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1. A study was made of the electrolyte changes which occur when frog muscles are immersed in a Ringer solution with 1/5 of the Na replaced by NH4Cl. Analyses were made in the solution and in the muscles for K and NH3, and the muscles were also analyzed for Cl, HCO3, and Na. Control muscles were immersed in normal Ringer's solution and similarly analyzed. 2. The amount of ammonia taken up was about equal to the K and Na lost. There was also a small increase in chloride content. The bicarbonate content was the same in both experimental and control muscles, indicating no change in the muscle pH due to the NH3 which penetrated. An increased loss of K due to the penetration of NH3 was also demonstrated by the use of radioactive K. 3. After 5 hours, the concentration of ammonia per gram of muscle is about the same as the concentration in the solution. After 4 or 5 days, the concentration in the muscle is about 1.5 times that in the solution. The inside to outside NH3 ratio is about equal to the corresponding H ion ratio, but is much less than the K ratio. 4. The rate of penetration of the NH3 is increased by a rise of temperature, by stirring the solution, and by decrease in the concentration of Na, K, Ca, or Mg in the solution; it is decreased by increasing the size of the muscles or by killing them with chloroform or boiling. 5. Liver, smooth muscle, skin, and kidney, in a few experiments, behaved much like muscle except that there was a formation of urea in the case of liver. 6. The injection of NH4Cl into anesthetized cats causes an increase in the level of K in the blood plasma.

The respiration of the isolated frog rod outer limb has been measured in the Cartesian diver. The outer limbs respire in Ringer solution without the addition of substrates, but the rate of respiration is increased by the addition of fructose diphosphate or succinate. The respiration is cyanide-sensitive, and therefore presumably mediated by the cytochromes. The QOO2 in 0.01 M fructose diphosphate is –1.0 µl. oxygen per mg. dry weight per hour at 20°C. This is lower than the QOO2 of whole frog retina, but comparable with it and many other tissues. The respiratory rate is independent of the state of dark adaptation (rhodopsin content) of the outer limbs. The metabolism of the outer limb is probably adequate to provide the DPN required for the maintenance of the rhodopsin concentration necessary for vision.

Intracellular records were made from fibers in the A-V conducting system of isolated toad hearts. The A-V region was perfused with Ringer's solution of various K and Na concentrations. Resting potential in 2.8 mM [K]o was about 60 mv. Over the range 0.28 to 28 mM, resting potential diminished with increasing [K]o. Spontaneous action potentials appeared when [K]o was increased to 11.2 mM, and when resting potential had fallen to about 40 to 50 mv. Changes in [Na]o over the range 22 to 110 mM had a little effect on resting potential, but there was a linear relation between the peak value of the action potential and log [Na]o Wenckebach periodicity was observed when [Na]o was lowered.

Working with sciatic nerves from the South American frog Calyptocephalella gayi we have been able to substantiate the finding of Ungar et al., that nerve proteins undergo a reversible denaturation concomitant with a train of nerve impulses. We also showed that 0.3 mM sodium azide is capable of impairing the reversibility of those changes. Furthermore, 10 meq/liter of calcium ion applied to extracts of resting nerves induce configurational rearrangements in the proteins similar to those produced during stimulation. We also proved that nerves previously kept in low calcium Ringer's solution do not show configurational changes upon stimulation. A tentative interpretation of the results described is that the configurational changes are a consequence of the extra influx of calcium ion due to nerve impulse propagation.

Isolated frog sartorii were immersed in Ringer's solution, which had a 2 mM magnesium concentration containing Mg28. The uptake of the radioactive magnesium was measured under steady state conditions. Although the biological variability was fairly large, it was observed that the uptake proceeded in three stages lasting respectively about 0.5, 30, and 300 minutes and accounting respectively for about 0.21, 0.71, and 0.67 millimole magnesium/kg. muscle. It was assumed that the first stage represents surface adsorption, the second stage represents extracellular water and connective tissue phases, and the third stage entry inside the cell. It is estimated that the maximum intracellular magnesium concentration is about 1.1 mM and that only about 0.6 millimole magnesium/liter intracellular water is exchanged per hour. The maximum energy required per hour to pump the magnesium out of the cell against the electrochemical gradient is calculated to be only 1.5 cal./kg. muscle. About 75 to 80 per cent of the magnesium in muscle is non-exchangeable and difficult to remove by diffusion. It appears from previous work that the exchangeable magnesium behaves similarly to exchangeable calcium.

The influence of polyvalent cations on the activation of end plate receptors has been studied in vitro on the sartorius muscle of the frog. In the absence of extracellular calcium, the sensitivity of the receptors to depolarizing quaternary ammonium salts was markedly reduced. Maximum receptor activation occurred in those fibers equilibrated in 1.8 mM calcium Ringer solution, with the response being reduced as the calcium concentration was raised or lowered. Magnesium was less efficient than calcium in regulating the sensitivity of the end plate receptors, the maximum receptor response occurring in those fibers equilibrated in 8 mM magnesium Ringer solution. In the presence of lanthanum the end plate response to carbamylcholine or acetylcholine was enhanced. Lanthanum increased the conductance change produced by carbamylcholine both in polarized and in potassium-depolarized fibers. The application of 10-2 mM lanthanum to the end plate increased MEPP's amplitude, rise time, and half-fall time by 19, 54, and 45%, respectively. The results suggest that polyvalent cations influence postjunctional membrane receptor processes in addition to their well-documented prejunctional action.

1. (1) An outline is given of some of the biologically important properties of inorganic ions and of the part played by different ion species in the metabolism of cells. 2. (2) The composition of about 100 physiological salines for use with fish, amphibia, mammals, birds, annelids, crustacea, insects and molluscs is given together with analyses of the blood composition of some of the species of animals most frequently used in physiological experiments.

The permeability of K channels to various cations is studied in myelinated nerve. Ionic currents under voltage clamp are measured in Ringer solution containing tetrodotoxin and a high concentration of the test ion. Reversal potentials for current in K channels are determined and used with the Goldman-Hodgkin-Katz equation to calculate relative permeabilities. The ratios P(Tl):P(K):P(Rb):P(NHNH4) are 2.3:1.00:0.92:0.13. No other ions are found to be measurably permeant including Li(+), Na(+), Cs(+), methylamine, guanidine, hydrazine, or hydroxylamine. The ratio P(Na)/P(K) is less than 0.01. Potassium conductance is depressed at pH values below 5.0. Leakage conductance is higher in K, Rb, Cs, NH(4), and Tl Ringer than in Na Ringer, but the selectivity sequence probably is not the same as for K channels. The hypothesis is offered that the narrowest part of the K channel is a circle of oxygen atoms about 3 A in diameter with low electrostatic field strength.

Caffeine increases resting calcium influx approximately threefold in normally polarized and in potassium-depolarized fibers of frog sartorius muscles. It does not affect the transient rapid increase in calcium influx that occurs at the beginning of a potassium depolarization. Calcium outflux in Ringer's solution, in zero calcium Ringer's solution, and in zero calcium Ringer's solution plus 0.004 M EDTA is also markedly increased by caffeine. The increased outflux reaches a rate which is approximately the same as the increased calcium influx. One interpretation of the findings is that caffeine reduces the binding of calcium both in the membrane and in the myoplasm; this increases the "permeability" to calcium and the ionic activity of calcium in muscle. This interpretation is consistent with the view that the contractile state of muscle is dependent at least in part on the thermodynamic activity of calcium in the muscle fibers.

The erythrocytes of some species are much damaged when handled in salt solutions, as in washing with the centrifuge after the ordinary method. The injury is mechanical in character. It may express itself in hemolysis only after the cells have been kept for some days. It is greatest in the case of dog corpuscles, and well marked with sheep and rabbit cells. The fragility of the red cells, as indicated by washing or shaking them in salt solution is different, not only for different species, but for different individuals. It varies independently of the resistance to hypotonic solutions. The protection of fragile erythrocytes during washing is essential if they are to be preserved in vitro for any considerable time. The addition of a little gelatin (⅛ per cent) to the wash fluid suffices for this purpose, and by its use the period of survival in salt solutions of washed rabbit, sheep, and dog cells is greatly prolonged. Plasma, like gelatin, has marked protective properties. Though gelatin acts as a protective for red cells it is not preservative of them in the real sense. Cells do not last longer when it is added to the fluids in which they are kept. Locke's solution, though better probably than Ringer's solution, or a sodium chloride solution, as a medium in which to keep red cells, is ultimately harmful. The addition of innocuous colloids does not improve it. But the sugars, especially dextrose and saccharose, have a remarkable power to prevent its injurious action, and they possess, in addition, preservative qualities. Cells washed in gelatin-Locke's and placed in a mixture of Locke's solution with an isotonic, watery solution of a sugar remain intact for a long time,—nearly 2 months in the case of sheep cells. The kept cells go easily into suspension free of clumps, they pass readily through paper filters, take up and give off oxygen, and when used for the Wassermann reaction behave exactly as do fresh cells of the same individual. The best preservative solutions are approximately isotonic with the blood serum. If the cells are to be much handled gelatin should be present, for the sugars do not protect against mechanical injury. Different preservative mixtures are required for the cells of different species. Dog cells last longest in fluids containing dextrin as well as a sugar. The mixture best for red cells is not necessarily best for leukocytes. A simple and practical method of keeping rabbit and human erythrocytes is in citrated whole blood to which sugar solution is added. In citrated blood, as such, human red cells tend to break down rather rapidly, no matter what the proportion of citrate. Hemolysis is well marked after little more than a week. But in a mixture of 3 parts of human blood, 2 parts of isotonic citrate solution (3.8 per cent sodium citrate in water), and 5 parts of isotonic dextrose solution (5.4 per cent dextrose in water), the cells remain intact for about 4 weeks. Rabbit red cells can be kept for more than 3 weeks in citrated blood; and the addition of sugar lengthens the preservation only a little. The results differ strikingly with the amount of citrate employed. Hemolysis occurs relatively early when the smallest quantity is used that will prevent clotting. The optimum mixture has 3 parts of rabbit blood to 2 of isotonic citrate solution. In the second part of this paper experiments are detailed which prove that cells preserved by the methods here recorded function excellently when reintroduced into the body.

The development of isometric twitch tension has been compared with the redevelopment of isometric tension in the fully active frog sartorius muscle following release. At 0°C the rate of rise of isometric twitch tension is the same as that for the muscle in the fully active state at the same tension but not until about 40 msec. after the stimulus and then only for a few milliseconds. The rates of rise of tension in the twitch and in the redevelopment of tension in the fully active muscle appear to be nearly the same at low tensions. Substitution of nitrate for chloride in the Ringer's solution bathing the muscle retards the development of tension during the early part of the contraction phase of the twitch and the effect reaches a maximum within 3 minutes after changing the solutions. These observations have been discussed in connection with some possible patterns of activation and the hypothesis has been advanced that the rate of activation of a sarcomere is determined mainly by the rate at which the transverse component of the link between excitation and contraction is propagated inwards from the periphery to the center of the fiber. This hypothesis has been discussed in relation to others concerning the nature of excitation-contraction coupling.

1. Frog nerves immersed in Ringer's solution lose on the average 23 per cent more potassium if the solution is equilibrated with pure nitrogen than if equilibrated with oxygen. 2. Tying off the ends of the nerves during immersion increased rather than diminished the loss of potassium. 3. There was some evidence that nerves tended to regain potassium if they were returned to oxygen after a period of anoxia. 4. Addition of acetylcholine to the solution increases the loss of potassium. 5. Equilibration of the solution and nerves with 20 per cent CO2 in O2 increases the loss of K from nerves in Ringer's solution but decreases it in frog blood.

The potassium exchange properties of glycerol-treated sartorius muscles of the frog were determined. Potassium (42K) uptake, efflux, and net flux were measured in the presence of glycerol and at various times after exposure to glycerol and return to isotonic Ringer solution. Potassium uptake was not altered by the presence of glycerol but was reduced on the average 53% after glycerol treatment. Efflux transiently increased in the presence of glycerol and was reduced 37% after glycerol removal. Consequently, there was a net loss of intracellular potassium as well as a gain of sodium. In contrast to the irreversible alterations of potassium exchange induced by glycerol treatment, action potentials with normal negative afterpotentials (N.A.P.) were elicited 4–5 hr after glycerol removal. The reappearance of the N.A.P. was associated with a return of the membrane potential to normal values (90 ± 2 mv). However, the response of these muscles to reduced extracellular potassium was anomalous. In K+-free Ringer solution the average resting membrane potential was 74 ± 3 mv and a positive afterpotential of 11 ± 3 mv was associated with the action potential.

The membrane potential of frog sartorius muscle fibers in a Cl- and Na-free Ringer's solution when sucrose replaces NaCl is about the same as that in normal Ringer's solution. The K+ efflux is also about the same in the two solutions but muscles lose K and PO4 in sucrose Ringer's solutions. The membrane potential in sucrose Ringer's solution is equal to that given by the Nernst equation for a K+ electrode, when corrections are made for the activity coefficients for K+ inside and outside the fiber. For a muscle in normal Ringer's solution, the measured membrane potential is within a few millivolts of EK. This finding is incompatible with a 1:1 coupled Na-K pump. It is consistent with either no coupling of Na efflux to K influx, or a coupling ratio of 3 or greater.

Studies were carried out on the isolated urinary bladder of the toad, Bufo marinus, in order to explain the dependence of active sodium transport on the presence of potassium, in the serosal medium. Attempts to obtain evidence for coupled sodium-potassium transport by the serosal pump were unsuccessful; no relation between sodium transport and uptake of K42 from the serosal medium was demonstrable. Rather, the predominant effect of serosal potassium appeared to be operative at the mucosal permeability barrier, influencing the permeability of this surface to sodium. The mucosal effects of serosal potassium were correlated with effects on cellular cation content. When sodium Ringer's solution was used as serosal medium, removal of potassium resulted in significant decrease in tissue potassium content, commensurate increase in tissue sodium content, and marked depression of mucosal permeability and sodium transport. When choline replaced sodium in the serosal medium, removal of potassium resulted in only slight alterations of tissue electrolyte content, and effects on mucosal permeability and sodium transport were minimal.

Electrical stimulation, either cathodal or anodal, of the monocellular electroplax preparation in Ca-free Ringer's solution results in a sustained depolarization which is determined by the amount of current passed through the cell. The membrane potential recovers only when Ca is added again. These changes take place at the innervated side of the electroplax only. This depolarization of the membrane is pH-dependent; it depolarizes more at pH 6.0 than at pH 9.0. The membrane does not depolarize and the action potential is not blocked within an hour in Ca-free solution unless the cell is stimulated. The sustained depolarization is not prevented or reversed by curare, tetracaine, physostigmine, tetrodotoxin, and tetraethylammonium. After stimulation, the outward K current remains unchanged regardless of whether Ca is present. In contrast, the inward current is dependent on Ca in the outside solution on the innervated membrane; in the absence of Ca following stimulation, the inward K current is decreased. The depolarization by carbamylcholine is reduced in Ca-free and increased in Mgfree Ringer's solution. In contrast to the depolarization induced by electrical stimulation, these carbamylcholine depolarizations may be reversed by washing with Ca-free or Ca- and Mg-free Ringer's solution.

In 1962 Frank (22) reported that the addition of any one of a number of divalent cations, including Ni, to a Ca-free Ringer solution prevented the rapid loss of contractility seen in the absence of external Ca. To investigate further the Ni-Ca substitution, studies were made of 45Ca and 63Ni exchange during contraction and at rest using frog striated muscle. In contrast to 45Ca, it was found that there is no increase of 63Ni uptake associated with a K contracture of the sartorius muscle. The rates of loss of 63Ni and 45Ca from resting toe muscles previously bathed in the respective radioisotopes are not significantly different. Resting and action potentials, after 1 hr in a Ringer solution with Ni replacing Ca, closely resemble these potentials in normal Ca-Ringer's solution. Studies on the syneresis of isolated myofibrils indicate that Ni cannot replace Ca in activating this reaction. It is suggested that Ca is required for at least two steps in E-C coupling: one is the spread of excitation at the sarcolemma and transverse tubular system; the second is the activation of actomyosin ATPase. Conceivably Ni can substitute for Ca in the former but not in the latter.

A chronograph is described for recording continuously the rates of many different kinds of rhythmic processes over long time periods. The rate is read directly from the length of a line of ink, drawn by a moving pen. Rates of beat of excised turtle's hearts in Ringer's solution have been recorded at 25°C. under constant conditions of temperature, pH, and oxygen supply for periods of 36 hours. Regular periodic variations in the fundamental rhythm are figured, as well as rates of extraordinary constancy. The effects of adrenalin, ephedrin, thyroxin, α and ß pituitary hormone insulin, nicotin, and atropin are described in the text.

A study has been made of the loss of excitability in a sodium-free medium and of the recovery of excitability in Ringer's solution by A fibers of normal frog nerves and of nerves in advanced stages of Wallerian degeneration. With normal nerves that are being kept in a sodium-free medium the number of conducting fibers does not undergo a readily detectable decrease in less than 1 to 2 hours; inexcitability of all the A fibers does not develop in less than 7 to 8 hours. During the development of inexcitability the speed of conduction of the still conducting fibers undergoes a progressive decrease; in advanced stages the speed of conduction is not more than one-fifth of the normal speed. The nerve fibers lose the ability to conduct rhythmic trains of impulses earlier than the ability to conduct single impulses. The recovery of excitability in Ringer's solution duplicates in a reverse order the sequence of changes that have been previously observed during the development of inexcitability. The rate of the recovery of excitability in Ringer's solution is higher than the rate of the loss of excitability in the sodium-free medium. With degenerating nerves the effect of the lack of sodium develops qualitatively in the same manner in which it develops with normal nerves. Degenerating nerve fibers, however, become inexcitable in a sodium-free medium earlier than normal fibers. The recovery of the excitability in Ringer's solution takes place in much the same manner in normal and in degenerating nerve fibers. The loss of excitability during Wallerian degeneration is a process that develops simultaneously, or practically so, throughout the entire length of the fibers. The nerve fibers retain a great deal of functional ability throughout the several days which precede the onset of inexcitability and then suddenly become inexcitable.

Infusion of isotonic Ringer's solution led to hypernatremia and plasma hyperosmolarity in diabetes insipidus dogs. This phenomenon suggests a safe and reliable test for diabetes insipidus which does not require collection of urine.

Frog sartorius muscles were made Na-rich by immersion in K-free sulfate Ringer's solution in the cold. The muscles were then loaded with Na(24) and the extracellular space cleared of radioactivity. When such Na-rich muscles were transferred to lithium sulfate Ringer's solution at 20 degrees C, Na efflux was observed to increase with time, to reach a maximum about 15 minutes after the transfer of the muscles to Li(2)SO(4), and then to decline. The decline in efflux from these muscles was proportional to ([Na](i))(8) over a considerable range of [Na](i). The membrane potential of Na-rich muscles was about -48 mv in K-free sulfate Ringer's at 4 degrees C but changed to -76 mv in the same solution at 20 degrees C and to -98 mv in Li(2)SO(4) Ringer's at 20 degrees C. By contrast, muscles with a normal [Na](i) showed a fall in membrane potential when transferred from K-free sulfate Ringer's to Li(2)SO(4) Ringer's solution. The general conclusions from this study are (a) that Na extrusion is capable of generating an electrical potential, and (b) that increases in [Na](i) lead to reversible increases in P(Na) of muscle fibers.

1. Embryonic chick and rat tissues preserved at temperatures ranging from –7° to +20° C. live longest at about +6° C. The duration of life under the most favorable conditions is less than twenty days. 2. The kind of isotonic medium used,—plasma serum, Ringer solution, or normal salt solution,—does not appreciably influence the period of survival. The quantity of medium in proportion to tissue is similarly without marked effect.

Skeletal muscles which have been soaked for 1 hr in a glycerol-Ringer solution and then returned to normal Ringer solution have a disrupted sarcotubular system. The effect is associated with the return to Ringer's since muscles have normal fine structure while still in glycerol-Ringer's. Karnovsky's peroxidase method was found to be a very reliable marker of extracellular space, filling 98.5% of the tubules in normal muscle. It was interesting to note that only 84.1% of the sarcomeres in normal muscle have transverse tubules. The sarcotubular system was essentially absent from glycerol-treated muscle fibers, only 2 % of the tubular system remaining connected to the extracellular space; the intact remnants were stumps extending only a few micra into the fiber. Thus, glycerol-treated muscle fibers provide a preparation of skeletal muscle with little sarcotubular system. Since the sarcoplasmic reticulum is not destroyed and the sarcolemma and myofilaments are intact in this preparation, of the properties of the sarcolemma may thus be separated from those of the tubular system.

Isolated perfused rabbit hearts have been used to determine the reflection coefficients, σ, of the heart capillaries to certain lipoid-insoluble substances. This was done by initially perfusing the heart with a Ringer solution containing no test molecule and then suddenly switching to a solution which differed from the original only by containing a small amount of test substance. This produced a loss of weight of the heart which was continuously recorded as a function of time. Taking the "zero" time rate of weight change and using an equation given by Kedem and Katchalsky reflection coefficients for urea, sucrose, raffinose, and inulin were obtained. These turned out to be 0.1, 0.3, 0.38, and 0.69 respectively. Using the approach of Durbin and Solomon equivalent pore radii were estimated to be about 35 Angstroms.

Summary. Action potentials were recorded inside and outside isolated frog muscle fibres in Ringer's solution and in moist air in order to correlate the different potentials and to study volume conduction The extracellular action potential recorded from isolated frog muscle fibres in Ringer's solution was diphasic with a first positive and a second negative phase. The duration of the potential measured at a distance of 30 μ from the surface of the fibre was 2.67 ± 0.14 msec (21.2° C). It increased slightly with increasing fibre circumference from 2.5 msec in a fibre of 150 μ circumference to 3.2 msec in a fibre of 350 μ, circumference. The amplitude increased proportionally to the square of the fibre circumference. The peak to peak amplitude was 0.8 to 2.9 m V 30 μ from the surface of the fibre. Near the fibre the amplitude decreased linearly with the logarithm of the distance from the axis of the fibre. At distances (d) exceeding 0.15 mm from the axis of the fibre the amplitude decreased as d-1,3. The peak of the intracellularly measured potential coincided with the negative peak of the extracellular potential with the fibre in Ringer's solution. This indicates that the extracellular action potential can be accounted for by means of the second derivative of the intracellular potential. A third positive phase of the volume conducted potential which is present in nerve disappears within the noise level for the muscle fibre in Ringer's solution because of the more protracted course of repolarization. The spread of the action potential along the fibre determined from the intracellularly recorded action potential was 8.8 ±0.9 mm (conduction velocity × duration of the potential from 50 per cent rise to 50 per cent fall). From the action potentials recorded at different distances from the fibre equipotential lines of the travelling potential field were constructed. By means of this field the charge passing the membrane during depolarization was determined to 1.4× 10-6 coulomb per cm2. The surface action potential from a fibre surrounded by a moist gas mixture had an amplitude of the same order of magnitude as the intracellularly measured action potential with the fibre surrounded by Ringer's solution. The time for rise from 20 to 100 per cent of crest was about half the rise time of the intracellularly recorded potential. The duration of the reconstructed monophasic potential from 50 per cent rise to 50 per cent full was slightly shorter than the intracellular action potential. The conduction velocity of the potential with the fibre in air was half that measured in Ringer's solution (2.41 ± 0.12 m per sec in Ringer's solution; 1.05 ± 0.05 m per sec in air at 21° C). From this difference in propagation velocity the impedance of the Ringer's film surrounding the fibre in air was estimated to 12 megohms per cm. The thickness of the Ringer's layer was about two μ.

The interaction between caffeine and calcium on the rate of desensitization of muscle postjunctional membrane (PJM) receptors during the sustained application of 0.27 mM carbamylcholine (CARB) has been studied in vitro on the sartorius muscle of the frog. The rate of PJM repolarization with CARB added to the solution bathing the muscle or the recovery of the effective transmembrane resistance (EMR) during the microperfusion of CARB directly onto the end-plate region of individual fibers was used as an index of the rate of desensitization. Caffeine (1.5 mM) increased the rate of PJM repolarization with bulk application of CARB in a 1.8 or 10 mM calcium Ringer solution but had no effect on PJM repolarization in a calcium-deficient, 4 mM magnesium Ringer solution. For EMR measurements the preparation was rendered mechanically quiescent by repeated challenges with isotonic KCl during an exposure of several hours to a calcium-free, 4 mM magnesium-1 mM EGTA Ringer solution. In these fibers, the microperfusion of 0.27 mM CARB together with 1.8 mM calcium plus 1.5 mM caffeine significantly increased the rate of EMR recovery above that observed in the absence of caffeine. Raising the calcium concentration to 10 mM had a similar effect; however, no additional increase was noted by the inclusion of 1.5 mM caffeine. It is suggested that the major role of caffeine in PJM desensitization is to increase the calcium permeability of the surface membrane. The transmembrane movement of calcium and the consequent intracellular accumulation of calcium is seen as a critical factor in controlling the rate of PJM desensitization.

Ca influx and efflux in single twitch muscle fibers were determined by the movement of 45Ca. The isotope was assayed by counting the center 1 cm of a fiber while it was in nonradioactive Rnger's solution. The average resting influx in 1.0 mM Ca Ringer's was 0.26 pM Ca/cm2. sec for 5 to 20 min influx periods. The average additional influx upon stimulation in 1.0 mM Ca was 0.73 pM Ca/cm2. twitch. The efflux after both resting and stimulated 45Ca influx can be described by a single exponential curve with an average time constant of 125 min. This relationship is an indication of Ca exchange with a single intracellular compartment. This compartment contains an estimated 47% of the total muscle Ca at 1.0 mM Ca. When the Ca in the Ringer was reduced to 0.5 mM Ca, both the resting and stimulated Ca fluxes decreased. When Ca was raised to 1.8 mM, the stimulated influxes increased but the resting influx did not.

Desheathed frog (R. pipiens) sciatic nerves were soaked in solutions that contained ouabain or NaN3, in K-free solutions, or in K- and Ca-free solutions. Then, the nerves were allowed to recover in standard Ringer's solution. At various times during the soaking periods, some nerves were analyzed for Na and K, and estimates were made of the influx and efflux of Na22. While a nerve was soaking in any one of the experimental solutions, the Na22 influx was increased, the Na content was rising, and the K content was falling by an equivalent amount. The rate coefficient for Na22 efflux was reduced by about 25 per cent by 0.05 mM ouabain and by about 50 per cent by 5 mM NaN3. Potassium-free solutions had little effect on the rate coefficient. It was concluded that the efflux of Na from frog nerve is dependent on the metabolism, but not on the external concentration of K. The equimolar exchange that is characteristic of the net movements of Na and K in frog nerve may not be due to the presence of a tightly coupled Na-K exchange pump, but may represent a constraint imposed by the requirement for electroneutrality.

Sodium transport and oxygen consumption were studied simultaneously in the short-circuited frog skin. Sodium transport was evaluated from I(o)/F, where I(o) is the short-circuit current measured with standard Ringer's solution bathing each surface and F is the Faraday constant. Oxygen tension was measured polarographically. Under a variety of circumstances the rate of oxygen consumption from the outer solution exceeded that from the inner solution, the ratio being constant (0.57 +/- 0.09 SD). Both I(o) and the associated rate of oxygen consumption J(ro) declined nonlinearly with time, but the relationship between them was linear, suggesting that the basal oxygen consumption was constant. For each skin numerous experimental points were fitted by the best straight line. The intercept (J(ro))(Io=0) then gave the basal oxygen consumption, and the slope dNa/dO(2) gave an apparent stoichiometric ratio for a given skin. The basal oxygen consumption was about one-half the total oxygen consumption in a representative untreated short-circuited skin. Values of dNa/dO(2) in 10 skins varied significantly, ranging from 7.1 to 30.9 (as compared with Zerahn's and Leaf and Renshaw's values of about 18). KCN abolished both I(o) and J(ro). 2,4-dinitrophenol (DNP) depressed I(o) while increasing J(ro) four- to fivefold. Anti-diuretic hormone stimulated and ouabain depressed both I(o) and J(ro); in both cases apparent stoichiometric ratios were preserved.

1. Experiments with perfused frog muscles and with isolated frog muscles immersed in Ringer's solution have failed to show any effect of curare in liberating potassium from muscle tissue. This makes it difficult to suppose that the paralytic effect of curare can be attributed to cation exchange between curare and K whereby a labile potassium compound needed for stimulation is removed from the neuromuscular junction. 2. Similar negative results were obtained with dihydro-ß-erythroidine and myanesin. 3. A small liberation of K from perfused muscle does result from treatment with acetylcholine. This is probably due to the contracture of the muscle since the effect is largely eliminated by previous treatment of the muscle with curare. The amount of potassium lost in this way from perfused muscles is too small to detect when muscles are analyzed after immersion in Ringer's solution with and without acetylcholine. It is concluded that there is no significant cation exchange between acetylcholine and K in muscle, but only a small loss of K due to the contracture produced by the acetylcholine.

1. The virulence of vaccine virus for the testicular tissues increases until its maximum is finally reached. The selective increase is not associated with any loss, reduction, or modification in its virulence for the skin. A highly potent testicular vaccine is also highly active for the skin. 2. The testicular strain of vaccine virus has no more tendency to localize in various organs than the ordinary skin strain. Both may localize in adjacent lymph nodes when introduced intravenously, subcutaneously, or intratesticularly in sufficiently large quantities, but other organs are not involved. 3. Intravenous inoculation of an excessive amount of a powerful vaccine virus (1 to 2 cc. of undiluted stock emulsion), irrespective of whether it is from the testis or the skin, will result in a generalized eruption over the entire body surface of rabbits. The eruption may be confluent on mucous membranes of the mouth, nostrils, genitalia, etc. Intratesticular or subcutaneous inoculations of the same virus fail to produce this effect. 4. Subcutaneous or intravenous introduction of much smaller quantities of the virus does not cause an appreciable local or general reaction in the rabbit. But the animals which have once received these injections become refractory to a subsequent vaccination as applied to the skin. It seems probable that an active immunity has been conferred. 5. Experiments on the viability and resistance of the testicular strain of vaccine virus indicate that the virus is best preserved when emulsified with Ringer's solution or 0.9 per cent saline solution. Distilled water, while apparently one of the best diluents, fails to keep the virus active as long as Ringer's or saline solutions. As would be expected, the lower the temperature is, the longer the virus retains its viability. At 18° or 37°C., the deterioration of the virus proceeds rapidly. However, a small part of the virus survives after many weeks' standing at 37°C. 6. Of the two most commonly employed chemical agents for the ripening (eliminating bacteria) process of the green vaccine pulp, glycerol and phenol, the latter is the less injurious. Phenol in concentration above 2 per cent destroys the virus within 24 hours at any temperature, but it has almost no injurious effect when used in 0.5 to 1 per cent. On the other hand, glycerol is a powerful vaccinicide. When used in full strength it destroys the virus within 24 hours, even at 4°C. In a concentration of 40 per cent, that ordinarily recommended for the ripening, the virus retains some of its virulence for about half a year at 4°C., while at higher temperatures the same concentration kills the virus within 1 to 2 months. The virus preserved in distilled water or Ringer's solution under similar temperature conditions remains more active during this period. From this it may be concluded that glycerol is not an indifferent agent, as is assumed by many, but a powerful vaccinicide when used in high concentrations. The injurious effect is markedly accelerated at 18° or 37°C. 7. The vaccine virus retains its virulence better in a sealed tube containing either hydrogen, nitrogen, or air than in an open receptacle. The virus deteriorates when placed in a sealed tube with oxygen or carbon dioxide. 8. Desiccation decreases to a considerable degree the virulence of the vaccine virus. In the dried state the virus retains its viability about as long as does the emulsion, but it is not protected from the deterioration caused by age under various conditions. 9. Iodine is a powerful disinfectant for the vaccine virus, but its sodium and potassium salts have no effect. Various bile salts destroy the vaccine virus when employed in sufficient concentration.

SUMMARY Two groups of patients undergoing partial gastrectomy in light halothanenitrous oxide anaesthesia with muscle relaxant and controlled ventilation were studied. One group (R group, 14 patients) received an average of 1700 ml of lactated Ringer's solution during surgery plus 1500 ml glucose/water on the day of operation. The other group (Gl group, 13 patients) received only 2000 ml glucose in water on the day of operation. The R group had a larger urinary output on the day of operation and on the first and second postoperative days. Endogenous creatinine clearance was within the normal range on the day of operation. Haematocrit decreased as expected from the operative blood loss, and there was not more retention of salt and water than expected to replace blood loss. The urinary K/NA ratio increased only slightly, indicating a small increase in aldosterone activity. Serum sodium and potassium decreased less in this group than in the Gl group. The Gl group suffered a fluid deficit on the day of operation as shown by haemoconcentration, negative fluid balance and a small urinary output. The low endogenous creatinine clearance indicates that the output on the day of operation was not large enough for normal renal clearance of metabolic waste products. This group excreted minimal amounts of NaCl in the urine postoperatively and showed “intolerance” to salt when this was given on the second postoperative day. The urinary K/Na ratio increased markedly, indicating an activation of the renin-angiotension-aldosterone system by uncorrected losses of blood and extracellular fluid during surgery. These findings are discussed in relation to recent reports on acute changes of functional extracellular fluid volume during surgery, the feasibility of replacing moderate blood losses with a balanced salt solution alone, and the pathogenesis and prophylaxis of postoperative renal failure. It is concluded that the administration of approximately 500 ml of lactated Ringer's solution per hour of operating time is a definite advantage. ZUSAMMENFASSUNG UND SCHLUSSFOLGERUNGEN Es wurde eine Vergleichsuntersuchung an zwei Gruppen von Patienten mit Magen-Teilresektionen in oberflachlicher Halothan-N20-Narkose mit Muskelrelaxans und kontrollierter Beatmung angestellt. Die eine Gruppe (Gruppe R, 14 Patienten) erhielt durchschnittlich 1700 ml Ringer-Laktat-Losung wahrend der Operation und 1500 ml Glukose in Wasser am Operationstag. Die andere Gruppe (Gruppe Gl, 13 Patienten) erhielt nur 2000 ml Glukose in Wasser am Operationstag. Die Gruppe R hatte sowohl am Operationstag als auch an den ersten beiden postoperativen Tagen eine grossere Harnausscheidung. Die endogene Kreatinin-Clearance blieb am Operationstag im normalen Bereich. Der Haematokrit fiel erwartungsgemass entsprechend dem operativen Blutverlust und die Salz- und Wasserretention war nicht starker als sie zum Ausgleich des Blutverlustes zu erwarten war. Der nur schwach erhohte K-Na-Quotient im Harn deutete auf einen geringen Anstieg der Aldosteronaktivitat hin. Serum -Na und -K waren in dieser Gruppe weniger vermindert als in der Gl-Gruppe. Die Gl-Gruppe litt am Operationstag an einem Flussigkeitsdefizit, wie sich in der Haemokonzentration, der negativen Flussigkeitsbilanz und der geringen Harnausscheidung zeigte. Die niedrige endogene Kreatinin-Clearance deutete darauf hin, dass die Ausscheidung am Operationstag nicht ausgiebig genug war, urn eine normale Nieren-Clearance der metabolischen Abfallstoffe zu gewahrleisten. Diese Gruppe schied postoperativ nur geringe Kochsalzmengen im Harn aus und zeigte eine «Salz-Intoleranz», wenn dieses am zweiten postoperativen Tag verabreicht wurde. Der K-Na-Quotient im Harn war deutlich erhoht, was auf eine Aktivierung des Renin-Angiotensin-Aldosteron-Systems durch die unkorrigierten Blut- und Extrazellularflussigkeitsverluste wahrend der Operation hinwies. Diese Befunde werden unter Hinweis auf neuere Literaturberichte uber akute Veranderungen des funktionellen EZF-Volumens wahrend Operationen, der Zulassigkeit des Ersatzes massiger Blutverluste durch ausgewogene Salzlosungen allein, sowie der Pathogenese und Prophylaxe des postoperativen Nierenversagens diskutiert. Dabei kommt der Autor zu dem Schluss, dass die Verabreichung von etwa 500 ml Ringer-Laktat-Losung pro Operationssrunde ein ausgesprochener Vorteil ist.

If a frog's sartorius muscle is soaked in a modified Ringer's solution, containing bromide, nitrate or iodide in place of chloride the tension and duration of a twitch (Kahn and Sandow) and its heat production (Hill and Macpherson) are largely increased in the order Br

Procaine, cocaine, pyribenzamine, antistine, and phosphate decrease the uptake of water by frog sartorii in a Ringer solution in which potassium has been substituted for sodium; all but the last two leave the swelling in hypotonic Ringer practically unaltered. They also decrease the depolarizing action of potassium. These effects are considered indicative of reduced membrane permeability to potassium.

1. Adrenalin 1­10−5 bis 1·10−8 kann am isolierten Froschherzen den Kalziummangel vollstandig ausgleichen. 2. Physostigmin erhoht am normal ernahrten isolierten Froschherzen die Hubhohe nur wenig, Frequenzabnahme ist niemals zu beobachten. Der absolute und relative. Kalziummangel kann zum Teil ersetzt werden. 3. Die Wirkung von Prostigmin ist verglichen mit der des Physostigmins gering und tritt sehr langsam ein; die Herzfrequenz wird gesteigert. 4. Tannin 1·10−6 vermag den Kalziummangel nur teilweise zu ersetzen. 5. Niedere Alkoholkonzentrationen von 0,01–0,05% verbessern sowohl am normal als auch am kalziumarm ernahrten Froschlerzen die Hubhohe. 0,1–0,5% Alkohol wirken schon lahmend. Alkoholkonzentrationen von 1–5% lahmen das normal, das kalziumarm und das uberreichlich mit Kalzium ernahrte Froschherz und zwar um so mehr, je starker der Kalziumgehalt von der Konzentration von 0,03% CaCl2 nach der einen oder anderen Seite abweicht.

After a 20 min initial washout, the rate of loss of radioactively labeled sodium ions from sodium-enriched muscle cells is sensitive to the external sodium and potassium ion concentrations. In the absence of external potassium ions, the presence of external sodium ions increases the sodium efflux. In the presence of external potassium ions, the presence of external sodium ions decreases the sodium efflux. In the absence of external potassium ions about one-third of the Na+ efflux that depends upon the external sodium ion concentration can be abolished by 10-5 M glycoside. The glycoside-insensitive but external sodium-dependent Na+ efflux is uninfluenced by external potassium ions. In the absence of both external sodium and potassium ions the sodium efflux is relatively insensitive to the presence of 10-5 M glycoside. The maximal external sodium-dependent sodium efflux in the absence of external potassium ions is about 20% of the magnitude of the maximal potassium-dependent sodium efflux. The magnitude of the glycoside-sensitive sodium efflux in K-free Ringer solution is less than 10% of that observed when sodium efflux is maximally activated by potassium ions. The inhibition of the potassium-activated sodium efflux by external sodium ions is of the competitive type. Reducing the external sodium ion concentration displaces the plots of sodium extrusion rate vs. [K]o to the left and upwards.

Isolated frog skin was bathed with a dilute solution containing 1 mm NaCl on the outside and with normal Ringer’s solution on the inner surface. Net Na flux was determined by simultaneous measurement of unidirectional fluxes with Na22 and Na24 and intracellular electrical potentials were examined with microelectrodes. There was a net inward transport of Na under both open-circuit and short-circuit conditions. The short-circuit current was approximately 15% greater than the net Na flux; the discrepancy could be accounted for by a small outward flux of Cl. The electrical potential profile did not differ greatly from that observed in skins bathed on the outside with normal Ringer’s solution. Under open-circuit conditions, there were usually several potential steps and under short-circuit conditions the cells were negative relative to the bathing solutions. Estimates of epithelial Na concentrations utilizing radioactive Na suggested that if all epithelial Na were in a single compartment, an active entry step would be necessary to allow a net inward Na transport. The results could also be explained by a series arrangement of Na compartments without necessarily postulating an active Na entry. The behavior of the potential profile suggested that this latter alternative was more likely.

In order to clarify whether or not the electronegative olfactory mucosal potentials (EOG) are generator potentials, the effects of changed ionic enviroment were studied. The EOG decreased in amplitude and in some cases nearly or completely disappeared, when Na(+) in the bathing Ringer solution was replaced by sucrose, Li(+), choline(+), tetraethylammonium(+) (TEA), or hydrazine. In the K(+)-free Ringer solution, the negative EOG's initially increased and then decreased in amplitude. In Ringer's solution with increased K(+), the negative EOG's increased in amplitude. When K(+) was increased in exchange for Na(+) in Ringer's solution, the negative EOG's decreased, disappeared, and then reversed their polarity (Fig. 6). Next, when the K(+) was replaced by equimolar sucrose, Li(+), choline(+), TEA(+), hydrazine, or Na(+), the reversed potentials recovered completely only in Na(+)-Ringer's solution, but never in the other solutions. Thus, the essential role of Na(+) and K(+) in the negative EOG's was demonstrated. Ba(++) was found to depress selectively the electropositive EOG, but it hardly decreased and never increased the negative EOG. Hence, it is concluded that Ba(++) interferes only with Cl(-) influx, and that the negative EOG's are elicited by an increase in permeability of the olfactory receptive membrane to Na(+) and K(+), but not to Cl(-). From the ionic mechanism it is inferred that the negative EOG's are in most cases composites of generator and positive potentials.

The influence of changes in ionic composition of the bathing solutions on intracellular electrical potentials in frog skin has been examined. When the skin bathed in SO4 Ringer's solution is penetrated with a microelectrode two approximately equal potential jumps were frequently observed and most experiments were carried out with the electrode located between these steps. Substitution of Cl for SO4 in the bathing solutions caused a decrease in PD across both the "outer" and "inner" barriers. When the skin was short-circuited an average intracellular potential of -18 mv was found with both Cl and SO4 Ringer's. With the skin in SO4 Ringer's, decrease in Na concentration of the outside solution caused a decrease in PD between the microelectrode and the outside solution which was approximately the same as the decrease in total skin PD. With SO4 Ringer's, an increase in K concentration in the inside solution caused a marked decrease in total skin PD. However, only 50 per cent of this change occurred at the inner barrier, between the microelectrode and the inside solution. The remainder of the change occurred at the outer barrier. This observation does not appear to be consistent with the model of the skin proposed by Koefoed-Johnson and Ussing (Acta Physiol. Scand., 1958, 42, 298).

Experiments were performed to test the applicability of permeability kinetics to whole frog sartorius muscle using K42 ions as tracers of potassium flux. The whole muscle was found to obey closely the kinetic laws expected to hold for single cellular units in which the potassium fluxes are membrane-limited and intracellular mixing is rapid enough not to introduce serious error. In a 5 mM K Ringer's solution, potassium efflux was very nearly equal to influx when the rate constant for K42 loss was applied to the whole of the muscle potassium. Over a fairly wide range of external potassium concentration, the assumed unidirectional fluxes measured with tracer K42 showed good agreement with net potassium changes determined analytically. The specific activity of potassium lost from labeled muscles to an initially K-free Ringer's solution was measured as a test of the adequacy of intracellular mixing. The results were those expected for a population of cells with uniformly distributed intracellular K42. A small deviation was encountered which can be attributed either to a dispersion of fiber sizes in the sartorius or to a possible small additional cellular compartment in each individual fiber. The additional cellular compartment, should it exist, contains from 0.5 to 1 per cent of the muscle potassium. This is evidently not large enough to interfere seriously with the applicability of permeability kinetics to the whole muscle.

The mechanical properties, as revealed by minute length changes, of isolated twitch fibers of the frog have been studied at rest and during low-level activation. Resting tension is 77 +/- 23 mN/cm(2) (mean +/- SD) at 2.2 microm sarcomere length.(1) The slope of the tension curve (DeltaP/DeltaL) recorded during a constant-speed length change of a resting fiber is initially large. At length changes exceeding about 0.18 % of the initial length of the fiber DeltaP/DeltaL falls abruptly and remains close to zero during the rest of the length change. The amplitude of the tension response is reduced after a length change and returns to normal in about 3 min. Hypertonic sucrose-Ringer solutions cause a small, maintained rise in tension up to 1.4-1.6 times normal osmotic strength. Higher sucrose concentrations cause relatively large, transient tension responses. The initial DeltaP/DeltaL is increased in moderately hypertonic solutions; it may be reduced in more strongly hypertonic solutions. Elevated [K](o) (range 10-17.5 mM) causes a marked reduction in DeltaP/DeltaL. In this range of [K](o) the reduction is not accompanied by changes in resting tension. Addition of 1-1.5 mM caffeine to the Ringer solution affects the resting tension very little but also reduces DeltaP/DeltaL. The results suggest that stiffness and tension development are not related in a simple way.