The effects of caffeine on sodium transport, membrane potential, mechanical tension and ultrastructure in barnacle muscle fibres

SUMMARY1. In single striated muscle fibres from the barnacle Balanus nubilu8 tension development following axial injection of caffeine (50 mM in 150 mM-KCl, pH 7.1) was used as an index of releasable Ca. It was shown that fibres incubated in 0 Ca (Na replaced) salines for up to 400 min gave ca. 15 % of the control tension response. Inclusion of 1 mM-La in the 0 Ca (Na) saline significantly reversed this decline.2. Estimates of the total fibre calcium assayed under the same experimental conditions indicated a 67 % loss of fibre Ca in 0 Ca, and only a 37 % loss in La-0 Ca media. No correction was made for the loss of calcium from the extracellular space.3. Experiments with 45Ca indicated that the efflux of calcium from this preparation was inhibited by 1 mM-La externally and that this effect was still significant even in the presence of 0 Ca (Na) salines. The caffeinestimulated efflux of Ca was also reduced by ca. 70 % in the presence of 1 mm La saline externally.4. The influx and efflux of 14C caffeine were shown to be rapid, and apparently passive. The diffusion coefficient for caffeine following intracellular injection was 2-4 + 0-2 x 10-6 cm2 sec-' at 18-22°C.5. There was no significant loss of 140La over a period of 2 hr following axial micro-injection into the fibres.6. In 140La uptake experiments there was a progressive increase in the La space over 10-5 hr, in contrast to the results with [3H]inulin, whose uptake saturated in ca. 1-5 hr. The probability of surface binding and the precipitation of La salts in the extensive extracellular space was suggested as an explanation.7. It is concluded that internal Ca within the sarcoplasmic reticulum, and not cleft or extracellular Ca is the most significant source for these caffeine-induced contractions. Fluxes across the surface membrane can however alter the internal Ca stores over longer periods of time.

Section: Discussionmentioning

confidence: 66%

Caffeine and the contractility of single muscle fibres from the barnacle Balanus nubilus

Ashley

Ellory

Griffiths

1977

“…However, microinjection of 10-M-Cyclic GMP into fibres from the same barnacle specimens resulted in a 72.7 + 14.5 %, n = 6 stimulation of the ouabain-insensitive Na efflux. Dependence of the response to injected cyclic GMP on external cations It is well established that stimulation of the Na efflux caused by injection of cyclic AMP (Bittar, Chambers & Schultz, 1976) and by external caffeine (Bittar et at. 1974) this kind.…”

Section: Lack Of Effect Of Injected Cyclic Gmp On Emmentioning

confidence: 99%

“…The microinjection of guanosine 3':5'-cyclic monophosphate (cyclic GMP) into barnacle muscle fibres is known to promptly stimulate the ouabain-insensitive Na efflux (Bittar, Hift, Huddart & Tong, 1974). The present paper describes a study which confirms this finding and provides information about the properties of the 0022-3751/79/4060-0711 $01.50 © 1979 The Physiological Society E. E. BITTAR AND D. M. SHARP cyclic GMP-stimulated component of the Na efflux, as well as the problem of how cyclic GMP is able to increase the efflux.…”

Section: Introductionmentioning

confidence: 99%

Stimulation by cyclic GMP of sodium efflux in barnacle muscle fibres.

Bittar¹,

Sharp²

1979

SUMMARY1. 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 (104 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 HEPES-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 104 M-cyclic GMP. 6. Injection of pure protein kinase inhibitor (1I 6 x 104 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 ouabaininsensitive Na efflux mainly by activating cyclic AMP-protein kinase rather than cyclic GMP-protein kinase.

“…Changes in the magnitude ofthe Na efflux were estimated on the basis of the fractional rate constant plot, as described by Bittar, Hift, Huddart & Tong (1974 …”

Section: Radio8odium Mea8urement8mentioning

confidence: 99%

Stimulation by injected guanosine triphosphate of the sodium efflux in barnacle muscle fibres.

Bittar¹,

Nwoga²

1982

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.