John A. Flatman

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 (EM) has been investigated in soleus muscles isolated from fed rats.

Nifedipine‐ and omega‐conotoxin‐sensitive Ca2+ conductances in guinea‐pig substantia nigra pars compacta neurones.

1. The membrane properties of substantia nigra pars compacta (SNc) neurones were recorded in guinea‐pig in vitro brain slices. 2. In the presence of tetrodotoxin (TTX) a Ca(2+)‐dependent slow oscillatory potential (SOP) was generated. Application of 0.5‐20 microM nifedipine abolished both spontaneous and evoked SOPs. A tetraethylammonium chloride (TEA)‐promoted high‐threshold Ca2+ spike (HTS) was little affected by nifedipine. On the other hand, omega‐conotoxin applied either locally or via the perfusion medium (1‐10 microM) blocked a part of the HTS, but it did not alter the SOP. 3. In normal medium nifedipine blocked the spontaneous discharge, decreased the interspike interval (ISI) recorded during depolarizing current injections and selectively reduced the slow component of the spike after‐hyperpolarization (AHP). omega‐Conotoxin decreased both the rising and falling slopes of the normal action potential, reduced the peak amplitude of the spike AHP, and, in some of the neurones, reduced the ISI during depolarization. The Na+ spikes recorded in Ca(2+)‐free medium were not altered by omega‐conotoxin. 4. The SOP was not blocked by octanol (100‐200 microM), amiloride (100‐250 microM), or Ni2+ (100‐300 microM). However, at 500 microM Ni2+ attenuated the SOP. 5. Application of apamin (0.5‐2.0 microM) induced irregular firing or bursting, abolished the slow component of the spike AHP and reduced its peak amplitude. In the presence of TTX and apamin long‐duration plateau potentials occurred, which were subsequently blocked by nifedipine. 6. In Ca(2+)‐free, Co(2+)‐containing medium TTX‐sensitive spikes and voltage plateaux were generated by depolarizing current pulses. It is suggested that a persistent Na+ conductance underlies the plateaux, which may be co‐activated during the SOP. 7. The results suggest that the Ca2+ currents underlying the SOP and the HTS are different and that they activate at least two Ca(2+)‐dependent K+ conductances. These conductances play major roles in the maintenance of spontaneous discharge and in control of membrane excitability.

Relations between excitability and contractility in rat soleus muscle: role of the Na+-K+ pump and Na+/K+ gradients

1 The effects of reduced Na+/K+ gradients and Na+‐K+ pump stimulation on compound action potentials (M waves) and contractile force were examined in isolated rat soleus muscles stimulated through the nerve. 2 Exposure of muscles to buffer containing 85 mM Na+ and 9 mM K+ (85 Na+/9 K+ buffer) produced a 54 % decrease in M wave area and a 50 % decrease in tetanic force compared with control levels in standard buffer containing 147 mM Na+ and 4 mM K+. Subsequent stimulation of active Na+‐K+ transport, using the β2‐adrenoceptor agonist salbutamol, induced a marked recovery of M wave area and tetanic force (to 98 and 87 % of the control level, respectively). Similarly, stimulation of active Na+‐K+ transport with insulin induced a significant recovery of M wave area and tetanic force. 3 During equilibration with 85 Na+/9 K+ buffer and after addition of salbutamol there was a close linear correlation between M wave area and tetanic force (r= 0·92, P< 0·001). Similar correlations were found in muscles where tetrodotoxin was used to reduce excitability and in muscles fatigued by 120 s of continuous stimulation at a frequency of 30 Hz. 4 These results show a close correlation between excitability and tetanic force. Furthermore, in muscles depressed by a reduction in the Na+/K+ gradients, β‐adrenergic stimulation of the Na+‐K+ pump induces a recovery of excitability which can fully explain the previously demonstrated recovery of tetanic force following Na+‐K+ pump stimulation. Moreover, the data indicate that loss of excitability is an important factor in fatigue induced by high‐frequency (30 Hz) stimulation.

Na+-K+ pump stimulation elicits recovery of contractility in K+-paralysed rat muscle

1. This study explores the role of active electrogenic Na(+)‐K+ transport in restoring contractility in isolated rat soleus muscles exposed to high extracellular potassium concentration ([K+]o). This was done using agents (catecholamines and insulin) known to stimulate the Na(+)‐K+ pump via different mechanisms. 2. When exposed to Krebs‐Ringer bicarbonate buffer containing 10 mM K+, the isometric twitch and tetanic force of intact muscles decreased by 40‐69%. The major part of this decline could be prevented by the addition of salbutamol (10(‐5) M). In the presence of 10 mM K+, force could be restored almost completely within 5‐10 min by the addition of salbutamol or adrenaline and partly by insulin. 3. In muscles exposed to 12.5 mM K+, force declined by 96%. Salbutamol (10(‐5) M), adrenaline (10(‐6) M) and insulin (100 mU ml‐1) produced 57‐71, 61‐71 and 38‐47% recovery of force within 10‐20 min, respectively. The effects of these supramaximal concentrations of salbutamol and insulin on force recovery were additive. Salbutamol and adrenaline produced significant recovery of contractility at concentrations down to 10(‐8) M (P < 0.005). 4. In soleus, the same agents stimulated 86Rb+ uptake and decreased intracellular Na+. These actions reflect stimulation of active Na(+)‐K+ transport and both showed a highly significant correlation to the recovery of twitch as well as tetanic force (r = 0.80‐0.88; P < 0.001). 5. The force recovery induced by salbutamol, adrenaline and insulin was suppressed by pre‐exposure to ouabain (10(‐5) M for 10 min or 10(‐3) M for 1 min) as well as by tetrodotoxin (10(‐6) M). 6. The observations support the conclusion that the inhibitory effect of high [K+]o on contractility in skeletal muscle can be counterbalanced by stimulation of active electrogenic Na(+)‐K+ transport, the ensuing increase in the clearance of extracellular K+ and in the transmembrane electrochemical gradient for Na+.

Relation between extracellular [K+], membrane potential and contraction in rat soleus muscle: modulation by the Na+-K+ pump.

An increased extracellular K+ concentration ([K+]0) is thought to cause muscle fatigue. We studied the effects of increasing [K+]0 from 4 mM to 8–14 mM on tetanic contractions in isolated bundles of fibres and whole soleus muscles from the rat. Whereas there was little depression of force at a [K+]0 of 8–9 mM, a further small increase in [K+]0 to 11–14 mM resulted in a large reduction of force. Tetanus depression at 11 mM [K+]o was increased when using weaker stimulation pulses and decreased with stronger pulses. Whereas the tetanic force/resting membrane potential (EM) relation showed only moderate force depression with depolarization from −74 to −62 mV, a large reduction of force occurred whenEM fell to −53 mV. The implications of these relations to fatigue are discussed. Partial inhibition of the Na+-K+ pump with ouabain (10−6 M) caused additional force loss at 11 mM [K+]0. Salbutamol, insulin, or calcitonin gene-related peptide all stimulated the Na+-K+ pump in muscles exposed to 11 mM [K+0] and induced an average 26–33% recovery of tetanic force. When using stimulation pulses of 0.1 ms, instead of the standard 1.0-ms pulses, force recovery with these agents was 41–44% which was significantly greater (P < 0.025). Only salbutamol caused any recovery ofEM (1.3 mV). The observations suggest that the increased Na+ concentration difference across the sarcolemma, following Na+-K+ pump stimulation, has an important role in restoring excitability and force.

The modulation of excitatory amino acid responses by serotonin in the cat neocortex in vitro

Summary1.The electrophysiological actions of excitatory amino acids and serotonin were investigated in slices from cat neocortexin vitro. Intracellular recordings were obtained from neurons (mainly in layer V) and the drugs applied extracellularly to the same neurons by microiontophoresis.2.Serotonin, and to some extent noradrenaline, facilitated the excitatory actions ofN-methyl-d-aspartate (NMDA), glutamate, and quisqualate but caused no changes in the passive neuronal membrane properties when presented alone. Serotonin had no effect on evoked excitatory postsynaptic potentials (EPSPs) or spike afterhyperpolarizations.3.The facilitatory effect of serotonin on the responses to NMDA was observed with both somatic and dendritic applications. It persisted during Mg2+ depletion and in the presence of tetrodotoxin and tetraethylammonium. The effect was attenuated by the serotonin antagonist cinanserin but not by methysergide. A possible underlying receptor modulation is discussed.

GYKI 52466, an inhibitor of spinal reflexes is a potent quisqualate antagonist

GYKI 52466 is a 2,3-benzodiazepine compound, which inhibits spinal reflexes in cats. Contrary to the classical benzodiazepines (e.g. diazepam), GYKI 52466 does not potentiate the inhibitory action of gamma-amino butyric acid, but seems to affect excitatory processes in the spinal cord directly.

Regulation of ClC-1 and KATP channels in action potential–firing fast-twitch muscle fibers

Action potential (AP) excitation requires a transient dominance of depolarizing membrane currents over the repolarizing membrane currents that stabilize the resting membrane potential. Such stabilizing currents, in turn, depend on passive membrane conductance (Gm), which in skeletal muscle fibers covers membrane conductances for K+ (GK) and Cl− (GCl). Myotonic disorders and studies with metabolically poisoned muscle have revealed capacities of GK and GCl to inversely interfere with muscle excitability. However, whether regulation of GK and GCl occur in AP-firing muscle under normal physiological conditions is unknown. This study establishes a technique that allows the determination of GCl and GK with a temporal resolution of seconds in AP-firing muscle fibers. With this approach, we have identified and quantified a biphasic regulation of Gm in active fast-twitch extensor digitorum longus fibers of the rat. Thus, at the onset of AP firing, a reduction in GCl of ∼70% caused Gm to decline by ∼55% in a manner that is well described by a single exponential function characterized by a time constant of ∼200 APs (phase 1). When stimulation was continued beyond ∼1,800 APs, synchronized elevations in GK (∼14-fold) and GCl (∼3-fold) caused Gm to rise sigmoidally to ∼400% of its level before AP firing (phase 2). Phase 2 was often associated with a failure to excite APs. When AP firing was ceased during phase 2, Gm recovered to its level before AP firing in ∼1 min. Experiments with glibenclamide (KATP channel inhibitor) and 9-anthracene carboxylic acid (ClC-1 Cl− channel inhibitor) revealed that the decreased Gm during phase 1 reflected ClC-1 channel inhibition, whereas the massively elevated Gm during phase 2 reflected synchronized openings of ClC-1 and KATP channels. In conclusion, GCl and GK are acutely regulated in AP-firing fast-twitch muscle fibers. Such regulation may contribute to the physiological control of excitability in active muscle.

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.

A dichtomy of the membrane potential response of rat soleus muscle fibres to low extracellular potassium concentrations

The effects of different extracellular potassium concentrations [K+]o on the resting membrane potential (EM) of rat soleus muscle fibres was assessed in the absence and presence of 10−3M ouabain. At concentrations of 3 mM K and below, the fibres could be divided into two significantly different and normally distributed populations on the basis of EM response: One group responded to a lowering of the [K]o with a graded hyperpolarization to between −80 and −103 mM. The second fibre population had a less negative EM (−70 mV) which did not respond to changes in [K]o between 0 mM and 3 mM. In the [K]o range, 0 mM to 5.9 mM, the mean EM of fibres treated with ouabain was only slightly less negative than the EM of the second fibre population. We conclude that this dichtomy of the mean EM at low [K]o reflects the presence of two fibre types with different electrochemical properties.