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Effect of guanosine triphosphate on the release and uptake of Ca2+ in saponin-permeabilized macrophages and the skeletal-muscle sarcoplasmic reticulum.

ABSTRACT: The effects of GTP, with or without polyethylene glycol (PEG), on the release and uptake of Ca2+ were examined by using saponin-treated macrophages and sarcoplasmic reticulum isolated from skeletal muscles. The application of GTP in concentrations in the range 0.1-10 microM induced a gradual, small but sustained release of Ca2+ from the saponin-treated macrophages. The addition of PEG to GTP markedly enhanced the GTP-mediated Ca2+ release. GTP at the same concentration ranges used for Ca2+ release decreased the amount of Ca2+ uptake, at a steady state, but stimulated the rate of Ca2+ accumulation in the presence of oxalate, the Ca2+-precipitating anion. The addition of PEG abolished the GTP-evoked stimulation of Ca2+ accumulation in the presence of oxalate. The stimulating effect on the rate of Ca2+ accumulation by GTP and its elimination by PEG were not due to changes in the permeability of oxalate by either GTP or PEG, or both. The Ca2+-releasing effect of GTP without PEG was enhanced by eliminating the uptake activity by decreasing the content of ATP. These results indicate that GTP has an inherent activity to release Ca2+ from non-mitochondrial intracellular stores of saponin-treated macrophages, and PEG enhances the GTP-mediated Ca2+ release, partly owing to its eliminating effect on GTP-stimulated Ca2+ uptake activity. These effects of GTP observed with saponin-permeabilized macrophages were not apparent in the isolated skeletal-muscle sarcoplasmic reticulum.

SUBMITTER: Hamachi T

PROVIDER: S-EPMC1147690 | biostudies-other | 1987 Feb

REPOSITORIES: biostudies-other

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Project description:Experiments were performed to characterize the properties of the intrinsic Ca(2+) buffers in the sarcoplasmic reticulum (SR) of cut fibers from frog twitch muscle. The concentrations of total and free calcium ions within the SR ([Ca(T)](SR) and [Ca(2+)](SR)) were measured, respectively, with the EGTA/phenol red method and tetramethylmurexide (a low affinity Ca(2+) indicator). Results indicate SR Ca(2+) buffering was consistent with a single cooperative-binding component or a combination of a cooperative-binding component and a linear binding component accounting for 20% or less of the bound Ca(2+). Under the assumption of a single cooperative-binding component, the most likely resting values of [Ca(2+)](SR) and [Ca(T)](SR) are 0.67 and 17.1 mM, respectively, and the dissociation constant, Hill coefficient, and concentration of the Ca-binding sites are 0.78 mM, 3.0, and 44 mM, respectively. This information can be used to calculate a variable proportional to the Ca(2+) permeability of the SR, namely d[Ca(T)](SR)/dt ÷ [Ca(2+)](SR) (denoted release permeability), in experiments in which only [Ca(T)](SR) or [Ca(2+)](SR) is measured. In response to a voltage-clamp step to -20 mV at 15°C, the release permeability reaches an early peak followed by a rapid decline to a quasi-steady level that lasts ~50 ms, followed by a slower decline during which the release permeability decreases by at least threefold. During the quasi-steady level of release, the release amplitude is 3.3-fold greater than expected from voltage activation alone, a result consistent with the recruitment by Ca-induced Ca(2+) release of 2.3 SR Ca(2+) release channels neighboring each channel activated by its associated voltage sensor. Release permeability at -60 mV increases as [Ca(T)](SR) decreases from its resting physiological level to ~0.1 of this level. This result argues against a release termination mechanism proposed in mammalian muscle fibers in which a luminal sensor of [Ca(2+)](SR) inhibits release when [Ca(T)](SR) declines to a low level.

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Effect of guanosine triphosphate on the release and uptake of Ca2+ in saponin-permeabilized macrophages and the skeletal-muscle sarcoplasmic reticulum.

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