The effect of ATP on calcium efflux in dialyzed barnacle muscle fibres.

Calcium efflux has been studied in barnacle muscle fibres under internal dialysis conditions. Prolonged dialysis of these fibres, with a medium free of ATP and containing 2 mM cyanide and 1 mM iodoacetate, causes the ATP in the perfusion effluent to fall to less than 20 micrometer. The mean calcium efflux from fibres dialyzed with EGTA buffered solution containing 0.3 micrometer ionized Ca and and no ATP is 0.6 pmol-cm-2-s-1. A two-fold stimulation of the calcium efflux is observed when ATP is added to fibres previously dialyzed with an ATP-free medium. Withdrawal of Na+ and Ca2+ from the external medium causes a marked drop in the Ca2+ efflux in the presence of internal ATP.     

Some aspects of sodium efflux from single barnacle muscle fibres

This mini-review attempts to summarize information about the efflux of 22Na from single barnacle muscle fibres, based on the use of the microinjection technique. The view is put forward that the Na efflux consists of three components: an ouabain-sensitive component, an ouabain-insensitive component (representing secondary active transport), and an Na-Na exchange diffusion component. Evidence is brought forward which supports the view that the ouabain-insensitive Na efflux is divisible operationally into 3 phases: (i) the cyclic nucleotide-sensitive phase, (ii) the Cai-sensitive phase, and (iii) the pHe-sensitive phase. It is shown how the barnacle muscle fibre preparation has yielded information about the validity of the cAMP-protein kinase hypothesis and how it can be used to shed some light on the post-translational mechanism of aldosterone action.     

Stimulation by octopamine of the sodium efflux in barnacle muscle fibres

1. The behaviour of the ouabain-insensitive Na efflux in barnacle muscle fibres towards dl-octopamine has been investigated.2. It is found that these fibres are quite often sensitive to external application of octopamine. A concentration as low as 10⁻⁹ M is effective.3. The kinetic results indicate that the stimulatory response develops within 5–10 min of exposure of the fibre to octopamine and is transitory in nature.4. The response to octopamine is greater in size in the presence of l-isoamyl-3-isobutylxanthine (IAX) than in the presence of l-propyl-3-methyl-7-(5-hydroxyhexyl)-xanthine (PMX). But neither IAX nor PMX stops the response from decaying.5. The response to octopamine is highly dependent on the presence of external Ca²⁺.6. The provisional conclusion is that the barnacle muscle fibre may be a useful preparation for studying the hormonal function of octopamine.     

Chloride fluxes in isolated dialyzed barnacle muscle fibers

Chloride outflux and influx has been studied in single isolated muscle fibers from the giant barnacle under constant internal composition by means of a dialysis perfusion technique. Membrane potential was continually recorded. The chloride outfluxes and influxes were 143 and 144 pmoles/cm²-sec (mean resting potential: 58 mv, temperature: 22°–24°C) with internal and external chloride concentrations of 30 and 541 mM, respectively. The chloride conductance calculated from tracer measurements using constant field assumptions is about fourfold greater than that calculated from published electrical data. Replacing 97% of the external chloride ions by propionate reduces the chloride efflux by 51%. Nitrate ions applied either to the internal or external surface of the membrane slows the chloride efflux. The external pH dependence of the chloride efflux follows the external pH dependence of the membrane conductance, in the range pH 3.9–4.7, increasing with decreasing pH. In the range pH 5–9, the chloride efflux increased with increasing pH, in a manner similar to that observed in frog muscle fibers. The titration curve for internal pH changes in the range 4.0–7.0 was quantitatively much different from that for external pH change, indicating significant asymmetry in the internal and external pH dependence of the chloride efflux.     

45Ca and (14C)EDTA efflux from dialyzed barnacle muscle fibers.

45Ca and 14C-labeled ethylenediamine-N, N'-tetraacetic acid (EDTA) effluxes were measured in internally dialyzed barnacle muscle fibers. In 45Ca experiments the internal ionized 45Ca was fixed at 0.2 muM with ethyleneglycolbis-(beta-aminoethylether)-N, N'-tetraacetic acid(EGTA). The 45Ca efflux was found to increase with internal CaEGTA from 0.05 pmol/cm2.s(CaEGTA equal to 0.02 mM) to 5.0 pmol/cm2.s(CaEGTAequal to 9.6 mM). To determine whether or not most of this increase in efflux was due to the exit of undissociated CaEGTA, comparable experiments were performed with Ca-[14-C]EDTA. Over the same range of internal calcium as studied in the 45Ca experiments, the Ca-[14-C]EDTA efflux was no more than 12% of the 45Ca efflux. We conclude that the exit of undissociated 45Ca cannot account for most of the 45Ca efflux nor can it account for the dependence of 45Ca efflux on internal CaEGTA. The experiments also demonstrated the existence of an endogenous pool of calcium, of 0.43 mmol/kg (about half the total calcium), which remained unexchanged during dialysis.     

Stimulation of cyclic nucleotides of calcium efflux in barnacle muscle fibers.

Internal application of 10⁻⁴, 10⁻⁵, 10⁻⁶ and 10⁻⁷M cGMP and cAMP caused an increase in ⁴⁵Ca efflux in barnacle muscle fibers. Stimulation by either nucleotide occurred in the absence of external calcium and could be prevented by external application of 10 mM procaine or by prior internal treatment of these fibers with EGTA. The results indicate that cyclic nucleotides increase calcium efflux by releasing calcium from internal stores.     

Cyclic AMP-stimulated chloride fluxes in dialyzed barnacle muscle fibers

Unidirectional chloride efflux and influx were studied in giant barnacle muscle fibers that were internally dialyzed. When cyclic 3'5'- adenosine monophosphate (cAMP) was included in the dialysis fluid, both unidirectional fluxes were stimulated by about the same amount. This stimulation was not associated with measurable changes either in membrane electrical conductance or with net movements of chloride. The stimulation required the trans-side presence of chloride. The stimulated flux was inhibited by the sulfonic acid stilbene derivatives 4-acetamido-4'-isothiocyanostilbene-2',2'-disulfonate (SITS) and 4,4'- diisothiocyanostilbene-2,2'-disulfonate (DIDS) or by furosemide. When cAMP was presented in high concentrations (10-5 M), the effect on chloride fluxes was characterized by a desensitization phenomenon. This desensitization was not the result of an increased amount of phosphodiesterase activity, but may be related to ATP and/or intracellular calcium levels. These results further support the hypothesis that the barnacle sarcolemma possesses a specialized chloride transport mechanism that largely engages in Cl-Cl exchange under conditions of normal intracellular pH.     

Sensitivity of the sodium efflux in barnacle muscle fibers to the microinjection of ATP

Sodium efflux in barnacle muscle fibers is promptly stimulated by internal application of ATP. This response is markedly augmented by pretreatment of the barnacle fiber with ouabain. ArP is found to be considerably less effective than ATP. It is suggested that the stimulatory response of the ouabain-insensitive Na efflux to the microinjection of ATP may be due to a significant rise in the sarcoplasmic cAMP concentration caused by the catalytic action of an adenyl cyclase system.     

Some observations on the behaviour of the sodium efflux in barnacle muscle fibres towards lithium ions

• 1.1. The behaviour of the Na efflux towards Li⁺ was studied using single barnacle muscle fibres as a preparation.
• 2.2. It is found that the Na efflux into Li⁺-ASW (artificial seawater) is reduced and that this effect is not fully reversed by returning back to Na⁺-ASW.
• 3.3. Preinjection of 100 mM-EGTA reduces the magnitude of the fall of the Na efflux into Li⁺-ASW.
• 4.4(a). The remaining Na efflux into Li⁺-ASW is further reduced by external application of 10⁻⁴ M-ouabain. (b) The remaining Na efflux in ouabain-poisoned fibres is reduced by replacing Na_e by Li⁺. However, some fibres show a rise rather than a fall.
• 5.5. Fibres loaded with NaCl (by injection) show a prompt and sustained stimulation of the Na efflux when Na_e is replaced by Li⁺. A similar but less pronounced response is often seen with ouabain-poisoned fibres.
• 6.6. Injection of LiCl (e.g. a 2 M-solution), causes a 20% fall in Na efflux. Subsequent replacement of Na_e by Li⁺ fails to bring about a fall in the remaining efflux.
• 7.7. Itis concluded that the Na efflux in these fibres consists of a Na-Na exchange diffusion component which is not mediated by the Na-K pump and that its operation is interrupted by injecting Li⁺. The relative size of this component is about one-fifth and not one-half of the Na efflux.

     

Effect of magnesium on calcium efflux in dialyzed squid axon

The Ca efflux mechanism located in the axolemma of the tropical squid Doritheutis plei is shown to be affected by the concentration of intracellular Mg (Mg_i). The removal of all of the Mg from, the experimental preparation causes an increase in Ca efflux. This effect seems to be more pronounced at low levels of internal ionized calcium and high levels of internal Na.     

ATP as a positive effector of the sodium efflux in single barnacle muscle fibers.

A study has been made of the mechanism by which the injection of ATPNa2 stimulates the ouabain-insensitive Na efflux in fibers from the barnacle, Balanus nubilus. The results of this study are as follows: ATPNa2 is found to be a more potent effector of the Na efflux in unpoisoned fibers than ATPMg on an equimolar basis, but not more potent than ADPNa2. In ouabain-poisoned fibers ATPNa2 and ATPMg are equipotent but the former is more potent than ADPNa2. The magnitude of the response to ATPNa2 injection into ouabain-poisoned fibers depends on: (i) the ouabain concentration used; (ii) the concentration of ATPNa2 injected, and (iii) the external Ca2+ concentration. Ouabain is without effect when it is applied at the time of ATPNa2 injection. Responsiveness to ouabain, however, is found to return if the glycoside is applied after complete decay of the response to ATP. Under these conditions, the effect of ouabain in fibers injected with ATPNa2 is significantly less than in fibers injected with ATPMg. Preinjection of EGTA in high concentrations fails to reduce the size of the response to ATPNa2 injection. Injection of Mg2+ following peak stimulation by ATP almost completely reverses the response. The response to Mg2+ is concentration-dependent. Ryanodine but not neomycin reduces the response to ATP. ATP gamma S is not as effective as ATPNa2. Nor is AMP-PNP consistently as effective as ATPNa2. Collectively, these results support the hypothesis that the response of the Na efflux to ATPNa2 injection involves the operation of the putative Na(+)-Ca2+ exchanger in the reverse mode and that a raised Cai2+ is not an absolute requirement. They also strongly suggest that two other governing factors are the Na+ gradient across the sarcolemma and the myoplasmic pMg. Mg2+ seems to act as an inhibitor.     

Influence of insulin on sodium efflux in barnacle muscle fibers

Summary The efflux of radiosodium in single muscle fibers from the barnacleBalanus nubilus was irresponsive to internal or external application of insulin. However, this was not the case with fibers isolated from a barnacle specimen pre-exposed overnight to a large dose of insulin. External application of insulin to pre-exposed fibers caused a decrease in the rate of decline of the radiosodium efflux and stopped the decline in the fractional rate constant for Na efflux. Such kinetics were interpreted as indicating that insulin acts either by releasing sequestered Na or abolishing the process of sequestration. Internal application of saline slowed the rate of decline but failed to completely abolish the mechanism of sequestration. Only in the presence of insulin was the fractional loss of Na each second constant. Internal application of insulin caused a prompt step-up in the rate of Na efflux, followed by a reduced efflux rate constant. This meant that injected insulin caused the release of sequestered Na, leading to partial saturation of the efflux. The response of the Na efflux to injected denatured insulin, though resembling that to native insulin was much smaller in size. Internal application of lysozyme produced a transitory step-up in the rate of Na efflux but failed to produce the kinetics observed with native or denatured insulin. Overnight exposure of the barnacle to a dose of denatured insulin failed to render the fiber sensitive to external and internal application of denatured or native insulinin vitro. Experiments with ouabain-poisoned fibers showed that external or internal application of native insulin caused stimulation of the remaining Na efflux. They also showed that a 10-fold increase in the concentration of ouabain failed to further reduce the ouabaininsensitive Na efflux. Microinjection of GTP into ouabain-poisoned fibers pre-exposed to insulin resulted in a striking rise in the remaining Na efflux. The magnitude of this effect was considerably greater than that in unexposed fibers. The response which was dose-dependent could be blunted by prior injection of CaCl₂. Similarly, the response to CaCl₂ injection could be blunted by prior injection of GTP. The evidence brought forward is compatible with the view that insulin acts by abolishing the mechanism of internal Na sequestration and by increasing the activity of the guanylate cyclase system.     

Increased sensitivity to injected 5'-guanylylimidodiphosphate of the sodium efflux in barnacle muscle fibres preexposed to aldosterone

1. A study has been made of the response to injected Gpp(NH)p of the ouabain-insensitive Na efflux in barnacle muscle fibres preexposed to aldosterone. 2. The response to injected Gpp(NH)p is not only greater in size than in unexposed fibres but also sustained. 3. Injection of MgCl2 following peak stimulation causes a partial reversal of the response. 4. Injection of ATPNa2 (and 5'-App(NH)p) leads to a sustained stimulatory response which is not significantly greater than that seen in unexposed fibres. 5. MgCl2 injection causes complete reversal of this response. 6. The response of preexposed fibres to injected CaCl2 in varying concentration and to injected cholera toxin is not significantly different from that seen in unexposed fibres. 7. This is also true of Gpp(NH)p when it is injected after peak stimulation by cholera toxin. 8. Prior application of verapamil (10(-4)M) drastically reduces the response to injected Gpp(NH)p. 9. The residual response is sustained but markedly reduced by injected Mg2+, Fe or Zn. 10. Injection of PKI following Gpp(NH)p reduces the response, provided PKI is also injected before Gpp(NH)p. By contrast, injection of R11 subunits causes a partial reversal if injected only once. 11. Imipramine and trifluoperazine, when applied externally (5 X 10(-5)M), cause almost complete reversal of the response. 12. The suggestion is made that the response to injected Gpp(NH)p is mainly due to activation of Ca2+-channels resulting in activation of the calmodulin/Ca-dependent form of adenylate cyclase and that the primary site of aldosterone action is at the level of the calmodulin form of adenylate cyclase.     

The effect of caffeine on calcium efflux and calcium translocation in skeletal and visceral muscle.

1. KCl-induced depolarization resulted in a large stimulation of the 45Ca efflux from both cockroach skeletal muscle and rat ileal smooth muscle. 2. Caffeine (10 mM) induced a large stimulation of 45Ca efflux from skeletal muscle, but a fall in the efflux from ileal muscle, especially if the efflux was previously stimulated by KCl depolarization. 3. Caffeine inhibited calcium uptake by skeletal muscle mitochondria and sarcoplasmic reticulum, was without effect on ileal muscle mitochondria, but significantly increased caclium binding by ileal muscle membrane vesicular preparations. 4. The induction of contractures and stimulation of 45Ca efflux in skeletal muscle by caffeine are clearly related to inhibition of intracellular calcium binding by the sarcoplasmic reticulum and mitochondria. 5. The relaxation of ileal muscle by caffeine and the inhibition of fibre calcium efflux correlate well with caffeine enhancement of intracellular calcium binding. These experiments suggest that the membrane vesicular compartment may be the main agency centrally involved in fibre calcium regulation in this muscle during the contraction-relaxation cycle.     

Characterization of the ATP-dependent calcium efflux in dialyzed squid giant axons

The magnitude of the activating effect of ATP on the Ca efflux was explored at different [Ca++]i in squid axons previously exposed to cyanide seawater and internally dialyzed with a medium free of ATP and containing p-trifluoro methoxy carbonyl cyanide phenyl hydrazine. At the lowest [Ca++]i used (0.06 micron more than 95% of the Ca efflux depends on ATP. At high [Ca++]i (100 micron), 50-60% of the Ca efflux still depends on ATP. The apparant affinity constant for ATP was not significantly affected in the range of [Ca++]i from 0.06 to 1 micron. Axons dialyzed to reduce their internal magnesium failed to show the usual activation of the Ca efflux when the Tris or the sodium salt of ATP was used. Only in the presence of internal magnesium is ATP able to stimulate the Ca efflux. Nine naturally occurring high-energy phosphate compounds were ineffective in supporting calcium efflux. These compounds were: UTP, GTP, CTP, UDP, CDP, ADP, AMP, CAMP, and acetyl phosphate. The compounds 2' deoxy-ATP and the hydrolyzable analog alpha,beta-methylene ATP were able to activate the Ca efflux. The nonhydrolyzable analog beta,gamma-methylene ATP competes with ATP for the activating site, but is unable to activate the Ca efflux. The results are discussed in terms of the specificity of the nucleotide site responsible for the ATP-dependent Ca efflux.     

Mode of action of theophylline on sodium efflux in barnacle muscle fibers

Summary The response of the Na efflux in unpoisoned barnacle fibers to 10mm theophylline is biphasic; i.e., inhibition is followed by stimulation. The stimulatory response is unaffected by ouabain. Fibers pretreated with ouabain show no transitory inhibition when 10mm theophylline is applied, but show prompt stimulation the magnitude of which is comparable to that observed with unpoisoned fibers. The same holds true for lower concentrations of theophylline. Prior injection of 500mm EGTA completely abolishes the biphasic action of 10mm theophylline. External application of 10mm theophylline following removal of external Ca²⁺ fails to bring about a biphasic effect. Ca²⁺ restoration, however, results in a moderate rise in the Na efflux. External application of 10mm theophylline stimulates the Na efflux into Ca²⁺-free artificial seawater (ASW) when the test fibers are pretreated with ouabain. Injection of the protein inhibitor of Walsh leads to reduced stimulation by 10mm theophylline of the Na efflux in unpoisoned fibers. Injection of the protein inhibitor of Corbin into unpoisoned fibers leads to reduced stimulation by 10mm theophylline. Injection of cAMP into ouabainpoisoned fibers, following internal application of Corbin's inhibitor and external application of 10mm theophylline, fails to cause a marked rise in the ouabain-insensitive Na efflux. Injection of Corbin's inhibitor into ouabain-poisoned fibers, following the onset of peak stimulation by 10mm theophylline, fails to reduce the Na efflux. Fibers injected with 1mm and 100mm EGTA and exposed to 10mm theophylline show a marked reduction in the response of the ouabain-insensitive Na efflux to injected cAMP when the concentration of theophylline is 10mm. A poor response to injected cAMP is also seen in fibers bathed in Ca-free ASW containing 10mm theophylline. Theophylline (10mm) fails to cause an enhanced stimulation of the ouabain-insensitive Na efflux into Ca-free 3mm-HEPES ASW or 10mm-Ca²⁺-3mm-HEPES ASW following the addition of protons to the bathing medium. An enhanced response is similarly not observed with injected cAMP following the addition of theophylline to the bathing medium. Injection of 8-fluorotheophylline, 3-isobutyl-1-methylxanthine and doxantrazole leads to a marked reduction in the response of the ouabain-insensitive Na efflux to injected cAMP. Contraction always takes place upon injecting these substances. These results are in keeping with the theory that theophylline acts chiefly by reducing myoplasmic pCa (pCa-log₁₀[Ca²⁺]), and that a reduced pCa leads to stimulation of the ouabain-insensitive Na efflux as the result of activation of the cGMP-dependent protein kinase system by newly formed cGMP.     

Some quantitative aspects of myoplasmic ATPMg and total internal ATP and ArP levels in resting barnacle muscle fibres

1. Flash height recorded following the injection of firefly into the external calibration medium depends on the concentration of K-glutamate used, e.g. 120 mM K glutamate reduces flash height by approximately 20 percent. 2. Suspending the fibre in air instead of artificial sea water (ASW) or replacement of NaCl in the bathing medium with Na-glutamate fails to alter flash height. 3. Firefly preparations from DuPont, Packard and SAI give similar myoplasmic ATPMg values viz. 1.1 mM. 4. Analysis of 36 fibres shows the following: myoplasmic ATP = 1.03 +/- 0.06 mM; total ATP (firefly method) = 5.26 +/- 0.12 mmol/kg water; total ATP (enzymic fluorimetry) = 6.27 +/- 0.13 mmol/kg water and ArP = 20.76 +/- 0.59 mmol/kg water. 5. Measurement of ATPMg in samples of myoplasmic aspirate gives a value that is greater than that obtained in situ. 6. Iodoacetate, whether applied externally or internally, reduces resting luminescence in a dose-dependent manner. It also reduces myoplasmic ATP and total ATP. 7. 2-Deoxy-d-glucose fails to reduce myoplasmic ATP but reduces total ATP. 8. Diethylpyrocarbonate, whether applied externally or internally, reduces myoplasmic ATP. It also causes a slow decline in ArP but little change in total ATP. 9. Injection of L-arginine causes a fall in resting luminescence in some fibres while in others it causes a prompt transitory rise. Injection of L-arginine also causes a fall in total ATP. 10. Collectively, these results suggest that the immediate buffering system in the myoplasm is ArP and that ATP supplied by glycolysis lies in a compartment, presumably the interfibrillar space, which is inaccessible to injected firefly.