmuscles prepared from the same animal were mounted in chambers surrounded by a thermostated water-jacket (Fig. 1). The muscles were supported on Sylgard® beds, with the muscle surface bearing the vascular pedicel facing upwards. In most of the experiments, the muscles were put under a constant tension of 2-3 g. Later measurements showed that when the muscles were pinned under tension, essentially the same results were obtained, and this procedure was therefore adopted. The fluid volume in the bath was maintained at 15 ml., and with a perfusion rate of around 3 ml./min, the buffer in contact with the muscles was found to have a Posof 470 mmHg and a pH of 7-38. The addition of drugs to the perfusate was done by continuous infusion of a concentrated solution. In order to bring the concentration in the bath to its equilibrium level more rapidly, a small volume of the stock solution of the drug was added directly to the bath. Control experiments with a radioisotope indicated that at the surface of the muscle, the concentration of an agent added to the bath in this way reached the equilibrium level within 1 min. Glass micro-electrodes were pulled from Jena glass tubes (o.d. 1.8 mm) containing a single capillary of the same glass. They were filled with a 3 M-KCI solution which was injected into the electrode shank from a syringe. Electrodes with a tip resistance of between 10 and 20 MQ were selected for the experiments. For a few experiments, in which the input resistance of the muscle fibres was measured, the electrodes were prepared for capacitive screening by spraying with graphite, which was then coated with an insulating lacquer (Engberg, Flatman & Lambert, 1975). The micro-electrodes were positioned vertically above the muscles with a Leitz
T. CLAUSEN AND J. A. FLATMAN
386
micromanipulator and fibre penetration was performed under visual control with a Zeiss stereodissecting microscope. The potential difference between the micro-electrode and an indifferent electrode (which made contact with the bath fluid via a bridge containing Krebs-Ringer bicarbonate buffer with 2 % agar) was measured using a high input impedance amplifier with a unity gain input stage (Eide, 1968) via non-polarizable half-cells. The electrode resistance was continuously monitored. Membrane conductance was calculated from measurements of the potential change caused by the intracellular injection of a constant current pulse through the recording electrode. At least twenty of the potential transients were averaged electronically. The potential change due to electrode resistance was balanced out by a bridge circuit. In some experiments, a double electrode technique was used to measure membrane conductance changes during drug action or membrane potential changes caused by current passage. 2-3 g weight AL Micro-electrode Soleus muscle Adjustable outlet
,~~~~~ ,X K/t
Superfusion fluid
inlet
_Z~\
Sylgard bed \ \
\~
Water-jacket with thermostat
'\ `\ '\ N\
\
`\ _\ `\
Fig. 1. Diagram of experimental setup for the measurement of membrane potential. A lateral view through one of the muscle chambers and the surrounding water-jacket. Two similar muscle chambers are placed side by side in the water-jacket. The superfusion fluid is pumped by a Meda® peristaltic pump from a reservoir in which the buffer is gassed with a mixture of 5 % CO2 and 95 % 02. The fluid is then pumped into the muscle chambers through a long length of narrow-gauge polyethylene tubing which is contained within the water-jacket, hence the fluid entering the muscle chamber is at temperature equilibrium with the water in the waterjacket. The level of the fluid in the bath and thus the bath volume is controlled by the bevelled suction tubes in the right side of the chamber. Muscle tension is maintained at a constant value by the weight and pulley system.
The output of the amplifier was visualized on a slow Y - t recorder and an oscilloscope screen. A storage oscilloscope was used to monitor the electrode resistance and also to allow the calculation of cell input resistance. In addition, an auditory signal was provided by which the behaviour of the electrode and the 'cleanness' of the cell penetrations could be judged. The experimental procedure was standardized as follows. The electrode was advanced slowly until a superficial fibre was penetrated. Only those penetrations in which the electrode resistance did not change by more than 10 % were accepted for later analysis. After each penetration the electrode was withdrawn, and moved laterally; usually, serial penetrations were made across the muscle in two regions midway between the vascular pedicel and either the proximal or the distal tendons.
CATECHOLAMINES AND ELECTROGENIC Na+ PUMP 387 The membrane potentials were recorded in about ten fibres, then, whilst the electrode was left in a cell and a stable potential was recorded, the drug under consideration was added to the muscle bath. When the membrane potential response had reached a new steady state level, the electrode was withdrawn, and the membrane potentials of a new series of ten cells in the same muscle were recorded in quick succession. The contralateral muscle acted as a control. The difference between the potential of the electrode in the bath and the potential recorded immediately following the cell penetration was considered to be the resting membrane potential of the muscle fibre.
Chemicai8, i8otope8 and hormone All chemicals used (except the toluene and the Triton-X-100) were of analytical grade. Bitartrate salts of ADR and NA were obtained from Rh6ne-Poulenc, Paris, and dissolved in redistilled water containing 0.001 M-HCl. In several series of experiments, sodium pyrosulphite was added to obtain more stable solutions. The response to the catecholamines was not modified by this compound. In the ion-flux studies catecholamines were added by pipetting small aliquots of concentrated solutions into the incubation media immediately before the tissues were added. Dibutyryl cyclic adenosine monophosphate (dbcAMP) and DL-isoprenaline were purchased from Sigma (St Louis). Salbutamol, thymoxamine and propranolol were gifts from the Glaxo Laboratories, William R. Warner & Co. Ltd, and ICI Ltd, respectively. 22Na (3000 mc/m-mole) and [U-14C]sucrose (400 mc/im-mole) were obtained from The Radiochemical Centre, Amersham, and 42K 100 mclm-mole) from the Danish Atomic Energy Commission, Isotope Laboratory, Riso. RESULTS
Cellular Na-K content Following 15 min of incubation in the presence of ADR (6 x 104 M), the Na content of the tissue compartment not available to [U-14C]sucrose was significantly decreased (Table 1). This was associated with an almost equimolar increase in K content. Both of these changes became more pronounced with time, and after 90 min, the intracellular K/Na ratio was increased threefold. Measurements performed with 22Na gave closely similar results. NA (6 x 106 M) produced almost the same change, and for both catecholamines it was found that the increased K/Na ratio could be maintained for at least 180 min in vitro (data not presented). It was not possible to detect any significant effects of ADR on sucrose space, wet weight or water content. Still, in view of the relatively small differences between the Na content of the whole tissue and that of the space available to sucrose it seemed desirable to obtain an alternative estimate of the change in the size of the intracellular Na pool. In an earlier study (Clausen & Kohn, 1977) it was demonstrated that following incubation in the presence of 22Na, the isotopic Na could be almost entirely removed from the extracellular space by washing the muscles in ice-cold buffer containing ouabain. No more than a few per cent of the label taken
388 T. CLAUSEN AND J. A. FLATMAN up into the cytoplasm was lost during the wash, and the results showed considerably smaller scatter than those based upon the simultaneous measurement of the spaces available to [22Na] and [14C]sucrose. TABLE 1. Effect of ADR on Na-K content in rat soleus muscle. Muscles were incubated for 90 min in 3 ml. Krebs-Ringer bicarbonate buffer containing 0-2 /zc/ml. of [LT-14C]sucrose. Where indicated, ADR (6 x 10-6 M) was present during the last 15 min or throughout the incubation period. After blotting, resection of the tendons and weighing, the muscles were homogenized in 4 ml. 5 % trichloroacetic acid. The extract was centrifuged and aliquots of the clear supernatant were taken for the measurement of 14C activity and Na-K content by flame photometry. All results are given as means + S.E. with the number of observations in parentheses. Experimental Na contents K contents conditions P P (Ismole/g wet wt.) (,umole/g wet wt.) Control 10X0± 2-0 (6) 82-2 + 0*6 (6) ADR present for 15 min 5-3 + 0 7 (6) < 0-05 86-2 + 0-7 (6) < 0*005 Control 9.9+ 1.4 (15) 84*2 + 0*6 (15) ADR present for 90 min 3-3+ 1-6 (14) < 0 001 89-2 + 1.0 (14) < 0 001
Table 2 shows the effects of catecholamines and other agents on the amount of isotopic Na taken up during 60 min of incubation at 300 C and retained during 60 min of washout in the cold. For the control muscles the results were similar to those obtained by measuring the amount of Na present in the space not available to sucrose (cf. Tables 1 and 2). Again, ADR and NA (6 x 10-6 M) produced a marked decrease. Salbutamol, a compound mainly acting via fl2-adrenoceptors, had virtually the same effect. DbcAMP and theophylline, alone or in combination, also produced a significant decrease which was blocked by ouabain. On the other hand, tetracaine (0.5 x 10-3 M), which inhibits Na influx without producing any stimulation of Na efflux (Dahl-Hansen & Clausen, 1973), induced almost exactly the same decrease in the amount of 22Na retained as ADR. In an attempt to account for the marked rise in the intracellular K/Na ratio induced by the catecholamines, the acute effects of these and some related compounds on Na-K transport were analysed in some detail. 22Na efflux and 42K influx Fig. 2 shows the effect of ADR (6 x 10-6 M) on the washout of 22Na from preloaded muscles. Within the first 10 min of exposure, the fraction of isotopic Na lost per min was increased by 83 %, and if it can be assumed that the cellular pool from which 22Na is released remains constant (around 9 ,umole/g tissue wet wt.), this increase would correspond to a rise in the
CATECHOLAMINES AND ELECTROGENIC Na+ PUMP 389 efflux rate of 0-4 pmole/g tissue wet wt./min. In the presence of ouabain
at a concentration (10-3 M) previously shown to produce maximal inhibition of active Na-K transport in rat soleus muscles (Clausen & Kohn, 1977), the stimulating effect of ADR was considerably smaller (21 %) and not statistically significant. TABiE 2. Effects of catecholamines, dibutyryl cyclic AMP, dbcAMP and theophylline on 22Na retention. Soleus muscles were incubated at 30° C for 60 min in Krebs-Ringer bicarbonate buffer containing 22Na (0.4 ptc/ml.) without or with the additions indicated. Then the muscles were washed 3 x 20 min in ice-cold buffer containing ouabain (10-3 M), blotted, weighed and counted. The 22Na activity retained following the wash in the cold was expressed as ,smole/g tissue wet wt, using the specific activity of 22Na in the incubation medium as a reference. All results are given as means ± S.E. with the number of observations in parentheses.
22Na taken up and retained during wash at 00 C Additions Control ADR (10-6 M) NA (10-6 M) Salbutamol (10-6 M) Tetracaine (5 x 10-4 M) Control dbcAMP (5 x 10-4 M) dbcAMP (2 x 10-3 M) Theophylline (2 x 10-3 M) dbcAMP (10-4 M) + theophylline (2 x 10-3 M) dbcAMP (5 x 10-4 M) + theophylline (2 x 10-3 M) Ouabain (10-3 M) Ouabain (10-3 M) + dbcAMP (2 x 10-3
(csmole/g wet wt.) 8-69+0-17 (10) 3-48+0-16 (5) 5-63±0-30 (5) 3-72+0-31 (5) 3-51+0-09 (5) 8-43+0-18 (9) 7-21 + 0-35 (4) 3-12+0-07 (9) 6-13±0-41 (4) 4-79+ 0-28 (4)
P < 0-001
< 0-001 < 0-001
383
THE EFFECT OF CATECHOLAMINES ON NA-K TRANSPORT AND MEMBRANE POTENTIAL IN RAT SOLEUS MUSCLE
BY TORBEN CLAUSEN AND JOHN A. FLATMAN From the Institute of Phy8iology, Univer8ity of Arhu8, Univer8itetaparken, DK-8000 Arhus C, Denmark (Received 29 November 1976) SUMMARY
1. The action of catecholamines on the transport and the distribution of Na and K and the resting membrane potential (EM) has been investigated in soleus muscles isolated from fed rats. 2. In a substrate-free Krebs-Ringer bicarbonate buffer adrenaline (ADR) (6 x 106 M) increased 22Na efflux by 83 %, 42K influx by 34 %, and Em by 10%. Similar effects were exerted by noradrenaline (NA), phenylephrine, salbutamol and isoprenaline. The effects of ADR on Na-K transport and EM were suppressed by ouabain (10-3 M) and propranolol (10-5 M), but not by thymoxamine (10-5 M) or tetracaine (10-4 M). 3. Following 90 min of incubation in the presence of ADR (6 x 106 M), the intracellular K/Na-ratio was increased threefold. NA produced almost the same change, and both catecholamines seem to induce a new steady-state distribution of Na and K which can be maintained for several hours in vitro. 4. The effect of ADR on 22Na efflux and EM could be detected at concentrations down to 6 x IO- and 6 x 10-10 M, respectively, and halfmaximum increase was obtained at around 2 x 10-8 M. NA was at least one order of magnitude less potent. 5. The effect of low concentrations of ADR on 22Na efflux was potentiated by theophylline (2 mM). When added together, dibutyryl-cyclic AMP and theophylline mimicked the action of ADR on 22Na efflux, 42K influx, Na/K content and EM. Ouabain (10-3 M) also suppressed the effect of dibutyryl-cyclic AMP and theophylline on Na-K transport. 6. Following the addition of ouabain (10-3M), EM rapidly dropped from a mean of -71 to -63 mV, and then showed a slow linear fall for up to 4hr. 7. The hyperpolarization induced by ADR was associated with a decrease in membrane conductance, 22Na influx and 42K efflux. The time course and the response to ouabain suggests that all of these effects are secondary to stimulation of the active coupled transport of Na and K.
384 T. CLAUSEN AND J. A. FLATMAN 8. It is concluded that in rat soleus muscle, the active Na-K transport is electrogenic and susceptible to stimulation by catecholamines via beta-adrenoceptors. This effect is mediated by adenyl cyclase activation and may account for the increase in EM and the intracellular K/Na ratio. INTRODUCTION
Several studies have demonstrated that catecholamines influence the transport of sodium and potassium as well as the resting membrane potential in striated muscle cells (for reviews, see Bowman & Nott, 1969; Daniel, Paton, Taylor & Hodgson, 1970). ADR was found to stimulate the accumulation of K in rabbit atria (Waddell, 1961), and the efflux of 24Na from frog atria (Haas & Trautwein, 1963). Dockry, Kernan & Tangney (1966) showed that isoprenaline favours the extrusion of Na and augments the resting membrane potential in rat soleus muscles, and, more recently, it was found that ADR stimulated the efflux of isotopic Na from frog sartorius muscle (Hays, Dwyer, Horowicz & Swift, 1974), and rat diaphragm muscle (Evans & Smith, 1973). In addition, it is evident from studies with avian slow muscle fibres that catecholamines induce hyperpolarization by an ouabain-suppressible mechanism (Somlyo & Somlyo, 1969). In soleus and extensor digitorum longus muscles of the guinea-pig, it was recently demonstrated that stimulation of f8-adrenoceptors with isoprenaline induces an increase in the resting membrane potential (Tashiro 1973). Each of these observations lent some support to the idea that the catecholamines induce a stimulation of an electrogenic active transport of Na and K in muscle cells. In order to reach a more integrated analysis of this possibility, it seemed desirable to obtain information about both active and passive fluxes of Na and K as well as the time course of changes in resting membrane potential and Na-K distribution across the plasma membrane in the same preparation. Furthermore, the possible regulatory role of these hormones in electrolyte homeostasis had to be assessed in experiments with concentrations close to the physiological range. In the present study, these questions were explored using the isolated rat soleus muscle, which in previous reports has already been well characterized with respect to hexose transport (Kohn & Clausen, 1971, 1972), Na-K transport, ouabain-binding and insulin action (Clausen & Kohn, 1977; Clausen & Hansen, 1974; Dahl-Hansen & Clausen, 1973). Part of the data presented in this article have briefly been described in preliminary reports (Clausen & Flatman, 1975; Wang & Clausen, 1976).
CATECHOLAMINES AND ELECTROGENIC Na+ PUMP 385 METHODS All experiments were performed with isolated soleus muscles obtained from fed females or male rats of the Wistar strain. All measurements of the distribution and transport of Na and K were performed using rats weighing from 60 to 70 g, and in order to allow direct comparison, most of the membrane potential recordings were performed using rats of the same size. In some series of experiments, however, where it was important to maintain stable membrane potential values, rats weighing between 150 and 250 g were used. Krebs-Ringer bicarbonate buffer, containing 1-27 mM-Ca and no metabolizable substrate, was used as the standard medium (Cohen, 1951). The buffer was continuously equilibrated with a mixture of 95 % 0° and 5 % C02, and apart from a few instances, all experiments were performed at 30° C in order to reduce the metabolic requirements and thus to ensure sufficient oxygenation of the muscles. The procedures for the preparation and incubation of the muscles have been described earlier (Kohn & Clausen, 1971). The measurements of the uptake and the washout of isotopic Na and K, Na-K contents and extracellular space were performed as described in recent reports (Dahl-Hansen & Clausen, 1973; Clausen & Kohn, 1977). Further details are given in the legends to Figures and Tables.
Mea8urements of membrane potentials The dissection technique was modified so that a small plate of bone was left attached to the proximal tendon and part of the calcaneus to the Achilles tendon. The muscles were quickly transferred to a petri dish containing Krebs-Ringer bicarbonate buffer at room temperature and under a stereomicroscope the small stainless-steel hooks used for fixing the muscles were passed through the two bone plates. No attempt was made to clean connective tissue from the surface of the muscles as this was found to lead to a decline of the membrane potential with time. Pairs ofmuscles prepared from the same animal were mounted in chambers surrounded by a thermostated water-jacket (Fig. 1). The muscles were supported on Sylgard® beds, with the muscle surface bearing the vascular pedicel facing upwards. In most of the experiments, the muscles were put under a constant tension of 2-3 g. Later measurements showed that when the muscles were pinned under tension, essentially the same results were obtained, and this procedure was therefore adopted. The fluid volume in the bath was maintained at 15 ml., and with a perfusion rate of around 3 ml./min, the buffer in contact with the muscles was found to have a Posof 470 mmHg and a pH of 7-38. The addition of drugs to the perfusate was done by continuous infusion of a concentrated solution. In order to bring the concentration in the bath to its equilibrium level more rapidly, a small volume of the stock solution of the drug was added directly to the bath. Control experiments with a radioisotope indicated that at the surface of the muscle, the concentration of an agent added to the bath in this way reached the equilibrium level within 1 min. Glass micro-electrodes were pulled from Jena glass tubes (o.d. 1.8 mm) containing a single capillary of the same glass. They were filled with a 3 M-KCI solution which was injected into the electrode shank from a syringe. Electrodes with a tip resistance of between 10 and 20 MQ were selected for the experiments. For a few experiments, in which the input resistance of the muscle fibres was measured, the electrodes were prepared for capacitive screening by spraying with graphite, which was then coated with an insulating lacquer (Engberg, Flatman & Lambert, 1975). The micro-electrodes were positioned vertically above the muscles with a Leitz
T. CLAUSEN AND J. A. FLATMAN
386
micromanipulator and fibre penetration was performed under visual control with a Zeiss stereodissecting microscope. The potential difference between the micro-electrode and an indifferent electrode (which made contact with the bath fluid via a bridge containing Krebs-Ringer bicarbonate buffer with 2 % agar) was measured using a high input impedance amplifier with a unity gain input stage (Eide, 1968) via non-polarizable half-cells. The electrode resistance was continuously monitored. Membrane conductance was calculated from measurements of the potential change caused by the intracellular injection of a constant current pulse through the recording electrode. At least twenty of the potential transients were averaged electronically. The potential change due to electrode resistance was balanced out by a bridge circuit. In some experiments, a double electrode technique was used to measure membrane conductance changes during drug action or membrane potential changes caused by current passage. 2-3 g weight AL Micro-electrode Soleus muscle Adjustable outlet
,~~~~~ ,X K/t
Superfusion fluid
inlet
_Z~\
Sylgard bed \ \
\~
Water-jacket with thermostat
'\ `\ '\ N\
\
`\ _\ `\
Fig. 1. Diagram of experimental setup for the measurement of membrane potential. A lateral view through one of the muscle chambers and the surrounding water-jacket. Two similar muscle chambers are placed side by side in the water-jacket. The superfusion fluid is pumped by a Meda® peristaltic pump from a reservoir in which the buffer is gassed with a mixture of 5 % CO2 and 95 % 02. The fluid is then pumped into the muscle chambers through a long length of narrow-gauge polyethylene tubing which is contained within the water-jacket, hence the fluid entering the muscle chamber is at temperature equilibrium with the water in the waterjacket. The level of the fluid in the bath and thus the bath volume is controlled by the bevelled suction tubes in the right side of the chamber. Muscle tension is maintained at a constant value by the weight and pulley system.
The output of the amplifier was visualized on a slow Y - t recorder and an oscilloscope screen. A storage oscilloscope was used to monitor the electrode resistance and also to allow the calculation of cell input resistance. In addition, an auditory signal was provided by which the behaviour of the electrode and the 'cleanness' of the cell penetrations could be judged. The experimental procedure was standardized as follows. The electrode was advanced slowly until a superficial fibre was penetrated. Only those penetrations in which the electrode resistance did not change by more than 10 % were accepted for later analysis. After each penetration the electrode was withdrawn, and moved laterally; usually, serial penetrations were made across the muscle in two regions midway between the vascular pedicel and either the proximal or the distal tendons.
CATECHOLAMINES AND ELECTROGENIC Na+ PUMP 387 The membrane potentials were recorded in about ten fibres, then, whilst the electrode was left in a cell and a stable potential was recorded, the drug under consideration was added to the muscle bath. When the membrane potential response had reached a new steady state level, the electrode was withdrawn, and the membrane potentials of a new series of ten cells in the same muscle were recorded in quick succession. The contralateral muscle acted as a control. The difference between the potential of the electrode in the bath and the potential recorded immediately following the cell penetration was considered to be the resting membrane potential of the muscle fibre.
Chemicai8, i8otope8 and hormone All chemicals used (except the toluene and the Triton-X-100) were of analytical grade. Bitartrate salts of ADR and NA were obtained from Rh6ne-Poulenc, Paris, and dissolved in redistilled water containing 0.001 M-HCl. In several series of experiments, sodium pyrosulphite was added to obtain more stable solutions. The response to the catecholamines was not modified by this compound. In the ion-flux studies catecholamines were added by pipetting small aliquots of concentrated solutions into the incubation media immediately before the tissues were added. Dibutyryl cyclic adenosine monophosphate (dbcAMP) and DL-isoprenaline were purchased from Sigma (St Louis). Salbutamol, thymoxamine and propranolol were gifts from the Glaxo Laboratories, William R. Warner & Co. Ltd, and ICI Ltd, respectively. 22Na (3000 mc/m-mole) and [U-14C]sucrose (400 mc/im-mole) were obtained from The Radiochemical Centre, Amersham, and 42K 100 mclm-mole) from the Danish Atomic Energy Commission, Isotope Laboratory, Riso. RESULTS
Cellular Na-K content Following 15 min of incubation in the presence of ADR (6 x 104 M), the Na content of the tissue compartment not available to [U-14C]sucrose was significantly decreased (Table 1). This was associated with an almost equimolar increase in K content. Both of these changes became more pronounced with time, and after 90 min, the intracellular K/Na ratio was increased threefold. Measurements performed with 22Na gave closely similar results. NA (6 x 106 M) produced almost the same change, and for both catecholamines it was found that the increased K/Na ratio could be maintained for at least 180 min in vitro (data not presented). It was not possible to detect any significant effects of ADR on sucrose space, wet weight or water content. Still, in view of the relatively small differences between the Na content of the whole tissue and that of the space available to sucrose it seemed desirable to obtain an alternative estimate of the change in the size of the intracellular Na pool. In an earlier study (Clausen & Kohn, 1977) it was demonstrated that following incubation in the presence of 22Na, the isotopic Na could be almost entirely removed from the extracellular space by washing the muscles in ice-cold buffer containing ouabain. No more than a few per cent of the label taken
388 T. CLAUSEN AND J. A. FLATMAN up into the cytoplasm was lost during the wash, and the results showed considerably smaller scatter than those based upon the simultaneous measurement of the spaces available to [22Na] and [14C]sucrose. TABLE 1. Effect of ADR on Na-K content in rat soleus muscle. Muscles were incubated for 90 min in 3 ml. Krebs-Ringer bicarbonate buffer containing 0-2 /zc/ml. of [LT-14C]sucrose. Where indicated, ADR (6 x 10-6 M) was present during the last 15 min or throughout the incubation period. After blotting, resection of the tendons and weighing, the muscles were homogenized in 4 ml. 5 % trichloroacetic acid. The extract was centrifuged and aliquots of the clear supernatant were taken for the measurement of 14C activity and Na-K content by flame photometry. All results are given as means + S.E. with the number of observations in parentheses. Experimental Na contents K contents conditions P P (Ismole/g wet wt.) (,umole/g wet wt.) Control 10X0± 2-0 (6) 82-2 + 0*6 (6) ADR present for 15 min 5-3 + 0 7 (6) < 0-05 86-2 + 0-7 (6) < 0*005 Control 9.9+ 1.4 (15) 84*2 + 0*6 (15) ADR present for 90 min 3-3+ 1-6 (14) < 0 001 89-2 + 1.0 (14) < 0 001
Table 2 shows the effects of catecholamines and other agents on the amount of isotopic Na taken up during 60 min of incubation at 300 C and retained during 60 min of washout in the cold. For the control muscles the results were similar to those obtained by measuring the amount of Na present in the space not available to sucrose (cf. Tables 1 and 2). Again, ADR and NA (6 x 10-6 M) produced a marked decrease. Salbutamol, a compound mainly acting via fl2-adrenoceptors, had virtually the same effect. DbcAMP and theophylline, alone or in combination, also produced a significant decrease which was blocked by ouabain. On the other hand, tetracaine (0.5 x 10-3 M), which inhibits Na influx without producing any stimulation of Na efflux (Dahl-Hansen & Clausen, 1973), induced almost exactly the same decrease in the amount of 22Na retained as ADR. In an attempt to account for the marked rise in the intracellular K/Na ratio induced by the catecholamines, the acute effects of these and some related compounds on Na-K transport were analysed in some detail. 22Na efflux and 42K influx Fig. 2 shows the effect of ADR (6 x 10-6 M) on the washout of 22Na from preloaded muscles. Within the first 10 min of exposure, the fraction of isotopic Na lost per min was increased by 83 %, and if it can be assumed that the cellular pool from which 22Na is released remains constant (around 9 ,umole/g tissue wet wt.), this increase would correspond to a rise in the
CATECHOLAMINES AND ELECTROGENIC Na+ PUMP 389 efflux rate of 0-4 pmole/g tissue wet wt./min. In the presence of ouabain
at a concentration (10-3 M) previously shown to produce maximal inhibition of active Na-K transport in rat soleus muscles (Clausen & Kohn, 1977), the stimulating effect of ADR was considerably smaller (21 %) and not statistically significant. TABiE 2. Effects of catecholamines, dibutyryl cyclic AMP, dbcAMP and theophylline on 22Na retention. Soleus muscles were incubated at 30° C for 60 min in Krebs-Ringer bicarbonate buffer containing 22Na (0.4 ptc/ml.) without or with the additions indicated. Then the muscles were washed 3 x 20 min in ice-cold buffer containing ouabain (10-3 M), blotted, weighed and counted. The 22Na activity retained following the wash in the cold was expressed as ,smole/g tissue wet wt, using the specific activity of 22Na in the incubation medium as a reference. All results are given as means ± S.E. with the number of observations in parentheses.
22Na taken up and retained during wash at 00 C Additions Control ADR (10-6 M) NA (10-6 M) Salbutamol (10-6 M) Tetracaine (5 x 10-4 M) Control dbcAMP (5 x 10-4 M) dbcAMP (2 x 10-3 M) Theophylline (2 x 10-3 M) dbcAMP (10-4 M) + theophylline (2 x 10-3 M) dbcAMP (5 x 10-4 M) + theophylline (2 x 10-3 M) Ouabain (10-3 M) Ouabain (10-3 M) + dbcAMP (2 x 10-3
(csmole/g wet wt.) 8-69+0-17 (10) 3-48+0-16 (5) 5-63±0-30 (5) 3-72+0-31 (5) 3-51+0-09 (5) 8-43+0-18 (9) 7-21 + 0-35 (4) 3-12+0-07 (9) 6-13±0-41 (4) 4-79+ 0-28 (4)
P < 0-001
< 0-001 < 0-001
