Search

Summary The paper describes an investigation into the increase in intracellular free Ca2+ and resting tension of barnacle muscle fibers when exposed to CO2. Isometric tension was recorded in isolated myofibrillar bundles prepared from barnacles and crabs. On replacement of a low relaxing bathing solution (free Ca2+∶20nm) at pH 7.1 with a similar one containing 100% CO2 and 130mm HCO3−, also at pH 7.1, the bundles developed a phasic contraction, which aequorin experiments confirmed was due to a release of Ca2+ from a store within the bundles. The source of this Ca2+ is tentatively identified as the sarcoplasmic reticulum (SR) for the following reasons: (1) prior exposure to 20mm caffeine depleted this Ca2+ store, (2) procaine (10mm) inhibited the response, and (3) the extracellular space or “clefts” and the mitochondria could be eliminated as possible sources. An effect of the CO2+HCO3t- on the free Ca2+/Mg2+ ratio in the bathing solution was excluded as a possible mechanism. The diuretic furosemide (1mm) enhanced the response to CO2+HCO3t-. Both furosemide and SITS (1–10mm), by themselves, also released Ca2+ in myofibrillar bundles. A scheme is put forward to explain these results: it is suggested that diffusion of dissolved CO2 into the SR produces an acidification of the SR lumen, which modifies either the Ca2+/-ATPase or the Ca2+-induced release process in such a way to release Ca2+.

1. Internal microinjection of the Ca‐sensitive photoprotein aequorin or the isotope 45Ca have been used to assess Ca movements in single muscle fibres from the barnacle Balanus nubilusand the crab Maia squinado.

1. An intracellular glass scintillator (Caldwell & Lea, 1973) has been used to obtain a continuous record of the influx of 45Ca into single muscle fibres of the barnacle, Balanus nubilus. 2. In the presence of intracellular EGTA (final concentration greater than 3 mM/kg), the scintillator detected an initial fast phase to the influx (half‐time = 18.3 min, compartment size = 4.1% fibre volume) followed by a slow, linear phase which gave a value for the Ca influx of 1.2 p‐mole . cm‐2 . sec‐1. The efflux of 45Ca was also measured with the scintillator by transferring a 45Ca‐loaded fibre into 45Ca‐free saline. Two exponential phases of efflux were detected with half‐times of 16.2 and 500 min. 3. The characterisitics of the fast phase of the influx and efflux are similar to those of the influx of the impermeant sucrose and inulin, suggesting that the fast phase represents exchange with the extracellular ‘cleft space’. This phase was insensitive to external La3+ (2 mM). 4. The slow phase is considered to represent the flux of Ca across the surface membrane. It was inhibited by external La3+ (2 mM) and stimulated by replacing external Na+ with Li+. 5. When EGTA‐injected fibres were depolarized using an axial, intracellular electrode the Ca influx, measured from the slow phase, was increased. At higher concentrations of intracellular EGTA (6‐‐22 mM/kg), the extra Ca influx due to a rectangular, depolarizing current pulse was proportional to the number of Ca spikes it produced. A single Ca spike gave an extra Ca influx of 19‐‐48 p‐mole . cm‐2. External D600 (5 x 10(‐4)M) inhibited both Ca spike and the extra Ca influx. 6. At lower intracellular EGTA concentrations (3.6‐‐11 mM/kg), a 50 mV depolarization of 250 msec duration gave a mean extra Ca influx of 80 p‐mole . cm‐2. The upper value was 145 p‐mole . cm‐2 and this would increase the total internal Ca by 4.1 micrometer/kg. It is calculated that if all this extra Ca was bound to the myofibrillar sites for tension, it would only produce 6.2% of the force expected for a similar depolarization in a fibre with no intracellular EGTA.

1. The relative isometric tension—pCa relationship has been determined for isolated bundles of barnacle myofibrils under a variety of ionic conditions using [Ca2+]‐buffered solutions which also contained an ATP regenerating system (creatine phosphate and creatine kinase).

1. In single striated muscle fibres from the barnacle Balanus nubilustension development following axial injection of caffeine (50 m Min 150 m M‐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 m M‐La in the 0 Ca (Na) saline significantly reversed this decline.

Thapsigargin has been reported to inhibit ATP-dependent Ca2+ uptake by isolated sarcoplasmic reticulum (SR) vesicles of vertebrate skeletal muscle fibres at nanomolar concentrations. There have been no reports confirming this effect in skinned muscle fibre preparations. We have examined the ability of thapsigargin to inhibit the uptake of Ca2+ by the SR in mechanically skinned fibres of frog iliofibularis muscles, using the size of the caffeine-induced contracture to assess the Ca2+ content of the SR. The SR was first depleted of Ca2+ and then reloaded for 1 min at pCa 6.2 in the presence and absence of thapsigargin. When 5 min were allowed for diffusion, a thapsigargin concentration of at least 131 µM was required to inhibit Ca2+ loading by 50%. In contrast, another SR Ca2+ uptake inhibitor, cyclopiazonic acid, was more effective, producing 50% inhibition at 7.0 µM and total inhibition at 50 µM. When cyclopiazonic acid (100 µM) was applied after, rather than during, Ca2+ loading, the caffeine-induced contracture was not changed. Thapsigargin (300 µM), on the other hand, caused some reduction in the peak amplitude of the caffeine-induced contracture when applied after Ca2+ loading. The poor effectiveness of thapsigargin in the skinned fibres, compared with in SR vesicles, is attributed to its slow diffusion into the skinned fibres, perhaps as a result of binding to myofibrillar components.