Sodium-selective micro-electrode study of apical permeability in frog skin: effects of sodium, amiloride and ouabain.

The intracellular sodium ion activity (aiNa), apical membrane potential (psi ac) and apical sodium electrochemical driving force (delta mu Na) in Rana temporaria skin were measured using double-barrelled sodium-sensitive micro-electrodes, in the presence of various apical sodium activities (aoNa), amiloride, ouabain, and during voltage clamp of psi ac. The permeability and specific conductance of the apical cell membrane to sodium entry (PaNa and GaNa respectively) were calculated from the Goldman-Hodgkin-Katz equation and the Nernst-Planck (electrodiffusion) permeability equations respectively. The roles of aoNa and aiNa in the control of apical sodium entry were studied. PaNa increased linearly with log decrease in aoNa between 79 and 0.01 mM. Under short-circuit conditions, aiNa remained constant over the aoNa range of 10-79 mM, but decreased when aoNa was lower than 10 mM, due to a fall in delta mu Na and GaNa. Amiloride decreased PaNa, GaNa and aiNa, a result analogous to that observed in spontaneous low-transporting skins. Ouabain inhibited sodium transport and increased aiNa before any changes in PaNa occurred. The latter decreased only when aiNa rose above 15 mM. Increasing delta mu Na by hyperpolarizing voltage clamp of the apical cell membrane elicited a saturable increase in aiNa. The opposite effect was elicited by depolarizing psi ac. Electrodiffusion appears to be the sole mode of apical sodium entry.

Intracellular ion activities in frog skin in relation to external sodium and effects of amiloride and/or ouabain.

Intracellular activities of sodium, potassium and chloride ions, aiNa, aiK, and aiCl were measured with ion-selective single-, double- and triple-barrelled micro-electrodes in skin and isolated epithelia of Rana temporaria bathed on both sides with normal or modified physiological saline. Apical and basolateral membrane potentials, psi ac and psi cs and resistance Ra and Rb respectively were also measured and from the latter the fractional resistance of the apical membrane, F(Ra) and voltage divider ratio, delta psi ac/delta psi cs were measured as criteria of satisfactory membrane penetration by the micro-electrodes. Under control conditions, aiNa was 12.3 +/- 0.8 mM, aiK was 70.3 +/- 22 mM and aiCl was 20.3 +/- 1.6 mM with psi ac averaging -38.0 +/- 3.2 mV. When 10(-4) M-amiloride was added to the apical bathing fluid aiNa fell within 10 min to 1.18 +/- 0.1 mM and aiCl to 5.2 +/- 0.9 mM, while aiK increased to 86.2 +/- 3.8 mM as measured from the basolateral border of isolated epithelia. The sodium transport pool of the skin was measured from the fall in aiNa in the presence of amiloride and could be expressed as 33 X 10(-9) mol cm-2 of epithelium. The mean rate of fall of aiNa under these conditions corresponded to an efflux rate at the basolateral border of 30.1 X 10(-9) mol cm-2 min-1 (48 microA cm-2) giving a half-time for turnover of the sodium transport pool of 33 s. Reduction of sodium concentration in the apical fluid from the normal 79 mM-Na to 10, 1 and 0.1 mM caused aiNa to fall in stages to 2 mM. Because psi ac increased in negativity to -101 mV in the process, this driving force for passive sodium accumulation, more than offset the increased sodium gradient opposing sodium influx across the apical border.

Rubidium influx into rat skeletal muscles in relation to electrical activity.

1. Rates of (86)Rb influx were compared in vivo over 2, 4 and 6 hr periods in various tonic and phasic muscles of rat following its I.P. injection. During the 2 hr period its influx rate into soleus was about 4 times that of the vastus with the EDL muscles at an intermediate rate. Uptake by diaphragm was fastest reaching equilibrium within 2 hr.2. Unilateral section of the sciatic nerve 48 hr before (86)Rb injection reduced isotope uptake into soleus to about 50% of its contralateral control muscle over a 4 hr period. In EDL muscles on the other hand nerve section increased influx by about 75% of control in conscious rats and more than doubled influx in anaesthetized rats.3. Tenotomy of soleus reduced (86)Rb influx to 40% of control, but tenotomy in EDL was without effect in influx.4. Uptake of urea into muscles within 5 min of its I.V. injection was used to determine the possibility of muscle blood flow determining (86)Rb influx. Accumulation of urea was not significantly different in control and denervated EDL muscles nor between soleus and vastus muscles in anaesthetized rats, so it seems unlikely that blood flow is important here.5. Membrane depolarization in response to addition of 30 mM rubidium to external bathing fluid was greater in the case of denervated than in control EDL muscles which was in keeping with the greater (86)Rb influx seen in the former muscles. The ouabain sensitivity of rubidium-induced depolarization in the denervated EDL muscles would suggest, however, that rubidium enters the fibres actively.

Accumulation of caesium and rubidium in vivo by red and white muscles of the rat.

1. Rats were given drinking water containing either 20 mM-CsCl or 20 mM-RbCl for a period of 2 weeks. Samples of blood were then taken from the rats under anaesthetic. They were immediately centrifuged and the plasma taken for analysis. Soleus muscles, diaphragm, extensor digitorum longus, white gastrocnemius and vastus lateralis muscles were then taken from the dead animals and these and the plasma were analysed for potassium, and for caesium or rubidium by means of the flame photometer.2. The concentrations of potassium and rubidium or caesium in the fibre water of these various muscles and in the samples of plasma water were then calculated.3. It was found that the red muscles including soleus and diaphragm generally tended to accumulate caesium and rubidium to a greater extent than did the white muscles such as the white gastrocnemius and vastus lateralis.4. When the concentration ratio [K](i)/[K](o) was divided into the ratio [Rb](i)/[Rb](o) for the different muscles, values of about 1.3 were obtained for the red muscles compared with values about 1.14 for white muscles.5. When in the case of the caesium-treated rats the ratio [K](i)/[K](o) was divided into the ratio [Cs](i)/[Cs](o) values ranged from 1.94 +/- 0.12 for the red soleus to 1.08 +/- 0.09 for the white gastrocnemius.6. When these values in the caesium-treated animals were plotted against the percentage of red fibres in the five muscle types (as obtained from the data of Sreter & Woo, 1963) the graph indicated that the white fibres had similar ionic gradients for Cs(+) and K(+) and that affinity for Cs(+) was confined to the red fibres.7. The membrane potential measured in soleus and extensor muscles immersed in plasma from the same animal was not significantly different from E(K) but was much less than E(Cs).8. These results are interpreted in terms of permeability differences between the slow red fibres and white twitch fibres.

Active transport of sodium and potassium in mammalian skeletal muscle and its modification by nerve and by cholinergic and adrenergic agents.

1. Active transport of Na(+) and K(+) by Na-rich extensor digitorum and soleus muscles of rat was found to be increased considerably when muscles were innervated during enrichment with Na(+) in K-free modified Krebs solution containing 160 mM-Na at 2 degrees C and recovery in a similar fluid with 10 mM-K and 137 mM-Na at 37 degrees C, bubbled with oxygen.2. Addition of acetylcholine (2.0 mug/ml.) to recovery fluid containing denervated extensors increased active transport, whereas addition of eserine (50 mug/ml.), decamethonium (0.1 mug/ml.) and to a lesser extent tubocurarine (0.26 mug/ml.) inhibited active transport. Blocking of nerve conduction in innervated extensor inhibited K(+) uptake more than Na(+) excretion.3. The membrane potential of Na-rich extensor muscles measured soon after re-immersion in recovery fluid was higher in denervated than in innervated muscles. In the latter it was close to the K-equilibrium potential (E(K)). It is suggested that denervation here makes the Na-pump electrogenic by decreasing K(+) uptake either by decreased permeability or by inactivating a K-pump. Evidence is presented that the latter is more likely.4. Addition of isoprenaline to Na-rich soleus muscles in recovery fluid increased active transport and reduced the membrane potential measured soon after re-immersion in recovery fluid. The Na-pump still remained electrogenic in the presence of isoprenaline. It was suggested that isoprenaline might also stimulate the Na-pump, perhaps through activation of lactic dehydrogenase.