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C C Ashley

Publications and source records attributed to C C Ashley.

At least 91 records · Page 5Linked to original sources
PubMed1981

Harmaline distribution in single muscle fibres and the inhibition of sodium efflux.

Harmaline, a known inhibitor of the (Na+ + K+)-ATPase in cell membranes, inhibited 50% of the 22Na efflux from barnacle muscle fibres at an extracellular concentration of 2.4 mM. Injected harmaline inhibited 50% of the efflux at an estimated intracellular concentration of about 8 mM . kg-1, assuming complete equilibration with no binding. Total fibre harmaline was measured in separate fibres by ultraviolet spectrophotometry. Fibres in 3 mM harmaline saline accumulated harmaline with a half-time of 17 min and a final total fibre concentration of 6-12 mM . kg-1. In harmaline-free saline this accumulated harmaline was lost exponentially with a half-time of 35 min; injected harmaline was lost exponentially from fibres with a half-time of 50 min. It is proposed that harmaline crosses the fibre membrane as the uncharged base and that its apparent accumulation against a concentration gradient is mainly due to intracellular binding with an additional contribution from a transmembrane ph gradient. It is concluded that, in fibres exposed to harmaline saline, the intracellular concentration can reach a sufficiently high value, as judged from the results of the injection experiments, to inhibit Na+ efflux at an interior-facing site on the fibre membrane. In contrast, harmaline appears to inhibit the Na+-dependent uptake of L-glutamate at an extracellular site.

Alkaloids↗
PubMed1981

Carbon dioxide or bicarbonate ions release Ca2+ from internal stores in crustacean myofibrillar bundles.

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+: 20 nM) at pH 7.1 with a similar one containing 100% CO2 and 13 mM 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 20 mM caffeine depleted this Ca2+ store, (2) procaine (10 mM) inhibited the response, and (3) the extracellular space or "clefts" and the mitochondria could be eliminated as possible sources. An effect of the CO2 + HCO3- on the free Ca2+/Mg2+ ratio in the bathing solution was excluded as a possible mechanism. The diuretic furosemide (1 mM) enhanced the response to CO2 + HCO3-. Both furosemide and SITS (1--10 mM), 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+.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1978

Free-calcium and tension responses in single barnacle muscle fibres following the application of L-glutamate.

The effect of the putative transmitter, L-glutamate, on free intracellular Ca2+, tension and membrane potential in single muscle fibres from the barnacle Balanus nubilus has been investigated. External application of L-glutamate (0.1--10 mM) resulted in a transient increase in free intracellular Ca2+, monitored by the Ca2+-activated protein aequorin. This increase in free intracellular Ca2+ was associated with membrane depolarization and force development, and was followed by a period of 'desensitization' in which the preparation was unresponsive to L-glutamate. This could be reversed by removing L-glutamate from the external saline. External application of a number of closely related compounds, including D-glutamate and L-aspartate, were ineffective for initiating the transient light response. The L-glutamate response was virtually abolished in Na-free (Li) medium and completely abolished in Ca-free (Na) medium. The responses to L-glutamate were not reduced in Mg-free medium. The fibre's response to 1mM L-glutamate was also inhibited by D-600 (10 micrometer) or by La3+ (1mM), suggesting that Ca was directly involved in the underlying ionic conductance changes brought about by this putative excitatory transmitter.

Aequorin↗
PubMed1978

Calcium ion regulation in barnacle muscle fibers and its relation to force development.

It is possible to use the photoproteins aequorin and obelin within the slowly responding striated muscle films to provide both qualitative and quantitive information as to the nature of the calcium-dependent reactions for force development. It seems that these reactions are not in equilibrium with the Ca2+ during a transient response. In addition, these experiments suggest that kinetically at least two calciums are required per functional unit for force development and that this active site is competed for by Mg2+, H+, and K+ ions.

Animals↗
PubMed1978

Calcium fluxes in single muscle fibres measured with a glass scintillator probe.

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.

Animals↗
PubMed1977

The effect of physiologically occurring cations upon aequorin light emission. Determination of the binding constants.

1. The effect of K+, Na+, Mg2+ and pH upon the rate of aequorin utilization has been investigated in the presence of Ca2+. 2. The aequorin light emission in a medium simulating the in vivo cationic conditions for barnacle muscle fibres indicates that two Ca2+ are apparently involved in this process for free calcium concentrations higher than approx. 10(-5) M. However, for free calcium concentrations lower than 10(-6) M, the intensity of light emitted by aequorin shows a steeper dependency upon [Ca2+] than the square low relationship, indicating that a third Ca2+ should be involved in the process of aequorin light emission, as it has been previously predicted (Moisescu, D.G., Ashley, C.C. and Campbell, A.K. (1975) Biochim. Biophys. Acta. 396, 133-140). 3. The inhibitory effect of physiologically occurring cations upon the aequorin light emission can be explained by the cooperative action of two cations, competing with Ca2+ for the reactive sites on aequorin. 4. At a given concentration, Na2+ was found to have a stronger inhibitory effect upon the aequoring light emission than K+. 5. The experiments indicate a strong interaction between Na+ and K+ in this inhibitory process, since for a given total concentration of monovalent cations, a mixture containing both Na+ and K+ has a larger inhibitory effect on the aequorin light response than solutions containing either Na+ or K+ alone. 6. All other interactions between K+, Na+, H+ and Mg2+ appear to be weak. 7. The reaction schemes used for the explanation of these and other published results on aequorin (Moisescu, D.G., Ashley, C.C. and Campbell, A.K. (1975) Biochim. Biophys, Acta 396, 133-140 and Blinks, J.R. (1973) Eur. J. Cardiol. 1, 135-142) are described, and the 'absolute' binding constants of all physiologically occurring cations for aequorin have been determined. 8. Based on these parameters one can make accurate quantitative predictions for the aequoring light response under a variety of ionic conditions, and this suggests that it is possible to determine absolute free calcium concentrations providing that the ionic composition of the solutions is known, and that the relative rate of aequorin utilization is higher than 0.005.

Aequorin↗
PubMed1977

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

1. In single striated muscle fibres from the barnacle Balanus nubilus 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 (45)Ca 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 caffeine-stimulated efflux of Ca was also reduced by ca. 70% in the presence of 1 mM La saline externally.4. The influx and efflux of (14)C 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) cm(2) sec(-1) at 18-22 degrees C.5. There was no significant loss of (140)La over a period of 2 hr following axial micro-injection into the fibres.6. In (140)La uptake experiments there was a progressive increase in the La space over 10.5 hr, in contrast to the results with [(3)H]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.

Adenosine Triphosphate↗
PubMed1977

Effect of changing the composition of the bathing solutions upon the isometric tension-pCa relationship in bundles of crustacean myofibrils.

1. The relative isometric tension-pCa relationship has been determined for isolated bundles of barnacle myofibrils under a variety of ionic conditions using [Ca(2+)]-buffered solutions which also contained an ATP regenerating system (creatine phosphate and creatine kinase).2. The results are in better agreement with the ;consecutive' scheme of reaction rather than with the ;independent' alternative (Ashley & Moisescu, 1972) for the co-operative action of two Ca(2+) ions in the process of tension activation in crustacean skeletal muscle.3. Variations in the pH of the activating solutions did have a marked effect on the relative tension-Ca curve, although no effect was observed on the absolute maximum value for isometric tension. A shift in pH by 0.5 u. in the range 6.6-7.6 shifted the Ca(2+)-activation curve by 0.5 log u. towards lower free Ca(2+) concentrations.4. Changes in the free Mg(2+) concentration of the activating solutions in the millimolar range produced a pronounced shift of the relative tension-pCa curve along the pCa axis. Increasing [Mg(2+)] from 1 to 5 mM shifted the curve by about 0.7 log u. to higher free Ca(2+) concentrations, without significantly modifying its steepness.5. Changes in the MgATP concentration of the activating solutions in the range of 1-13 mM had no significant effect on the relative tension-pCa relationship.6. Varying the K(+) concentration in the activating solutions was also observed to have a marked effect upon the tension-pCa relationship in barnacle. An increase in the K(+) concentration from 90 to 170 mM shifted the curve by some 0.6 log u. towards higher free Ca(2+) concentrations.7. Cooling the standard activating solutions from room temperature to +4 degrees C made no apparent difference to the relative tension-pCa relationship, but decreased significantly the absolute tension responses.8. The results presented show that tonicity by itself has a marked effect upon the absolute steady-state tension levels in isolated bundles of myofibrils.9. Maximum isometric tension in this preparation was not simply related to ionic strength, or to the monovalent cation concentration, but it depended, as well, upon the anionic composition of the activating solution. In addition, a change in ionic strength of 25 mM over the range of 245-270 mM did not appear to modify the relative tension-pCa relationship.10. The effect of the physiologically occurring cations H(+), K(+), Mg(2+) upon the relative isometric tension-pCa relationship can be accounted for on the basis of a model of competitive inhibition between these cations and Ca(2+) for the functional unit for tension. This inhibitory effect appears to involve at least one H(+), one Mg(2+) and two K(+) per each Ca(2+) ion participating in the activation process of the functional unit for tension.

Adenosine Triphosphate↗