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1. The effects of graded concentrations of caffeine on the Na efflux were investigated. External application of 10 mM caffeine usually caused a biphasic response, viz. a fall, followed by a rise in the Na efflux. 1 and 5 mM caffeine usually caused stimulation. Only the stimulatory phase of this response depended on the presence of external Ca(2+).2. Internal application of 100 mM caffeine caused a small rise in the Na efflux, the magnitude of which was independent of external Ca(2+) and comparable to that obtained with external application of 1 mM caffeine. This action, however, could be greatly augmented by pre-treating the fibre with 5 x 10(-5)M ouabain.3. The rise in Na efflux caused by external application of 10 mM caffeine could be greatly augmented by pre-treating the fibre with 5 x 10(-5)M ouabain. The observed stimulatory response was biphasic, more so in the absence of external Ca(2+). Restoration of external Ca(2+) following the onset of the second stimulatory phase resulted in further rise of the Na efflux. Measurements of the Na efflux during treatment with graded concentrations of ouabain and 10 mM caffeine showed that the rate coefficient for Na efflux varied with the ouabain concentration in the range 10(-8)-10(-4)M. Measurements of the ouabain-insensitive Na efflux before and during treatment with 10 mM caffeine in bathing media containing varying concentrations of Ca, disclosed the existence of two Ca(2+)-thresholds, one in the 0-2.5 mM range and the other in the 12.5-15 mM range.4. Comparisons were made between the effects on the Na efflux of 10 mM caffeine followed by external acidification, and external acidification, followed by 10 mM caffeine. The magnitude of the response of the ouabain-insensitive Na efflux to external acidification before treatment with 10 mM caffeine was greater than that found when external acidification followed external application of the alkaloid. It also was considerably greater than that of the response to external application of 10 mM caffeine before external acidification.5. External application of 10 mM procaine prevented 10 mM caffeine from stimulating the Na efflux, and from inducing contractures. Internal application of 100 mM-EGTA reduced the response of the Na efflux to 10 mM caffeine, and also prevented the fibre from contracting. External application of 10(-4)M diphenylhydantoin reduced the response of the Na efflux to 10 mM caffeine but failed to prevent the development of contractures.6. Internal application of 0.05 M-cGMP, cAMP or its dibutyryl derivative caused a large rise in the Na efflux. The magnitude of the effects observed in ouabain-poisoned fibres was often greater than that in unpoisoned fibres. Internal application of 2.5 units/ml. phosphodiesterase beforehand failed to reduce the magnitude of the stimulatory response to injected cyclic nucleotides. Injected phosphodiesterase also failed to reduce the response of the Na efflux to 10 mM caffeine.7. External application of 10 mM caffeine to unpoisoned and ouabain-poisoned fibres caused a fall of approximately 10 mV in the membrane potential. In unpoisoned fibres this effect was transitory. The response of the membrane potential to internal application of graded concentrations of CaCl(2) was biphasic. When low concentrations of CaCl(2) were used the membrane potential underwent a small rise but when high concentrations were used the opposite was found. These results could not be repeated with graded concentrations of MgCl(2).8. The effects of graded concentrations of caffeine on tension development were also studied. Strong contractures were observed with caffeine concentrations as low as 4 mM, while peak tetanus tension was usually exceeded with 7-8 mM concentrations. The tension-external Ca(2+) curve was sigmoidal in shape.9. Electron microscopic studies showed that 10 mM caffeine in ASW caused little or no distension and disorganization of cisternal fine structure. Such structural changes, however, were far more pronounced in fibres suspended in Ca(2+)-free ASW and then treated with 10 mM caffeine in Ca(2+)-free ASW. Fibres soaked in Ca(2+)-free ASW had ruptured mitochondria and mitoplasts, whereas those additionally treated with 10 mM caffeine had relatively intact mitochondria.10. The main conclusions drawn from this work are: (i) that caffeine stimulates the ouabain-insensitive Na efflux (and inhibits the transport enzyme) by raising the internal free Ca(2+) concentration; (ii) that in the presence of inhibition of the transport enzyme, the magnitude of the stimulatory response to 10 mM caffeine depends not only on the external Ca(2+) concentration but mainly on the residual level of activity of the transport enzyme; (iii) that the Ca(2+)-sensitive and CO(2)-sensitive components of the ouabain-insensitive Na efflux, though not the same, may overlap at the level of the plasma membrane or share a common metabolic step away from the membrane; (iv) that cyclic nucleotides participate in the control of the magnitude of the ouabain-insensitive Na efflux, and that the phosphodiesterase system under the present experimental conditions does not seem to be involved in the mechanism underlying the stimulatory action of caffeine; (v) that the membrane potential changes caused by caffeine fail to explain the stimulatory response of the Na efflux, as well as contracture of these fibres; (vi) that the contractile machinery in these fibres is considerably more sensitive to caffeine than that in vertebrate muscle and the muscles of other arthropods, and (vii) that the Ca(2+) released by the sarcoplasmic reticulum which leads to a biphasic response of the Na efflux is the result of cisternal disorganization caused by caffeine.

Bittar, E. E., S. S. Chen, B. G. Danielson AND E. Y. Tong. An investigation of the action of diphenylhydantoin on sodium efflux in barnacle muscle fibres. Acta physiol. scand. 1973. 89. 30–38. Sodium efflux from barnacle fibres is suppressed by external but not by internal application of diphenylhydantoin (DPH). DPH (10-4 M) at pH 7.8 reduces the Na efflux by 32 %, a value slightly less than half that obtained with 10-4 M ouabain. Ouabain has the ability to abolish the remaining Na efflux, whereas DPH incompletely abolishes the ouabain-insensitive Na efflux. The onset of inhibition by DPH is slower than that by ouabain. At lower external pH the onset of peak action by DPH is more rapid than at pH 7.8. The sensitivity of the Na efflux to DPH is unaffected by external K. However, the residual efflux from fibers poisoned with ouabain following K removal is insensitive to DPH. DPH prevents the Na efflux from responding to external acidification. It also has the power to almost completely reverse the effect of external acidification on the Na efflux. It is provisionally concluded that the T-system is the site of DPH action.

1. The cation composition of single barnacle muscle fibres following damage by axial insertion of a microsyringe has been measured. The Na and Ca contents of these fibres were raised. 2. Electronmicroscopic studies of fibres following insertion of a microsyringe indicated that the damage done resulted in tubular obstruction of the T-system. 3. Fibres loaded with radiosodium by micro-injection showed that the Na* efflux declined exponentially with time, but that in most fibres the slope ratio of d/dt ln [Na*]1 to d/dt (ln d[Na*]i/dt) was less than unity. Injections of distilled water deep in the fibre failed to influence the course of the Na* efflux. 4. K removal reduced the Na efflux by 47%. However, a few fibres displayed very little K-dependence. 5. When measured in fibres already soaked in a K-free medium for long periods the sodium efflux consisted of a brief rapid phase, followed by a slow phase of Na loss. 6. In the presence of 30 m M-K, there was little or no rise in the Na efflux. Raising the external K to 50 or 100 m M caused a marked rise in the Na efflux. Raising the external K to 30 m M in the absence of external Ca2+ led to a rise in the Na efflux. A high K solution always caused shortening of these fibres. 7. Internal application of 1 M or 1 m M-CaCl2 often caused a significant rise in the Na efflux. 8. Internal application of 2·5 or 5 M saline caused a prompt and large fall in the Na efflux. In the presence of high K saline-loaded fibres failed to contract. 9. Internal application of 0·5 M-ATP stimulated the Na efflux. A larger effect was not observed in fibres pre-treated with 2 M-MgCl2. Internal application of 0·5 M-ArP was without effect. 10. The results indicate that the barnacle fibre is a suitable preparation for the study of Na fluxes by means of the micro-injection technique. They also indicate that the mechanism regulating the Na efflux is not quite the same as that found in squid axon or frog muscle.

Attempts were made to characterize the ouabain-insensitive Na efflux in barnacle muscle fibers. Lowering the external pH to 5.8, raising the external K to 30 mM or microinjecting 1 M CaCl 2 led to a marked rise in the Na efflux from fibers previously poisoned with 5×10 −5 M ouabain. External application of ouabain following stimulation of the Na efflux by acidifying the bathing medium was without effect. Internal application of 2 M MgCl 2 following inactivation of the ouabain-sensitive component of the Na efflux was also without effect.

Measurements of radiosodium efflux were carried out on single fibres from the barnacle Balanus nubilus or B. aquila, using the micro-injection technique. 10-4 M ouabain caused an average fall of 68% of the Na efflux, the minimal effective concentration being ca. 10-7 M. Internal application of 4 × 10-3 M ouabain was without effect. Abolition of the ouabain-sensitive component with ouabain, followed by raising the external K concentration to 30 mM, resulted in a marked rise in the residual Na efflux. Stimulation of the ouabain-insensitive Na efflux was also seen after reducing the external pH, as well as after microinjecting CaCl2 but not MgCl2. Uncoupling of the active transport system by K removal, followed by external application of ouabain, and subsequent acidification of the bathing medium, led to stimulation which was considerably greater than that taking place when the Na efflux was uncoupled by K removal and then stimulated by external acidification.

BITTAR, Caldwell and Lowe 1 showed that the efflux of Na from single crab muscle fibers is usually exponential and sensitive to the removal of external K ions. These findings have now been extended to muscle fibers from the barnacle Balanus nubilus , and confirmed. Barnacle fibers are about 3–5 cms long and 1–2 mm wide, hence are easily cannulated and ideal for microinjection experiments. The following communication describes experiments meant to shed some light on the part played by external pH in the Ussing mechanism. The results of this work show quite clearly that Na efflux into K-free artificial sea water is markedly stimulated by a low external pH and that the process of stimulation is reversible.

Acidification of the bathing medium in the absence of external bicarbonate ions was without effect on the Na efflux. By contrast, acidification in the presence of different concentrations of HCO 3 − led to stimulation of the Na efflux. Measurements of the pCO 2 indicated that stimulation of the pump always took place when the pCO 2 reached approximately 40 mm Hg.

Earlier experiments with barnacle muscle fibers had indicated that ouabain is a more powerful inhibitor of the Na pump than diphenylhydantoin (DPH), and that the ouabain-insensitive Na efflux is markedly stimulated by lowering the external pH, raising the external K concentration or by microinjecting CaCl 2 . In the present work the effect of acidification of the bathing medium before and after external application of DPH was investigated. This revealed that (i) the action of DPH is pH-dependent, and (ii) DPH blocks or inactivates the proton-sensitive component of the Na efflux.

Isometric tension measurements carried out on uncannulated, intact barnacle muscle fibers show that the minimal concentration of caffeine which produces contracture is 2mM and that which produces maximal tension is 30mM. Pretreatment of barnacle fibers with 3mM-caffeine renders these fibers less sensitive to a second challege e.g. with 30mM-caffeine, the amplitude of the contractile response being time-dependent. Caffeine-treated fibers fail to relax completely. Isotonic tension measurements indicate that the residual tension caused by caffeine is dose-dependent. Significantly, the tension caused by caffeine is independent of the external Ca 2+ .

A DESCRIPTION of the response of the sodium pump of single muscle fibres of the crab Maia squinado to 8-lysine vasopressin1 has shown that the natriferic action of the hormone was governed by the pH differential across the fibre membrane. In an attempt to elucidate further the nature of hormonal actions, these investigations have been extended to include insulin. The results reported here support the view2–4 that insulin when applied externally leads to an increase in the efflux of sodium from skeletal muscle, and they provide evidence that phleomycin, a DNA-polymerase inhibitor, is somehow able, in fibres previously exposed to the hormone in vivo, to prevent the reduction of sodium efflux by injected insulin.

Diphenylhydantoin is shown to reduce the Na efflux in single bernacle muscle fibers. The magnitude of the inhibiting effect is 38%, which is slightly more than half that of ouabain. The results are explained by supposing that the action of diphenylhydantoin on the transport enzyme is incomplete or that its main point of action is the T-system.

External application of 10−4 M ethacrynic acid at pH 7.8 is usually without effect on the Na efflux into a 10 mM HCO3− solution. At an external pH of 6.8, however, 10−3 M ethacrynic acid causes a gradual rise in the loss of Na into a solution the initial HCO3- concentration of which is 5 mM. Lowering the external pH of this solution to 5.8 results in a reversal of the slow rise in Na loss. External application of 10−5 M ethacrynic at pH 6.9 and pH 6.3 is unaccompanied by a rise in the Na efflux into a Ca2+-free solution the initial HCO3− concentration of which is 5 mM. External application of 10−3 M ethacrynic acid following stimulation of the Na efflux into a Ca2+-free solution by reducing the external pH from 7.8 to 6.3 results in a large fall in the rate of loss of Na. These results indicate that the CO2-sensitive Na efflux is the principal site at which ethacrynic acid acts.

Nachweis, dass eine Microinjektion von Troponin-C den Na-Ionen-Ausfluss aus Einzelfasern des Entenmuschel-Muskels steigert.

Es wurde gezeigt, dass die Natrium-Ausscheidung in der Muskelfaser von Entenmuskeln auf aldosteron reagiert.

Nachweis, dass Thymol sowie Coffein in der Barnakel-Muskelfaser eine primare Hemmung mit nachfolgender Stimulierung des Natrium-Auswartsstromes hervorruft.

Few are the cases of hemothorax 1-3 associated with pancreatitis, and fewer still are those documented in the world literature. For this reason the following case is chronicled. Report of Case A Negro male, age 31, was admitted to the District of Columbia General Hospital on May 12, 1960, with the presenting complaint of dyspnea at rest of about 1 week's duration. He had been in good health until some 3 weeks before entry when he noticed left pleuritic pain, dyspnea on exertion, and a cough productive of minimal mucoid sputa. During the next 2 weeks this symptomcomplex progressed, punctuated by dyspnea at rest and inability to retain solid foods. The pleuritic pain seemed to coexist with epigastric and left hypochondrial pain which decreased in severity upon sitting. There was no back pain. The past history was significant in that his alcohol intake was moderate, limited chiefly to beer. The