F A C U L T Y OF M E D I C I N E
A. GENERAL PHARMACOLOGY (1) Ionic Discrimination in Muscle
Workers: (Mrs) Shirley E. Simon, B. M. Johnstone, K. H. Shankly and Patricia Tasker
This work is a continuation of that reported previously. The con- cept of a three phase system for muscle was put forward in earlier reports. This postulate was originally enunciated to describe results obtained for the distribution of inorganic ions across the cell mem- brane at the steady state. Our knowledge of thc mechanism of ionic discrimination has been extended and confirmed by the measurement of the rates of exchange of the various ions across the membrane.
These studies have included measurements of the efflux rates of L i+. Na+, T, SO., + , Cs+ and Rb+. In thc case of L i+ thc ionic move- ments were followed analytically, using a flame spectrophotometer.
The exchange rates of Na+, Cs+ and Rb+ were also followed by this method and, i n addition, tracer techniques using radioactive isotopes of thc ions were employed. 3sSO., and 1 3 1I were used to measure the effluxes of these ions.
An attempt was made to correlate the fast moving fraction of the various effluxes with the amount of material derived f r o m thc extra- cellular space, as measured with 1 3 1I-taggcd albumin. I t was found that thc amount of fast moving material was too great to be derived exclusively f r o m this space, and must include intracellular material.
It has been inferred from these studies that, contrary to currently held opinion, the cell membrane cannot be considered the principal rate limiting step in the outward movement of the ions considered.
Further work in this field is in hand, and it is hoped to determine thc simultaneous rates of efflux of l ; iI-tagged albumin and " N a from the one muscle, in order to characterize that portion of the
" N a efflux which is truly extracellular. This should permit an accurate estimate of the rate of movement of this ion across the membrane. A method of separating the gamma radiations of these isotopes with tlie aid of a single channel pulse analyser is being developed.
(2) High Energy Phosphate Compounds in Muscle
Workers: G. P. Brincr, R. Prater, (Mrs) Shirley E. Simon and Patricia Tasker
A correlation of ion shifts with the levels of adenosine triphosphate and creatine phosphate in muscle has been carried out.
A new technique for the accurate and rapid estimation of A T P was developed. The adenosine polyphosphates are separated by par- tition chromatography, localized under ultra-violet light, eluted, and
P H A R M A C O L O G Y 199
the adenine moiety estimated by measuring the absorption in the ultra-violet by means of a Bcckman spectrophotometer. Creatine phosphate was estimated in thc same muscle extract using the method of Ennor and Rosenberg. Thc inorganic ions were also esti- mated in the same extract sample, so that a correlation of the results f r o m individual muscles was possible.
The effect of variation of thc external ionic environment, and of a small number of metabolic inhibitors was determined. The ions were investigated at various levels, and included K+. Rb+, Cs+ and L i+.
It was found that K+ and R b+ brought about decreases in the levels of high energy phosphate compounds only at an external level of com.cq./l. Higher and lower levels were ineffective. Cs+ caused losses of creatine phosphate at all levels tested, and Li'1" was without effect on thc ATP and creatine phosphate content.
This work has been correlated with that carried out bv Miss Muller on the respiration and glycolysis of muscle under similar experimental conditions.
It is intended to pursue this work further. The rate of exchange of the phosphate groups of A T P will bc determined with tracer techniques, and correlated with the rates of exchange of the inorganic ions.
(3) Lithium Balance Studies in Man
Workers: (Mrs) Shirley E. Simon, F. H . Shaw and S. Gershon Work has been carried out in conjunction with the clinical re- search team of this Department to estimate thc level of L r in the plasma and the red cells in response to constant doses of L i+. Data obtained from normal subjects has been compared with that obtained from cancer patients. I n vilro experiments of the rate of exchange of L r across thc red cell membrane have also been carried out.
Similar studies on L i+ balance in tumour bearing mice are in hand.
(4) Metabolic State and Ionic Content Worker: Margaret Muller
Thc correlation of ionic shifts in toad muscle with thc metabolic status of the tissue was further studied. In particular, thc effects of the alkali metal ions, lithium, rubidium and caesium were investi- gated to complete the work previously done with sodium and potassium.
With different ionic environments, oxygen uptakes were measured using a Warburg apparatus and, after each experiment, the muscle was analysed for lactic acid (by the method of Barker and Sum- merson) and for the ions sodium and potassium, and lithium, rubidium, caesium when present.
200 F A C U L T Y OF M E D I C I N E
Ionic movements, either with or against a gradient; changes in respiration and in aerobic glycolysis were thus related.
The main finding was that the oxygen consumption increased as the concentration of external potassium and rubidium increased, reaching a maximum at about 20 meq/litre, it then declined again.
(5) Kinetics of Efflux of Ions from Muscle Worker: (Mrs) Clare G. Johnstone
The results of a series of measurements of the rate of efflux of lithium and sodium ions and radio-iodinated human serum albumin from toad muscle were analysed graphically and statistically. Each graph was separated into two or three components which were com- pared with each other and with thc theoretical behaviour of simple model systems.
The factors affecting the rate of efflux are the rate of diffusion and the extent of chemical combination in the fibre interior, the membrane and the extracellular space, and the efficiency of stirring in the outside medium.
The component due to simple diffusion from the extracellular space has been identified, and relationships so far obtained for lithium indicate that the method may be used to determine the relative importance of the other factors listed.
(6) Membrane Potentials and Ionic Contents of Amphibian Muscle Worker: B. M. Johnstone
Work has continued on a critical examination of the relationship between membrane potential and internal ions of muscle cells.
Contrary to current theory, no correlation has been found between membrane potential and internal concentrations of sodium, potas- sium or chloride ions. Possible sources of error have been reduced by measuring the extracellular space of individual muscles and checking of asymmetry potentials in the measuring apparatus.
In order to check the generality of our findings, similar experi- ments were attempted on acities tumour cells. These cells were im- mobilized in agar and gave anomalous membrane potentials, the cytoplasm being apparently slightly positive. The use of acities cells was discontinued due to thc difficulties of the technique and, with the co-operation of C.S.L., hyla cells are being used instead.
When the rate of ionic efflux from a muscle is of similar order to that of free diffusion, efficient stirring of the external medium is necessary. A stirring apparatus has been devised to do this on a semi-micro scale and checked with agar models.
It has been shown to eliminate errors due to this cause for intervals down to one minute.