Beta 2-adrenoceptors mediate the stimulating effect of adrenaline on active electrogenic Na-K-transport in rat soleus muscle.

The relative role of beta 1- and beta 2-adrenoceptors in mediating the stimulating effect of adrenaline on active electrogenic Na-K-transport has been assessed in experiments on rat soleus muscles in vitro and in vivo. 2 In the rat isolated soleus muscle, adrenaline (10(-6) M) increases the resting membrane potential (EM) by 5.8 mV and stimulates 22Na-efflux and ouabain-suppressible 42K-uptake by 91 and 94%, respectively. 3 All of these effects are completely blocked by propranolol (10(-5) M), whereas the beta 1-selective adrenoceptor antagonist, metoprolol, was found to be at least 50 times less potent. 4 The beta 2-adrenoceptor agonist, salbutamol, was at least 100 times as potent as H133/22 (a beta 1-selective agonist) in stimulating 22Na-efflux and 42K-influx. 5 In experiments performed under pentobarbitone anaesthesia, the intravenous injection of adrenaline (5 microgram) or salbutamol (0.5 to 50 microgram) led to a rapid and marked increase in the EM of the exposed soleus muscle. This hyperpolarizing effect could not be accounted for by the concomitant, relatively modest change in extracellular K.

The response of cat spinal motoneurones to the intracellular application of agents with local anaesthetic action.

QX-222 (the trimethyl analogue of lignocaine), methylxylocholine, lignocaine and pentobarbitone were iontophoresed intracellularly into cat lumbosacral motoneurones. Iontophoresis and recording was either from a triple-barrelled microelectrode unit or from two separately advanced microelectrodes. QX-222 and methylxylocholine caused a very slow reversible block of the current-evoked and antidromic action potentials (AP) with no significant change of membrane potential (EM). Lignocaine had a minimal blocking effect on the AP. No change, or only a small decrease, of membrane slope conductance (GM) was seen when the APs had been totally abolished. QX-222 and methylxylocholine reduced the massive GM increase evoked by the passage of large depolarizing currents and converted the post-current hyperpolarization (time constant 120-150 ms) into a depolarization of similar time course. It is suggested that the quaternary local anaesthetics can reduce the fast and slow voltage-dependent potassium conductances. Both agents totally blocked AP generation without decreasing the magnitude of the Ia e.p.s.p. It is suggested that intracellularly iontophoresed QX-222 (on account of its low lipid solubility) could be used as a pharmacological tool to block specifically the active Na and channels in only the cell impaled by the microelectrodes.

Excitation of substantia nigra pars compacta neurones by 5-hydroxy-tryptamine in-vitro.

Incoming serotonergic fibres are known to make direct synaptic contact with dopamine-containing neurones in the substantia nigra pars compacta (SNc). However, the effects of 5-HT (5-hydroxytryptamine) on these cells have not been thoroughly investigated. In the present study we show that application of 10-50 microM 5-HT increases the firing frequency of SNc neurones in-vitro, and produces inward rectification in a voltage region negative to -50mV. This effect is sensitive to extracellular Cs+, but not to Ba2+, and has similar properties as the intrinsic inward rectifier current, Ih. Antagonists of the 5-HT1A and 5-HT2 receptors were inefficacious. It is concluded that 5-HT excites SNc neurones via an enhancement of the conductance underlying Ih.

Sustained extracellular potentials in the cat spinal cord during the microiontophoretic application of excitatory amino acids.

Sustained negative potentials were recorded in the ventral horn of the cat spinal cord during current balanced extracellular iontophoresis of excitatoyr amino acids. The potentials (referred to a distant indifferent electrode) were measured by an extracellular microelectrode. These focal potentials (FPs) were evoked by DL-homocysteate, L-glutamate, N-methyl-D-aspartate and kainate. These FPs are not an artifact of extracellular microiontophoresis. Their time course is correlated with the depolarization of spinal motoneurones by excitatory amino acids. During iontophoresis of kainate, FPs can be as large as 50 mV and can be recorded for up to 1 mm from the site of drug application. The FP and depolarization caused by kainate were usually irreversible. The depolarization of motoneurones evoked by excitatory amino acids is very much larger when recorded as a 'transmembrane potential' (i.e. the potential of an intracellular electrode minus the potential of a local extracellular electrode) rather than as a 'classical' intracellular potential (i.e. referred to a distant reference electrode). Possible mechanisms for the generation of the FP are discussed. It is suggested that FP may be recorded routinely during microiontophoretic studies employing extracellular recording of neuronal activity. The application of the FP as a measure of cell depolarization during pharmacological studies of excitatory amino acids and agents that block their action is discussed.

A comparison of extracellular and intracellular recording during extracellular microiontophoresis.

A technique is described in which a central recording microelectrode can be moved independently of a concentrically arranged multibarrelled electrode prepared for microiontophoresis. Recordings were made from cat spinal motoneurones during microiontophoretic applications of excitatory amino acids and biogenic amines with the central electrode placed first extracellularly and then intracellularly. Recording were also made from one of the iontophoretic barrels. Both intra- and extracellular electrodes were used to record action potential firing, the ventral root field (VRF) evoked by antidromic ventral root stimulation and the membrane potential (EM). They were also used to record 'focal potentials' evoked by the extracellular application of drugs to nearby neurones. The firing pattern evoked by extracellular iontophoretic applications of DL-homocysteate and glutamate was not altered significantly following impalement of the cell by the recording microelectrode. Excitatory amino acids usually caused a reduction of the VRF negative wave and evoked an additional late positive wave. These VRF changes recovered at the same rate as the extracellularly recorded, negative 'focal potentials' (Flatman and Lambert, 1979). Iontophoretic applications of biogenic amines caused small increases, small decreases, or no change of the VRF negative wave. Variable responses were also seen during intracellular recording: hyperpolarization, no response and, occasionally, depolarizations were recorded. It is concluded that, during the drug action, VRF changes are difficult to interpret and are a poor index of drug-evoked changes in neuronal excitability or EM.

The induction and modification of voltage-sensitive responses in cat neocortical neurons by N-methyl-D-aspartate.

The actions of the excitatory amino acid, N-methyl-D-aspartate (NMDA), on layer V neurons of cat sensorimotor cortex were examined in an in vitro slice preparation using current clamp, single electrode voltage clamp (SEVC), and ionic substitution techniques. Low doses of NMDA evoked a slow depolarization with a net decrease of input conductance. Larger doses additionally evoked repetitive firing, rhythmic depolarization shifts (DSs), low-threshold calcium spikes (in the presence of TEA+) and bistable membrane potential behavior. Ionic substitution experiments suggested that entry of both Ca2+ and Na+ ions contributed to the NMDA responses. Attention was focused on the NMDA response with Ca2+ entry blocked. Examination by SEVC revealed that, in both normal cells and in the presence of several blocking agents, NMDA induced a highly voltage-dependent inward ionic current which could result in a region of negative slope conductance on the cell's current-voltage relation. The development of this current seems capable of accounting for all aspects of the observed response, including the DSs and low-threshold Ca2+ spikes. Substitution of TEA+ for most external Na+ (with Ca2+ entry blocked) largely eliminated the NMDA responses and corresponding ionic current. Our results in neocortical neurons are compared to those recently obtained in cultured murine neurons.

Multiple actions of N-methyl-D-aspartate on cat neocortical neurons in vitro.

The potent excitatory amino acid receptor agonist, N-methyl-D-aspartate (NMDA), was applied to cat neocortical neurons in an in vitro slice preparation. NMDA evokes a slow depolarization with a net input conductance decrease, repetitive firing, rhythmic depolarization shifts and bi-stable membrane potential behavior. Use of blocking agents, ion substitution and voltage clamp indicates that NMDA induces a highly voltage-dependent TTX-resistant inward sodium current which accounts for much of the NMDA response.

Combined effects of adrenaline and insulin on active electrogenic Na+-K+ transport in rat soleus muscle.

Both beta 2-adrenoreceptor stimulants (such as adrenaline and salbutamol) and insulin can increase active Na+-K+ transport and hyperpolarise skeletal cells. Thus, adrenaline and insulin, which are otherwise antagonistic regulators of several metabolic processes, have one action in common, namely, stimulation of active ion translocation. This is especially interesting as cyclic AMP stimulates Na+-K+ transport, whereas a lowering of the cytoplasmic concentration of cyclic AMP has been proposed as an early signal in the action of insulin. Here we report the results of experiments in which the active Na+-K+ transport and membrane potential (EM) of rat soleus muscles were studied during the action of supramaximal doses of insulin and beta 2-adrenoreceptor stimulants, alone and in combination. We conclude that the stimulant action of insulin on active electrogenic Na+-K+ transport is unlikely to be evoked by a lowering of the intracellular concentration of cyclic AMP.

The effect of catecholamines on Na-K transport and membrane potential in rat soleus muscle.

1. The action of catecholamines on the transport and the distribution of Na and K and the resting membrane potential (E(M)) has been investigated in soleus muscles isolated from fed rats.2. In a substrate-free Krebs-Ringer bicarbonate buffer adrenaline (ADR) (6 x 10(-6)M) increased (22)Na efflux by 83%, (42)K influx by 34%, and E(M) by 10%. Similar effects were exerted by noradrenaline (NA), phenylephrine, salbutamol and isoprenaline. The effects of ADR on Na-K transport and E(M) 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 10(-6)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 (22)Na efflux and E(M) could be detected at concentrations down to 6 x 10(-9) and 6 x 10(-10)M, respectively, and half-maximum 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 (22)Na efflux was potentiated by theophylline (2 mM). When added together, dibutyryl-cyclic AMP and theophylline mimicked the action of ADR on (22)Na efflux, (42)K influx, Na/K content and E(M). 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(-3)M), E(M) 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, (22)Na influx and (42)K 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.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 E(M) and the intracellular K/Na ratio.

Effects of insulin and epinephrine on Na+-K+ and glucose transport in soleus muscle.

To identify possible cause-effect relationships between changes in active Na+-K+ transport, resting membrane potential, and glucose transport, the effects of insulin and epinephrine were compared in rat soleus muscle. Epinephrine, which produced twice as large a hyperpolarization as insulin, induced only a modest increase in sugar transport. Ouabain, at a concentration (10(-3) M) sufficient to block active Na+-K+ transport and the hyperpolarization induced by the two hormones, did not interfere with sugar transport stimulation. After Na+ loading in K+-free buffer, the return to K+-containing standard buffer caused marked stimulation of active Na+-K+ transport, twice the hyperpolarization produced by insulin but no change in sugar transport. The insulin-induced activation of the Na+-K+ pump leads to decreased intracellular Na+ concentration and hyperpolarization, but none of these events can account for the concomitant activation of the glucose transport system. The stimulating effect of insulin on active Na+-K+ transport was not suppressed by amiloride, indicating that in intact skeletal muscle it is not elicited by a primary increase in Na+ influx via the Na+/H+-exchange system.

Measurement of force and both surface and deep M wave properties in isolated rat soleus muscles.

In isolated soleus muscles of 4-wk-old rats, M wave parameters were recorded with surface and deep recording electrodes and examined in relation to both twitch and tetanic force. Addition of ouabain (10(-5) M for 16 min) to isolated muscles caused an approximately 40% decrease in twitch amplitude and area (P < 0.01) that was associated with a 98% decrease in surface M wave amplitude, a 78% decrease in deep M wave amplitude (both P < 0.001), a 98% decrease in surface M wave area (P < 0.01), 48% of which occurred within 60 s of addition of ouabain (P < 0.05), and a 55% decrease in deep M wave area (P < 0.05). The decrease in twitch parameters on addition of ouabain was most closely correlated with deep M wave area (r = 0.92). Direct tetanic stimulation at a frequency of 30 Hz resulted in an initial potentiation of M waves, which was not seen at a frequency of 90 Hz. Instead, 90 Hz stimulation resulted in a prompt decrease in tetanic force that was correlated with a decrease in both deep M wave amplitude (r = 0.94; P < 0.01) and deep M wave area (r = 0.96; P < 0.01). It is concluded that simultaneous surface and deep recordings involving area and amplitude are fundamental to analysis of the effects of pharmacological agents on muscle performance and the use of M waves as predictors of muscle excitability.

The variation in action of excitatory amino acids in relation to distance of iontophoretic application to spinal motoneurones.

Intracellular recordings were made from lumbosacral motoneurones of barbiturate-anaesthetized cats. DL-homocysteate (DLH) and L-glutamate were iontophoresed extracellularly over a range of distances from the impaled motoneurone. Movement of the iontophoretic electrode unit was controlled by a micromanipulator which was advanced independently of that moving the intracellular electrode. Depolarizations to DLH were first detected at a greater distance from the impaled motoneurones (mean, 383 micron) than depolarizations to L-glutamate (mean, 165 micron). As the point of application approached the soma of the motoneurone, depolarizations developed more rapidly, were larger and the latent period of the L-glutamate depolarization became shorter. Dendritic 'hot-spots' of the depolarizing action of L-glutamate were not detected.

The actions of excitatory amino acids on motoneurones in the feline spinal cord.

1. Combined recording or ionophoretic electrodes of the concentric type were used to investigate the depolarizing responses of DL-homocysteate (DLH) and L-glutamate in cat lumbar motoneurones. 2. Typically, DLH responses were slow both in onset and recovery, while glutamate responses were fast in onset and recovery and were frequently accompanied by a post-response hyperpolarization. 3. DLH responses (smaller than those necessary to evoke firing) were accompanied by a stable decrease in GM. This decrease was usually more than could be accounted for by anomalous rectification of the membrane. 4. Small glutamate responses were accompanied by either a small decrease, no change or a small increase in GM. There was a biphasic change in GM during large responses: GM decreased during the rising phase and early part of the response plateau and thereafter increased as the depolarization was maintained. It is proposed that the high conductance state during glutamate application (but not the depolarization itself) is a manifestation of glutamate uptake. 5. Firing evoked by DLH was stable during very long applications of the drug. Firing evoked by glutamate was usually of short duration, despite the maintained depolarization. 6. No reversal potential for the DLH responses could be demonstrated, but the responses decreased in size both with hyperpolarization and depolarization of the membrane. A 'null point' of the response in the negative direction was found to be approximately -95 mV. 7. DLH resonses were insensitive to changes in the internal Cl concentration. When the external K concentration was increased by K+ ionophoresis, the DLH responses became smaller. It is concluded that the DLH response is probably mediated via a decrease in K+ conductance and that the availability of this conductance channel is potential dependent. 8. Changes in the sizes of evoked potentials (e.p.s.p.s, i.p.s.p.s and a.h.p.s) with DLH and glutamate responses were investigated. The size of each of these evoked potentials was inversely related to GM during the responses; thus they all showed stable increases during DLH responses. E.p.s.p.s recorded during DLH were of longer half-width and time-to-peak than the control, but there was no change in the maximum slope (V.sec-1). When e.p.s.p.s decreased in size with glutamate the time-to-peak remained constant. 9. Acidic amino acids have been implicated as natural excitatory transmitters. The consequence of our results for the mechanism of excitatory transmission is therefore discussed.