Phosphate metabolites in single barnacle muscle fibers investigated by phosphorus-31 nuclear magnetic resonance

doi:10.1016/0305-0491(86)90163-X

Comparative Biochemistry and Physiology Part B: Comparative Biochemistry

Volume 83, Issue 4, 1986, Pages 875-879

https://doi.org/10.1016/0305-0491(86)90163-X Get rights and content

Abstract

1.
1. A study has been made of phosphate-containing metabolites in single barnacle muscle fibers using phosphorus-31 nuclear magnetic resonance.
2.
2. Spectra from single fibers (∼50 mg in wet weight) show major resonances from sugar phosphates, inorganic phosphate, arginine phosphate and the α, β and γ phosphorus atoms of ATP.
3.
3. The approximate “free” concentration of each metabolite was determined by integration of the spectrum, using a sample of 1 M-methylene diphosphonic acid as a reference. A notable feature of the results obtained is that the concentrations of SP&P_i in freshly dissected fibers are low.
4.
4. Time-dependent changes in ³¹P-NMR spectra indicate that ArP declines fairly slowly, while SP and P_i rises. The half-life of ArP at 26°C turns out to be about 8 hr. ATP remains relatively constant for the first 8 hr but disappears following the disappearance of ArP. As the intensity of the P_i resonance increases with time, it broadens and moves upfield, suggesting internal acidosis.
5.
5. These results demonstrate that ³¹P-NMR can provide useful information about metabolism and its regulation in single barnacle muscle fibers.

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NMR studies of metabolism in perfused organs
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Intracellular pH transients in giant barnacle muscle fibers
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There are more references available in the full text version of this article.

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<sup>1</sup>H NMR relaxometry as an indicator of setting and water depletion during cement hydration
2013, Cement and Concrete Research
Citation Excerpt :
By monitoring the relaxation decay of protons, Korb et al. [14] measured the longitudinal relaxation time T1 as a function of magnetic field strength to provide microstructure evolution information during ongoing hydration. Results reported by Nestle et al. [15] explained how to determine the degree of hydration by transverse relaxation experiment (T2 is also frequently applied in NMR techniques as shown in [7,16,17]). Furthermore, results reported by Faure et al. [18] showed that the evolution of the optimum T1 value according to time is correlated with well-acknowledged 4-stage hydration process since early age: dissolution, induction, setting and hardening.
Proton nuclear magnetic resonance relaxometry has been used to detect setting and microstructure evolution during cement hydration. NMR measurements were performed since casting, during setting and until hardening (from 0 to 3 days). The mobility of water molecules was assessed by an analysis focused on the diagram of longitudinal relaxation time T₁ generated by an Inversion Recovery sequence. The initial stiffening of the solid network was identified by an analysis of the relaxation rate 1/T₁. The kinetics of water depletion was investigated by using a simple one-pulse acquisition sequence. In parallel, conventional techniques (Vicat needle and temperature monitoring), as well as numerical simulations of hydration, were used to complement and validate these NMR results. Cement pastes and mortars with different water-to-cement ratios made of grey or white OPCs were tested. Furthermore, the effects of the addition of sand, super-plasticizer and silica fume on the hydration kinetics were investigated.
Concerning stimulation by injected fluoroaluminate of the sodium efflux in barnacle muscle fibers
1993, BBA - Biomembranes
Single barnacle muscle fibers from Balanus nubilus were used primarily to examine the validity of two ideas: first, that the injection of KF stimulates the ouabain-insensitive Na⁺ efflux, and that this action is potentiated by adding AlCl₃ (Al) in a low concentration to the solution of KF prior to injection. And second, that the injection of a KF-AlCl₃ solution into ouabain-poisoned, $K^{±} - depolarized$ fibers elicits a stimulatory response resembling that obtained by injecting GTP. The results of this study are as follows: injection of 0.5 M KF into unpoisoned fibers causes a sustained rise in the resting Na⁺ efflux. However, injection of a 0.5 M KF, 10⁻³ M AlCl₃ solution leads to a reduced rather than an augmented response. Whereas injection of 0.5 M KF into ouabain-poisoned fibers elicits a marked stimulatory response, the injection of 0.5 M KF, 10⁻³ M AlCl₃ reduces the remaining Na⁺ efflux. Injection of KF-AlCl₃ in equimolar concentrations, e.g., 0.25 M, elicits a response that is significantly larger than that obtained by injecting 0.25 M KF. A dose-response curve indicates that a 0.2 M solution of fluoroaluminate probably represents an optimal concentration. Injection of 0.3 M KF following peak stimulation by injecting 0.3 M AlCl₃ completely reverses this response to Al. In sharp contrast, injection of a 0.3 M KF, 0.3 M AlCl₃ mixture following peak stimulation by injecting 0.3 M AlCl₃ is ineffective. Injection of KF into ouabain-poisoned, K⁺ depolarized fibers does not always cause sustained stimulation of the remaining Na⁺ efflux. But injection of KF-AlCl₃ in equimolar concentration always seems to cause a delayed sustained stimulatory response. Sustained stimulation is also observed after injecting Na₂GTP. Although these results provide evidence in support of the hypothesis that the primary point of action of equimolar KF-AlCl₃ solutions following injection into barnacle fibers is the membrane adenylate cyclase system, they raise doubts about the validity of the concept that trace amounts of Al are required for KF to act as a positive effector of this system.
The reduction of the inhibitory effect of aluminum on Na<sup>+</sup> efflux in barnacle muscles fibers by preinjecting phosphate
1993, BBA - Biomembranes
The object of the present study was to test the hypothesis that the pre-enrichment of single muscle fibers from the barnacle Balanus nubilus with inorganic phosphate may protect the basal Na efflux from the inhibitory effect of Al injection. This approach was adopted in the light of evidence that the preinjection of ATP fails to stop the Na efflux in unpoisoned fibers from falling following the injection of Al. The results of the experiments are as follows: (i) Preinjection of K₂HPO₄ into unpoisoned fibers reduces the magnitude of the inhibitory effect on the basal Na efflux of injected Al in a dose-dependent manner but fails to completely stop it from occuring. (ii) Injection of K₂HPO₄ following Al into unpoisoned fibers fails to arrest or reverse the inhibitory effect of injected Al. (iii) Injection of K₂HPO₄ in a concentration as high as 0.5 M is without effect on the course of the basal Na efflux. (iv) Injection of K₂HPO₄ into ouabain-poisoned fibers fails to stop Al from stimulating the ouabain-insensitive Na efflux. Injection of K₂HPO₄ following peak stimulation by injecting Al is also without effect. (v) Injection of K₂HPO₄ in a concentration as high as 0.5 M is without effect on the course of the ouabain-insensitive Na efflux. Collectively, the results obtained with unpoisoned ‘hypersensitive’ fibers are consistent with the view that a significant fraction of the injected inorganic phosphate binds Al³⁺, and hence protects the basal Na efflux from the untoward action of Al³⁺.
The ability and inability of ATP to stop aluminum from reducing the sodium efflux in unpoisoned barnacle muscle fibers
1992, BBA - Biomembranes
A study has been made in single barnacle muscle fibers with the object of determining whether ATP is able to protect the resting Na efflux from the effects of injected aluminum (Al) and whether Al is able to reduce or abolish the stimulatory action of ATP on the efflux. The results of the experiments show that neither ATPMg nor ATPNa₂ preinjection stops Al from reducing the basal Na efflux in unpoisoned fibers which undergo a large fall (hypersensitive fibers). Preinjection of Al into such fibers reduces or abolishes the stimulatory response of the Na efflux to ATP injection. In less hypersensitive fibers, however, ATPMg is protective. This is also true of ATPNa₂ preinjection in both classes of fibers showing stimulation. Injection of a mixture of AlCl₃-ATPNa₂ into unpoisoned fibers causes less inhibition than AlCl₃ injection. The hypothesis that both ATPMg and ATPNa₂ are protective is also supported by the results obtained with ouabain-poisoned fibers: (i) Al injection after ATP fails to reverse the stimulatory response to ATP, while ATP injection afer Al exerts only a small or no effect. (ii) Mg²⁺ injection fails to reverse the stimulatory response to Al injection in poisoned fibers. And (iii) Anti-proteolysis agents e.g. leupeptin and pepstatin, upon preinjection, do not alter the kinetic results obtained by injecting Al into unpoisoned and ouabain-poisoned fibers.
An investigation of the sensitivity of the ouabain-insensitive sodium efflux in single barnacle muscle fibers to pentachlorophenol
1991, Toxicology and Applied Pharmacology
The aim of the present work was to explore the possibility that pentachlorophenol (PCP) influences the behavior of the resting Na efflux in single muscle fibers from the barnacle, Balanus nubilus. It is shown here that PCP causes a transitory rise in the Na efflux in both unpoisoned and ouabain-poisoned fibers and that the response is dose-dependent, the minimal effective concentration in ouabain treated fibers being less than 10⁻⁶ m. The efficacy of PCP is significantly greater than that of 2,3,4-trichlorophenol. 2,3-Dichlorophenol is ineffective. This is also the case with phenol. The magnitude of the response to PCP is a function of external pH. Lowering pHe increases the response. The response has an absolute requirement for external Ca²⁺ and is a sigmoidal function of external Ca²⁺ concentration. Since treatment of these fibers with PCP in high concentration leads to prompt contraction, experiments were designed to determine whether the observed rise in ouabain-insensitive Na efflux is due to a fall in myoplasmic pCa and whether trigger Ca²⁺ originates from the bathing medium. The results obtained show that prior injection of ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetic acid (EGTA) or 1,2-bis(2-aminophenoxyethane-N,N,N′,N′-tetraacetic acid (BAPTA) leads to a drastic reduction in the response to PCP. They also show that prior external application of verapamil or devapamil stops the response to PCP from occurring. Both Cd²⁺ and Co²⁺ are also effective but only temporarily. Last, the effects of ryanodine and 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) were tested, since the former is known to block the sarcoplasmic reticulum Ca²⁺ release channel, and the latter to impair the action of agents known to release Ca²⁺ from internal depots. Both ryanodine and TMB-8 are found to reduce the response to PCP. Taken together, these observations support the hypothesis that PCP stimulates the ouabain-insensitive Na efflux by increasing the internal free Ca²⁺ and that the increase in internal Ca²⁺ is due to the entry of trigger Ca²⁺ from the outside via Ca²⁺ channels, as well as release of Ca²⁺ is due to the entry of trigger via its channel. They also indicate that the efficacy of PCP depends on the 5 Cl atoms present in its aromatic ring and pH_e.
Protection by GTP from the effects of aluminum on the sodium efflux in barnacle muscle fibers
1991, BBA - Biomembranes
The idea that guanine nucleotides act as chelators of Al³⁺ and that Al interrupts the mechanism by which GTP or Gpp(NH)p stimulates the Na efflux in single muscle fibers from the barnacle Balanus nubilus has been tested. As a rule, injection of GTP or Gpp(NH)p into unpoisoned and ouabain-poisoned fibers produces a rise in the ²²Na efflux that is usually transitory in nature. Fibers preinjected with GTP show a fall in the Na efflux following the injection of AlCl₃ in an equimolar concentration. If, however, the concentration of Al for injection is halved, then GTP is found to be fully protective. Fibers preinjected with AlCl₃ show little or no response to the injection of GTP. This is also the case with ouabain-poisoned fibers. Ouabain-poisoned fibers preinjected with GTP also show little or no response to the injection of AlCl₃. The stimulatory response to the injection of AlCl₃ into fibers preinjected with 0.5 M GTP is dose-dependent. A graded response is also found when 0.5 M AlCl₃ is injected into fibers preinjected with GTP in varying concentrations. Gpp(NH)p is fully protective against the inhibitory effect of Al injection in unpoisoned fibers. Further, Gpp(NH)p abolishes the biphasic effect of Al injection on the ouabain-insensitive Na efflux. To strengthen the argument that GTP acts as a chelator of Al, a solution mixture of 0.5 M GTP/0.5 M AlCl₃ (pH 1–2) was injected into unpoisoned fibers. This is found to lead to a smaller fall in the resting Na efflux than that obtained by injecting AlCl₃ alone or injecting AlCl₃ after GTP. It is thus quite clear that the barnacle muscle fiber is a useful preparation for studies of this type.

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Comparative Biochemistry and Physiology Part B: Comparative Biochemistry

Abstract

References (20)

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

Comp. Biochem. Physiol.

Analysis of phosphate metabolites, the intracellular pH, and the state of adenosine triphosphate in intact muscle by phosphorus nuclear magnetic resonance

J. biol. Chem.

NMR studies of metabolism in perfused organs

Phil. Trans. R. Soc. Lond. Ser. B

³¹P Nuclear-magnetic-resonance studies of energy metabolism in tissue from the marine invertebrate Tapes watlingi

Eur. J. Biochem.

Intracellular pH transients in giant barnacle muscle fibers

Am. J. Physiol.

Analysis of intact tissue with ³¹P NMR

A. Rev. Biophys. Bioeng.

Analysis of living tissue by phosphorus-31 magnetic resonance

Science

Phosphorus nuclear-magnetic-resonance studies of compartmentation in muscle

Biochem. J.

Phosphodiesters in muscular dystrophies

Ann. N.Y. Acad, Sci.

New non-destructive biophysical methods for studying cell ion content transport and metabolism

Cited by (11)

<sup>1</sup>H NMR relaxometry as an indicator of setting and water depletion during cement hydration

Concerning stimulation by injected fluoroaluminate of the sodium efflux in barnacle muscle fibers

The reduction of the inhibitory effect of aluminum on Na<sup>+</sup> efflux in barnacle muscles fibers by preinjecting phosphate

The ability and inability of ATP to stop aluminum from reducing the sodium efflux in unpoisoned barnacle muscle fibers

An investigation of the sensitivity of the ouabain-insensitive sodium efflux in single barnacle muscle fibers to pentachlorophenol

Protection by GTP from the effects of aluminum on the sodium efflux in barnacle muscle fibers

Phosphate metabolites in single barnacle muscle fibers investigated by phosphorus-31 nuclear magnetic resonance

Abstract

Comp. Biochem. Physiol.

J. biol. Chem.

NMR studies of metabolism in perfused organs

Phil. Trans. R. Soc. Lond. Ser. B

31P Nuclear-magnetic-resonance studies of energy metabolism in tissue from the marine invertebrate Tapes watlingi

Eur. J. Biochem.

Intracellular pH transients in giant barnacle muscle fibers

Am. J. Physiol.

Analysis of intact tissue with 31P NMR

A. Rev. Biophys. Bioeng.

Analysis of living tissue by phosphorus-31 magnetic resonance

Science

Phosphorus nuclear-magnetic-resonance studies of compartmentation in muscle

Biochem. J.

Phosphodiesters in muscular dystrophies

Ann. N.Y. Acad, Sci.

New non-destructive biophysical methods for studying cell ion content transport and metabolism

³¹P Nuclear-magnetic-resonance studies of energy metabolism in tissue from the marine invertebrate Tapes watlingi

Analysis of intact tissue with ³¹P NMR