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1. A study has been made of the response of the Na efflux to injected guanosine triphosphate (GTP) in barnacle muscle fibres pre-exposed to aldosterone. 2. (i) Injection of GTP into unpoisoned fibres causes a transitory stimulation. By contrast, injection of GTP into ouabain-poisoned fibres causes a sustained stimulation. Little or no fall in Em is recorded following GTP injection. (ii) The stimulatory response to GTP of aldosterone pre-exposed ouabain-poisoned fibres differs from that seen in unexposed, ouabain-poisoned fibres, in that its magnitude is greater and always sustained. 3. The magnitude of the response to GTP depends on external Ca and pH but not external Na. The response itself is not seen at 0 degrees C. 4. (i) Verapamil reduces the size of the response to GTP only if applied before GTP. Injection of EGTA, Fe, Zn and Co partially abolishes the residual response. (ii) Injection of MgCl2 almost completely reverses the response to GTP. KCl is ineffective. 5. These results are explained by supposing that removal of internal Mg and trace elements by GTP leads to activation of phosphoprotein phosphatase.

1. External application of 10(-4) M digitoxigenin caused an 80% fall in the Na efflux in barnacle muscle fibres. The observed inhibition was irreversible and could be produced by as little as 10(-7) M digitoxigenin. 2. Internal application of 10(-2) M digitoxigenin lowered the Na efflux by 46% but was without effect following addition of external ouabain or K removal. 3. External application of digitoxigenin (or ouabain) following internal application of the aglycone was without effect on the Na efflux. 4. The efflux of [3H]digitoxigenin was fairly rapid, whereas that of [3H]ouabain was slow. 5. Inhibition of the Na efflux by internal application of digitoxigenin is probably the result of an action of the aglycone on the external side of the membrane following its leakage from the fibre interior.

1. Single muscle fibres from the barnacle Balanus nubilus have been studied to provide information about the mode of action of aldosterone on Na transport in a symmetric cell. 2. Neither internal nor external application of aldosterone modified the course of the Na efflux. However, fibres pre-exposed overnight to a dose of aldosterone showed a biphasic response to external aldosterone. The first phase was prompt in onset and consisted of a falling rate constant for Na efflux becoming a constant. This has been interpreted as indicating that aldosterone acts by releasing sequestered or bound Na. The second phase was delayed in onset with the average latent period being 68 min. It consisted of a step-up in the rate of Na efflux, followed by a transitory rise in the efflux rate constant. This second phase was dose-dependent, the minimum concentration being 10(-9) M. 3. Internal application of aldosterone in a concentration as low as 10(-10) M promptly stopped the rate constant for Na efflux from further falling but failed to cause delayed stimulation. 4. The response of the Na efflux into Li-ASW following external application of aldosterone was bi-phasic. The magnitude of the delayed stimulation was comparable to that found in controls. 5. No correlation between latent period or size of the internal Na bound fraction, and the magnitude of the delayed stimulation could be established. 6. Internal application of actinomycin-D, alpha-amanitin or cordycepin shortly before application of external aldosterone caused complete abolition of the delayed stimulation. 7. Overnight pre-exposure of the barnacle to actinomycin-D caused complete abolition of the falling rate constant for Na efflux, as well as the delayed stimulation caused by external aldosterone. 8. Internal application of spironolactone SC-14266 shortly before external application of aldosterone caused complete abolition of the biphasic response to the steroid. 9. It is concluded that (i) barnacle fibres can be made sensitive to aldosterone, (ii) the biphasic effects on the Na efflux depend on prior induction of RNA, (iii) the first effect caused by internal or external aldosterone involves mobilization of sequestered Na, (iv) the results obtained with spironolactone are in keeping with the current view that aldosterone interacts with a receptor system before causing de-repression. 10. The implications of the finding that aldosterone releases sequestered Na are briefly touched upon.

1. A study has been made of the nature of the delayed stimulation caused by external aldosterone in barnacle fibres pre-exposed to aldosterone. 2. (i) Microinjection of 0-5 M-ATPMg2- caused only a small but prompt rise in the Na efflux. (ii) Microinjection of 0-5 M-ATPMg2- followed by external application of 10(-5)M aldosterone greatly augmented the magnitude of the delayed stimulation. The response was dose-dependent, as well as dependent on the concentration of external K+ and H+, but not Na+, Ca2+ or Mg2+. (iii) External application of 10(-5) M aldosterone for 30 min followed by its withdrawal from the bathing medium failed to bring about delayed stimulation. By contrast, fibres into which ATP had been injected showed delayed stimulation under these conditions. 3. Microinjection of actinomycin-D or spironolactone SC-14266 into fibres into which ATP had been injected followed by external application of aldosterone resulted in complete abolition of the delayed stimulation. 4. Delayed stimulation was reduced whether ATP had been injected or not by prior external application of 10(-4)M ouabain or internal application of 8 x 10(-2)M ethacrynic acid. It was completely abolished by prior application of ouabain externally and ethacrynic acid internally, or only 10(-4)M diphenylhydantoin externally. 5. (i) Microinjection of atractyloside or bongkrekic acid caused a substantial fall in the resting Na efflux. Bonkrekic acid proved more powerful than atractyloside. Microinjection of 0-05 M-ATPMg2- into fibres poisoned with 2-0 x 10(-2)M bongkrekic acid completely restored the Na efflux.

The efflux of radioactive sodium injected into single muscle fibres from the leg muscles ofMaia squinado(Herbst) has been studied. The efflux is reduced by the sodium-pump-inhibitor ouabain and by removal of the external potassium. An increase in external potassium leads to a transient increase in sodium efflux which is then followed by a decline to a value well below the normal level. Externally applied caffeine and injected calcium can, under certain conditions, cause a lowering of the sodium efflux which may be associated with contraction of the fibre. The injection of lactate and pyruvate did not affect the efflux. The results are discussed in terms of the possible effects of the structure of the fibre on the efflux.

1. A study has been made of the feasibility of monitoring myoplasmic ATPMg in single barnacle muscle fibres by microinjections DuPont luciferase-luciferin. 2. Injection of DuPont luciferase-luciferin into a cannulated fibre results almost immediately in light emission the intensity of which reaches a maximum within 2 sec and then decays in two phases, the first rapidly owing to product inhibition, and the second less rapidly (the resting phase). The greater the peak flash is, the more rapid the rate of decline of the first phase. The entire kinetic picture bears a close resemblance to that observed with a light reaction mixture. 3. (i) Injection of dehydroluciferin before luciferase-luciferin reduces peak flash in a dose-dependent manner. (ii) Injection of 0.5 m-MgSO4 shortly after the onset of peak flash fails to raise the resting level of luminescence. By contrast, injection of 10 mM-EDTA promptly reduces resting luminescence. Injection of 10 mM-EGTA increases resting luminescence transitorily. (iii) Injection of NaCl and KCl in graded amounts reduces resting luminescence in a dose-dependent manner. 4. Reducing (or increasing) the pH of the external medium (10 mM-HCO3-ASW) shortly before injecting luciferase-luciferin results in a reduction in peak flash. The pHe optimum for light emission lies in the region of 7.8. 5. Injection of pyrophosphate (PPi) shortly after luciferase-luciferin causes a dual effect: PPi in low concentration stimulates light output, whereas PPi in high concentration reduces light output. Injection of PPi shortly before luciferase-luciferin results in a reduced peak flash. 6. Injection of CoA shortly after luciferase-luciferin increases resting luminescence in a dose-dependent manner. Both the growth and decay phases of light emission following the injection of CoA differ from the characteristic phases observed following the injection of luciferase-luciferin. Additionally, CoA causes a sustained increase in resting luminescence, but fails to modify the kinetic picture if injected prior to luciferase-luciferin. 7. Injection of ATPMg shortly after old DuPont luciferase-luciferin leads to a characteristic growth and decay curve which is indistinguishable from that caused by luciferase-luciferin. However, this is not true if ATPMg is injected after new DuPont luciferase-luciferin. The addition of 10(-4) M-ATPMg to the myoplasm produces a peak flash comparable to that produced by injecting old DuPont luciferase-luciferin. Application of the non-competitive inhibition equation fails to provide a reasonable estimate of myoplasmic ATPMg, presumably because it is difficult to arrive at a reliable Km value for ATPMg. Injection of new DuPont luciferase-luciferin twice in succession leads to identical flash heights. 8. Calibration performed with a solution containing 120 mM-K glutamate permits an estimate to be made of myoplasmic ATPMg if fibres are injected with luciferase-luciferin so that flash height is known beforehand. This approach leads to a myoplasmic ATPMg value of 1.2 +/- 0.1 mM (S.E...

1. A study has been made of the mechanism by which injected disodium GTP stimulates the ouabain-insensitive Na efflux in single muscle fibres from the barnacle, Balanus nubilus. 2. Injection of GTPNa2 causes a stimulatory response which is usually transitory and almost completely reversed by injecting MgCl2 (but not KCl). 3. Injected 5'-guanylylimidodiphosphate, Gpp(NH)p, mimics this action of GTP but the reversal seen with injected Mg2+ is less pronounced. 4. (i) Pre-treatment of these fibres with verapamil reduces the size of the stimulatory response to GTP and Gpp(NH)p. (ii) Pre-injection of protein kinase inhibitor (PKI) or regulatory subunits reduces the response as well. (iii) Pre-treatment with imipramine or trifluoperazine reduces the response to injected GTP; in combination with verapamil, a greater reduction in response is seen. 5. Injection of EDTA leads to a stimulatory response which is transitory. This response is largely abolished by verapamil. 6. Injection of cholera toxin leads to a sustained stimulatory rather than a transitory response. GTP or Gpp(NH)p when injected following peak stimulation by cholera toxin leads to a moderate sustained stimulation. 7. These results support the view that the stimulatory response to injected GTPNa2 is the result of activation of Ca2+ channels and of increased availability of GTPMg and that these two conditions bring about activation of adenylate cyclase and hence activation of cyclic AMP-protein kinase by newly formed cyclic AMP.

1. The ouabain-insensitive Na efflux in barnacle muscle fibres is promptly stimulated by injection of cyclic GMP. The minimal effective injected concentration is found to be about 10(-7) M. This effect of cyclic GMP could not be mimicked by injecting 5'-GMP. 2. External application of ouabain (10(-4) M) to fibres not pretreated with ouabain during the stimulatory response to cyclic GMP causes some inhibition of the Na efflux indicating that cyclic GMP does not cause appreciable inhibition of the Na:K pump. 3. The magnitude of the stimulatory response to injected cyclic GMP depends on the external Ca2+ concentration, as well as pHe but not on the Na+, K+ or Mg2+ concentration. It also depends on pHi, since acidification of HCO3-containing ASW leads to a greater enhancement of the response to cyclic GMP than is observed with acidified HERPES-ASW. 4. Stabilization of myoplasmic pCa by injecting 100 mM-EGTA before or after cyclic GMP fails to alter the magnitude of the response to the nucleotide. Enrichment of the fibre with Mg2+ at the time of injection of cyclic GMP leads to a reduced response. No change in response, however, is seen when the internal free Mg concentration is suddenly reduced by injecting 0.05 M-pyrophosphate with cyclic GMP. 5. Injection of cyclic GMP-dependent protein kinase stimulatory modulator before cyclic GMP fails to enhance the response to the nucleotide. The same is true of the phosphodiesterase inhibitor protein. However pre-injection of 10(-2) M-papaverine enhances the response to a subsequent injection of 10(-3) M-cyclic GMP. 6. Injection of pure protein kinase inhibitor (1.6 x 10(-4) M) before 10(-3) M-cyclic GMP reduces the response to the nucleotide. 7. The argument is put forward that injected cyclic GMP stimulates the ouabain-insensitive Na efflux mainly by activating cyclic AMP-protein kinase rather than cyclic GMP-proton kinase.

1. A study has been made of the behaviour of the radiochloride efflux in single muscle fibres from the barnacle, Balanus nubilus. 2. In the majority of the fibres studied, the fractional rate constant for 36Cl efflux is a constant and unaffected by the injection of distilled water (approximately 0 . 3 microliter. in volume). 3. Acidification of the HCO3(-)-containing medium causes stimulation of the Cl efflux, the threshold value being pH 7 . 0. The magnitude of the response is a logarithmic function of the external H+ and HCO3 concentration over a wide concentration range. 4. (i) Total replacement of the external Cl and NO3 fails to alter the course of the Cl efflux. However, the magnitude of the response to acidification is reduced to a marked degree. (ii) Replacement of the external Na by Li reduces not only the Cl efflux but also the size of the response to acidification. 5. Injection of HCl, HCO3 or KCl fails to alter the Cl efflux. Injection, however, of 4 M-KCl or NaCl causes a fall in the efflux. 6. 10( 4)M-ouabain is ineffective. It also fails to alter the response of the Cl efflux to acidification. 7. (i) Injection of cyclic AMP stimulates the Cl efflux in a dose-dependent manner, but only transitorily. (ii) Preinjection of pure protein kinase inhibitor causes a marked reduction in the magnitude of the response to cyclic AMP. 8. Preinjection of pure protein kinase inhibitor fails to affect the response to external acidification. 9. (i) Pretreatment externally with ethacrynic acid reduces the response to external acidification. (ii) External application of 4-acetoamineo-4'-isothiocyano-2,2'-stilbene disulphonate (SITS) reduces the resting Cl efflux. It also abolishes completely the response to acidification. (iii) The effect of 4,4'-diisothiocyano-2,2'-stilbene disulphonate (DIDS) resembles that of SITS. (iv) Injection of H2DIDS fails to reduce the resting efflux but tends to reduce the magnitude of the response to acidification. 10. (i) 5 X 10(-4) M-benzolamide is without effect on the basal Cl efflux. (ii) Benzolamide in high concentration reduces the magnitude of the response to acidification. This occurs within a rather narrow concentration range. 11. (i) A sudden reduction in environmental temperature from 24 to 0 degrees C causes a marked fall in the Cl efflux. (ii) Acidification of the artificial sea water at 0 degrees C stimulates the efflux. 12. The present experiments have led to evidence which is consistent with the view that the Cl efflux is modulated by at least two distinct mechanisms: one is responsive to acidification when HCO3 as buffer is present and involves participation of a benzolamide-sensitive component presumably lying in the fibre membrane. The other is responsive to injection of cyclic AMP, and, and probably involves cyclic AMP-protein kinase.

Die infolge der verzogerten Wirkung von Ethacrynsaure nur naherungsweise erfassbare Hemmung der Abgabe von Na+ durch Oozyten der Krote (Bufo bufo) lasst sich exakter bestimmen, wenn die chemische mit einer Temperaturbehandlung kombiniert wird.

In Muskelfasern der Entenmuschel (Balanus nubilus undBalanus aquila) betragt der Natrium Diffusions-Koeffizient 2,2×10−6 cm2/sec.

1. A study has been made of sodium efflux from single muscle fibres of the crab Maia squinado, and of the effect of inhibitors and uncouplers on this efflux, using the microsyringe technique. 2. (i) Externally applied ethacrynic acid or injected ethacrynic acid produces 50% inhibition. The inhibitory state due to externally applied ethacrynic acid is shown to be sensitive to 40 mM-K+ in the bathing medium. (ii) Internally applied chlorpromazine causes 40% inhibition. (iii) Internally applied ethyl alcohol causes a slight increase or a slight decrease in Na efflux or no effect. (iv) Internally applied antimycin A produces a net inhibitory effect of almost 50%. (v) Internally applied rotenone produces a 60% inhibitory effect while fibres that fail to contract initially show about a 90% fall in efflux. (vi) The response pattern to internally applied oligomycin is similar to that of antimycin but the net inhibitory effect is only 25%. (vii) Internally applied atractylate produces 45% inhibition. (viii) Internally applied gramicidin A produces about 40% inhibition. (ix) Internally applied valinomycin is without an effect, but in the presence of 40 mM-K+ in the external medium there is marked inhibition of Na efflux. 3. The evidence put forward suggests that the Na pump of the Maia fibre is divided into two parts, one the Na+-K+-ATPase, driven by ATP and arginine phosphate (ArP), and the other, an electrogenic mechanism, driven by respiration and possibly direct coupling of glycolytic energy. The process of reverse electron flow can be invoked as a reasonable explanation of some of the observations made.

Die Befunde ergeben, dass einzelne Muskelfasern der Entenmuschel durch Mikroinjektion mit45Ca aufgeladen werden konnen. Eine Umspulung der Muskelfasern mit einer coffeinhaltigen Losung bewirkt ein starkeres Ausstromen des Calciums als eine Mikroinjektion des Coffeins in die Fasern direkt.

Nachweis, dass Coffein zunachst den Auswartstrom von Na-Ionen aus Einzelfasern der Bernakelmuskeln verringert, ihn hernach aber zu steigern vermag.

Nachweis, dass der Auswartsstrom von Natrium aus Muskelfasern der ArthropodenBalanus nubilus undBalanus aquila in kunstlichem, kaliumfreiem Meerwasser durch Mikroinjektion von Kalziumchlorid aktiviert wird.

Es werden Analysenresultate uber den Ionengehalt von einzelnen Muskelfasern der Entenmuschel,Balanus nubilus, mitgeteilt. Es wurden Unterschiede nachgewiesen in der Ionenkonzentration verschiedener Fasern, die von demselben Muskelbundel isoliert worden waren.

Die dargestellten Experimente zeigen, dass die Methode, welche durch Microinjection die Muskelfasern von Barnakel mit Na-22 beladt, keinen Schaden bewirkt, was die Efflux-Verhaltnisse fur Na anbelangt; zudem wird mit dieser Methode keine besonders durchlassige Muskelmembran erzeugt.

ONE of the major unsolved problems in clinical nephrolog is the way cellular electrolyte disturbances develop in uraemic patients. The studies of Bergstrom1 have shown abnormalities in the electrolyte content of the skeletal muscle of patients suffering from acute or chronic uraemia. Other lines of evidence suggesting toxic sequelae come from the studies of Kuroyanagi, Kurisu, Sugiyama and Saito2, Morgan and Morgan3, Giovannetti, Balestri and Cioni4, and Welt, Sachs and McManus5, using red blood cells. Welt et al. have found that the activity of the sodium–potassium–ATPase of erythrocytes obtained from uraemic subjects is reduced and the internal sodium concentration of these cells increased. This is the pattern that would be expected to accompany the direct action of toxins on the sodium transport system of erythrocytes.

1. Ethacrynic acid (10−3 M) at pH 7·4 caused approximately 83% inhibition of Na efflux from single dissected toad oocytes. 2. Less inhibition was found at concentrations below 10−3 M but higher concentrations did not cause greater inhibition. 3. At an external pH of 7·0 the onset of inhibition was more rapid than at pH 7·4. 4. Ouabain (10−3 M) produced only 30% inhibition of the Na efflux, and the subsequent application of ethacrynic acid largely inhibited the remaining efflux. 5. Application of ouabain, after ethacrynic acid had produced some 80% inhibition, caused only a further 9% drop in efflux. 6. In uninhibited cells, Na efflux was found to be greater at pH 8·0, and less at pH 7·0 than at 7·4.

An einzelnen Oozyten vonBufo bufo wird gezeigt, dass der Natriumtransport in kaliumfreier Ringerlosung gegenuber einer Zunahme des ausseren pH relativ intensiv ist.

Much study has been devoted to the subject of the pathogenesis of fever (1). VENULET and co-workers (2—5) have investigated the peripherial mechanism of action of lipopolysaccharide isolated from E. Coll and have shown that the pyrogen response in rabbits is accompanied not only by hyperglycemia but by a decrease in blood lactic acid. These workers, moreover, have localized the cellular site of action of pyrogen in that the activity of mitochondrial succinic dehydrogenase in liver cells is increased at the peak of the temperature rise. The succinic dehydrogenase activity was measured by the method of SLATER and BONNER involving the blockage of cytochrome activity by cyanide and the acceptance of H 2 by methylene blue (6). In another series of experiments the response of rabbits to pyrogen was studied in association with the action of Lysergic acid diethylamide (LSD) which is a central stimulator as well as 2,4-Dinitrophenol (DNP) which is a peripheral stimulator (7). The results indicate that the O g consumption of liverslices increases following the use of DNP but the hyperthermic response itself is temporarily abolished in the presence of CQ4-induced liver damage. In the case of LSD, its effect is maintained in spite of liver damage and the O2 consumption remains unchanged. The implication here ist that pyrogen exerts a peripheral effect. — The above data suggests that the pyrogen test might be used to distinguish between the various forms of liver disease (8). For example, it would be predicted that patients with diffuse liver damage such as that occuring in infectious hepatitis would react less intensely to pyrogen than normal humans. With this in mind, we studied the pyrogen response in three groups of humans: Ten human subjects free of liver disease, eleven patients with infectious hepatitis and eleven patients with cirrhosis. Pyrogen was given intravenously in a dose of one microgramm. The lipopolysaccharide was isolated by the method of PALMER and GERLOUGH (9), as modified by WESTPHAL, LUDERITZ, EICHENBERGER and KEIDERLING (10). Half hourly temperatures were recorded until the temperature began to level off. Blood for liver function tests was drawn before and after the use of pyrogen (bromsulphaleine, SGPT, SCOT, alkaline phosphatase, thymol turbidity, cephalin floculation and bilirubin tests). The table shows the mean temperature rise in the three groups of people studied together with the SGOTand SGPT-values. The mean temperature rise for the normals is 1,45°, for the cirrhotics 1,85° and for the hepatitics 1,03°. — It was noted by us, too, that most of the hepatitics, unlike the other patients, failed to complain of chills, headache und muscle aches. In summary, the pattien with hepatitis responds to E. Coli pyrogen less intensely than normal humans, while the cirrhotic responds in exagerated fashion. Why this is so with the cirrhotic is perhaps a matter of succinic dehydrogenase activity and we plan to measure the activity of this enzyme in liver biopsy specimens before and after pyrogen administration.

Experimente an einzelnen Oozyten vonBufo bufo zeigen, dass Nigericin den Natriumefflux anregt, indem gebundenes Natrium freigesetzt wird.

INSULIN applied in vitro reduces the internal sodium concentration of rat skeletal muscle1–2 and stimulates the active transport of sodium in sodium-loaded frog muscle3 and in toad bladder4. External application of insulin to single crab muscle fibres in vitro can cause a striking rise in the efflux of sodium5. Insulin stimulates protein synthesis in skeletal muscle in the presence of actinomycin D6,7 and, because it has been shown that this inhibitor does not stop the hormone from stimulating the sodium pump5, it is difficult to decide whether the primary action is on the efflux mechanism or induction of de novo protein synthesis. This communication describes the behaviour of another single symmetric cell, the toad oocyte, towards insulin.

Es wird gezeigt, dass die Mikroinjektion von Athacrynsaure [2,3-Dichloro-4(2-methylenbatyryl)-phenoxyacetsaure] in die isolierte Muskelfaser der Entenmuschel nur dann den Natriumausstrom herabsetzt, wenn die Badelosung vorher angesauert wird.