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C C Ashley

Publications and source records attributed to C C Ashley.

At least 73 records · Page 4Linked to original sources
PubMed1989

Rapid relaxation of single frog skeletal muscle fibres following laser flash photolysis of the caged calcium chelator, diazo-2.

Diazo-2 is a calcium chelator based on BAPTA [(1989) J. Biol. Chem., in press], whose electron withdrawing diazoacetyl group may be rapidly (2000 s-1) converted photochemically to an electron donating carboxymethyl group by exposure to near ultraviolet light, producing an increase in its calcium affinity (Kd changes from 2.2 microM to 0.073 microM) without steric modification of the metal binding site. Photolysis of a 2 mM solution of this compound with a brief flash of light from a frequency-doubled ruby laser (347 nm) caused single skinned muscle fibres from the semitendinosus muscle of the frog Rana temporaria to relax with a mean half-time of 60.4 +/- 5 ms (range 30-100 ms, n = 15) at 12 degrees C, which is faster than the relaxation observed in intact muscles (half-time 133 ms at 14 degrees C [(1986) J. Mol. Biol. 188, 325-342]) and similar to the rate of the fast phase of tension decay in intact single fibres (20 s-1 at 10 degrees C [(1982) J. Physiol. 329, 1-20]).

Animals↗
PubMed1989

Free calcium at rest during "catch" in single smooth muscle cells.

Tension and intracellular free calcium concentration [( Ca2+]i) were measured simultaneously in single smooth muscle cells isolated from the anterior byssus retractor muscle (ABRM) of Mytilus edulis that were loaded with the fluorescent Ca2+ indicator fura-2. Electrical stimulation evoked a transient elevation of [Ca2+]i associated with a "catch" contraction. During the catch state, however, [Ca2+]i was effectively at its resting level and was unaffected by 5-hydroxytryptamine, which induced a rapid relaxation from catch. The results indicate that a maintained high [Ca2+]i is not required for the maintenance of catch tension in intact ABRM and that there was no significant change in [Ca2+]i upon abolition of catch.

Animals↗
PubMed1989

Effects of 5-hydroxytryptamine (serotonin) and forskolin on intracellular free calcium in isolated and fura-2 loaded smooth-muscle cells from the anterior byssus retractor (catch) muscle of Mytilus edulis.

Effects of 5-hydroxytryptamine (5-HT) and forskolin on intracellular free calcium concentration [( Ca2+]i) were studied in suspensions of fura-2 loaded smooth-muscle cells from the anterior byssus retractor 'catch' muscle of Mytilus edulis. The successive addition of 5 mM carbachol (CCh) and 100 mM KCl to the suspension evoked a transient elevation of [Ca2+]i from the resting value of 124 +/- 2.7 nM (mean +/- SE, n = 18) to 300-400 nM, which was associated with contraction. The change in [Ca2+]i induced CCh was concentration-dependent with the EC50 of 10(-5) M. The resting [Ca2+]i was unaffected by 10 microM 5-HT. The change in [Ca2+]i induced by 5 mM CCh was suppressed by 5-HT from 167 +/- 14.0 (n = 11) to 124 +/- 14.9 (n = 8) nM whereas that induced by 100 mM KCl was enhanced from 321 +/- 31.9 to 405 +/- 17.6 nM (n = 8). 5-HT applied during the decaying phase of the CCh response caused a rapid decline in [Ca2+]i. In both the responses to CCh and KCl, the falling phase was accelerated by 5-HT. 10 microM forskolin, a potent activator of adenylate cyclase, mimicked the effects of 5-HT as did a membrane-permeant cyclic AMP analogue, 8-parachlorophenylthio cyclic AMP (cpt-cAMP). Application of 100 microM cpt-cAMP partially suppressed the Ca2+i response to CCh and enhanced that to KCl. D-Tubocurarine (500 microM) added during the decaying phase of the response induced by 100 microM CCh, caused a rapid decline in [Ca2+]i similar to that caused by both 5-HT and forskolin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Ca-induced Ca release from the sarcoplasmic reticulum of isolated myofibrillar bundles of barnacle muscle fibres.

Isometric tension and aequorin light were recorded from isolated myofibrillar bundles (diameter 0.2 mm) of barnacle muscle fibres to examine Ca release from the sarcoplasmic reticulum (SR). Transfer of a bundle from a pCa 6.7 solution to a pCa 5.8 solution, both buffered with 0.1 mM EGTA, resulted in a phasic increase in myofibrillar free Ca2+ which was superimposed on a slow rise to a steady level and a fast rise in tension. The peak of the free Ca2+ response was higher than the free Ca2+ in the bulk solution. Treatment of the bundle with the detergent Brij to destroy the SR membranes abolished the phasic rise in Ca2+ and considerably reduced the amplitude of contraction. A second challenge of a bundle to the pCa 5.8 solution without prior reloading of the SR Ca store gave a much reduced phasic component. When a pCa 5.8 solution with 1.0 mM EGTA buffering was used, the phasic rise in myofibrillar free Ca2+ could not be detected and the rise in tension was four times slower than with 0.1 mM EGTA. The results are consistent with the operation of a Ca-induced Ca release mechanism in the SR membrane of this crustacean muscle.

Aequorin↗
PubMed1989

Correlation of blood pressure in normotensive and hypertensive individuals with platelet but not lymphocyte intracellular free calcium concentrations.

1. Fifty-two normotensive and essential hypertensive subjects were studied. Intracellular free calcium concentration [( Ca2+]i) was measured in lymphocytes (37 subjects) and platelets (18 subjects) by means of the fluorescent indicators, quin 2 and fura-2. In 31 subjects, plasma ionized calcium concentration was also measured. 2. There was a positive correlation between platelet [Ca2+]i and systolic blood pressure (r = 0.485, P less than 0.05), diastolic blood pressure (r = 0.542, P less than 0.02) and mean blood pressure (r = 0.534, P less than 0.02). 3. No statistically significant relationship was observed between plasma ionized calcium and blood pressure. 4. No relationship was found between lymphocyte [Ca2+]i and blood pressure, or between lymphocyte [Ca2+]i and plasma ionized calcium. 5. There was no relationship between [Ca2+]i of lymphocytes and platelets measured simultaneously from the same subject. 6. These findings reconcile previous conflicting reports and show a relationship between platelet but not lymphocyte [Ca2+]i and blood pressure in man.

Adult↗
PubMed1989

Spontaneous oscillations and agonist-evoked changes in Ca2+ in cultured smooth muscle cells.

In confluent A10 aortic smooth muscle cells, at 37 degrees C and in the presence of 1 mM extracellular Ca2+ ([Ca2+]o), the resting intracellular free calcium [Ca2+]i) was found to be 111 +/- 6 nM (n = 17), and in single cells it was 82.4 +/- 7.6 nM. In the presence of 1 mM EGTA, in confluent cells, resting [Ca2+]i was 88.1 +/- 11 nM. The confluent cells showed spontaneous transients and elevations in [( Ca2+]i) as well as maintained oscillations. The oscillations had a periodicity of 6-9 s and were not present in single cells. One nanomolar and 2 microM vasopressin elevated [Ca2+]i to between 0.7 and 3 microM. In the presence of extracellular Ca2+, the initial transient was followed by a plateau phase that declined slowly; in EGTA, this plateau phase was not present but could be restored by adding back Ca2+. Although A10 cells show spontaneous action potentials, oscillations in [Ca2+]i have not been reported. The plateau phase of the vasopressin response appears to be mediated by Ca2+ influx, whereas the initial transient is consistent with the release of stored Ca2+.

Arginine Vasopressin↗
PubMed1988

Carbachol and KCl-induced changes in intracellular free calcium concentration in isolated, fura-2 loaded smooth-muscle cells from the anterior byssus retractor muscle of Mytilus edulis.

Intracellular free calcium concentration [( Ca2+]1) was measured in suspensions of fura-2 loaded smooth-muscle cells isolated from the anterior byssus retractor muscle of Mytilus edulis. Successive application of 5mM carbachol (CCh) and 100mM KCl to the cells transiently elevated [Ca2+]1 from the resting value of 124 +/- 4.5nM (mean +/- S.E., n = 14) to 295 +/- 15.3 and 383 +/- 20.5 nM, respectively. The response to CCh was concentration-dependent with an ED50 of 10(-5) M. Under the microscope, 67 +/- 3.0 and 83 +/- 1.3 % of fura-2 loaded cells contracted on the addition of 5mM CCh and 100mM KCl, respectively. In Ca2+ -free sea water, the CCh induced change in [Ca2+]1 was partially suppressed whereas that induced by KCl was completely abolished, suggesting an agonist-evoked release of stored Ca2+.

Animals↗
PubMed1988

Transient kinetics and time-resolved X-ray diffraction studies in isolated single muscle fibres.

The timing of events associated with the contraction and relaxation of the force cycle is described in isolated single arthropod muscle fibers using the fluorescently labelled derivatives of the Ca2+ binding sub-unit of troponin TnC. The kinetics of the subtracted fluorescence (490-410 nm) response from injected TnCDANZ, labelled at the Ca2+ specific sites, shows a rapid rise which is some 90% complete at 50% force consistent with rapid Ca2+ binding to this sub-unit. Subsequently the TnCDANZ fluorescence decays 2x more slowly, at 12 degrees C, than force consistent with a slower release of this bound Ca2+. In fibers injected with both aequorin and TnCDANZ, the aequorin kinetics are essentially unaltered compared to control fibers in the presence of 10-100 microM TnCDANZ. The peak of the aequorin response occurs some 150-170 msec in front of the TnCDANZ peak and the T 1/2 for light decay is faster than either force or TnCDANZ decay, but there is a 'tail' to the aequorin light response (elevated free Ca2+) well into the relaxation phase, seen both in cannulated and intact muscle fibers. The kinetics of the fluorescence of TnCIAANS, labelled of the Ca2+-Mg2+ sites, shows a slow decrease (T 1/2 1.8 sec) and subsequent increase (T 1/2 2.5 sec) in fluorescence consistent with a slow loading and unloading of these sites with Ca2+ during a tetanus. Time resolved X-ray diffraction from intact muscle fibers indicate that forces of up to 600 kN/m2 can be developed at sarcomere lengths of 8-10 micron. Force shows a marked sarcomere dependency while the aequorin response is relatively insensitive. At these high forces, there is a marked change in intensity of the first actin layer line (A2 at 38 nm), consistent with S1 (cross-bridge) attachment, which has a T 1/2 for rise of 125-150 msec.

Actins↗
PubMed1987

The kinetics of calcium binding to fura-2 and indo-1.

The kinetics of Ca2+ dissociation from fura-2 and indo-1 were measured using a stopped-flow spectrofluorimeter. The dissociation rate constants were 84 s-1 and 130 s-1, respectively, in 0.1 M KC1 at 20 degrees C. The rate constants were insensitive to pH over the range 7.0 to 8.0. The second order association rate constants were estimated indirectly to be in the region of 5 X 10(8) M-1 X s-1 and thus approach the diffusion-controlled limit. The results demonstrate that these new generation indicators are well-suited to measure rapid changes in concentration of intracellular Ca2+.

Benzofurans↗
PubMed1987

Evidence for Na+/Ca2+ exchange in isolated smooth muscle cells: a fura-2 study.

Isolated smooth muscle cells (SMC) from guinea pig taenia coli were employed. Suspension of cells were externally loaded in saline with the fluorescent calcium indicators quin-2/AM or fura-2/AM at 20-40 microM or 4 microM respectively, resulting in an estimated intracellular concentration of 100-200 microM for quin-2 or 10-20 microM fura-2 (free acid). On addition of 100 microM carbachol or high K+o (80 mM) depolarization, fura-2 loaded cells contracted (104 +/- 47 micron, n = 121 rest: 39 +/- 13 micron, n = 59 contracted) identically to control (103 +/- 35 micron, n = 232 rest: 39 +/- 16 micron, n = 89 contracted) cells, whereas quin-2 loaded cells were unresponsive to these protocols and there was no significant length change. The Ca2+i of fura-2 loaded cells was 100 +/- 18 nM (mean +/- SD, n = 15) and was not significantly different from quin-2 loaded cells 107 +/- 26 nM (n = 13). Treatment of fura-2 loaded cells with 100 microM ouabain saline for 10-60 min progressively elevated the Ca2+i to a mean of 266 +/- 83 nM (n = 15). Reduction of Na+o (96% Li+ replaced) significantly increased Ca2+i to 317 +/- 77 nM (n = 8). After pretreatment with ouabain (100 microM), Na+o replacement (Li+) increased Ca2+i at a significantly faster rate [3.6 nM min-1 (control) cf. 19.8 nM min-1 (ouabain)].

Animals↗
PubMed1986

Fura-2 diffusion and its use as an indicator of transient free calcium changes in single striated muscle cells.

Two questions bearing on the use of fura-2 to measure transient changes in intracellular Ca2+ concentration have been addressed. To investigate fura-2 intracellular binding, the amounts of fura-2 and [14C]glycine in Balanus nubilus myofibrillar bundles after loading were determined and their intracellular apparent diffusion constants measured. No significant fura-2 immobilisation occurs under the conditions used. The apparent diffusion constant for fura-2 in aqueous solution was determined. The relationship between half-time for relaxation of force and fura-2 fluorescence transients, and intracellular fura-2 concentration, in voltage-clamped single muscle fibres was examined. Significant buffering of the Ca2+ transient occurred at fura-2 concentrations above approximately 6 microM.

Animals↗
PubMed1986

An examination of the ability of inositol 1,4,5-trisphosphate to induce calcium release and tension development in skinned skeletal muscle fibres of frog and crustacea.

We have examined the ability of inositol 1,4,5-trisphosphate (InsP3) to cause contractions of mechanically skinned muscle fibres of frog and barnacle. InsP3 (10-500 microM) did not cause any tension development in 25 frog skinned fibres and 26 barnacle myofibrillar bundles, although contractions could be readily evoked by caffeine and by replacement of an impermeant anion by Cl-, treatments known to release calcium from the sarcoplasmic reticulum (SR). Four barnacle bundles did give responses to InsP3. InsP3 did not modify responses to caffeine or calcium-induced calcium release. Free Mg2+ was lowered to 40 microM and 15 mM D-2,3-diphosphoglycerate was added in order to inhibit the possible breakdown of InsP3 by inositol trisphosphatase. Neither measure revealed a response to InsP3. Arsenazo III absorbance measurements failed to detect any binding of Mg2+ (0-0.5 mM) by 0.35 mM InsP3 in our solutions. Inhibitors of SR calcium uptake (cadium, quercetin, furosemide), omission of EGTA from the solution and varying the temperature from 4 degrees to 22 degrees C also failed to reveal a response of frog skinned fibres to InsP3. The nucleotide GTP, which has been reported to enhance InsP3-induced calcium release from rat liver microsomes, had no effect at 50 microM on the response of frog fibres to InsP3. It is concluded that under conditions in which other calcium release mechanisms operate well, InsP3 is relatively ineffective at releasing calcium from the SR in amounts sufficient to induce contraction. Although we have been unable to find evidence to support the proposed role of InsP3 as an essential link in excitation-contraction coupling of skeletal muscle, we cannot entirely reject its role if essential cofactors are lost in the skinned preparations.

2,3-Diphosphoglycerate↗
PubMed1986

Na+/Ca2+ exchange in isolated smooth muscle cells demonstrated by the fluorescent calcium indicator fura-2.

Fura-2, a novel fluorescent indicator of cytoplasmic calcium concentrations ([Ca2+i]), was 'loaded' into smooth muscle cells isolated from guinea pig taenia coli. Resting cells maintained a stable [Ca2+i] of 107 +/- 26 nM (n = 13), which could be perturbed with ionomycin. [Ca2+i] was elevated by stimulation of the cells with carbachol or 50 mM KCl. Reduction of the plasmalemmal Na+ concentration gradient by inhibition of the Na+/K+-ATPase with ouabain markedly elevated [Ca2+i]; this elevation was dependent on extracellular Ca2+. [Ca2+i] was also increased by replacement of the extracellular Na+ with an organic cation.

Animals↗
PubMed1985

Measurement of free Ca2+ changes and total Ca2+ release in a single striated muscle fibre using the fluorescent indicator quin 2.

The fluorescent Ca2+ indicator, quin 2, has been used in isolated striated muscle fibres. There is a distinct quin 2 fluorescence peak at lambda 500 nm upon excitation at lambda 339 nm after axial injection of the potassium salt of quin 2, pH 7.1. Single voltage-clamp or current clamp electrical stimulation resulted in a distinct transient change in the fluorescence at lambda 500 nm which was not observed at lambda 400 nm, the peak of the fibre autofluorescence. Ca2+ buffering is marked at high quin 2 concentrations (greater than or equal to 400 microM) producing a slow decay of force and fluorescence. At lower concentrations (8-30 microM) of quin, the decay of force is within the range observed in non-injected control fibres. A Kd of 457 nM at 5 mM free Mg2+ suggests an upper resting free Ca2+ concentration of 310 nM at 12 degrees C.

Aminoquinolines↗
PubMed1985

Kinetic investigations in single muscle fibres using luminescent and fluorescent Ca2+ probes.

The Ca2+-sensitive photoprotein aequorin and the Ca2+-dependent fluorescent indicators quin 2 and TnCDANZ have been used to investigate contractile processes in single crustacean muscle fibres. The investigations with quin 2 indicate that the free Ca2+ rises to a maximum value before peak force as with aequorin light (approximately 200 msec delay at 12 degrees C) and subsequently decays more slowly, unlike the majority of the aequorin signal, although an aequorin 'tail' signal remains. The resting quin 2 fluorescence from the cell suggests an upper limit of 348 nM for the resting calcium concentration. Experiments with TnCDANZ indicate that this fluorescence response rises rapidly but then the rate of rise slows to reach a maximum value at a time when peak force is achieved and then the fluorescence signal decays more slowly than force. The latter result implies that Ca2+ is attached to the Ca2+-specific sites of TnC when externally recorded force is small.

Aequorin↗
PubMed1984

Fluorescence changes from single striated muscle fibres injected with labelled troponin C (TnCDANZ).

A fluorescently labelled derivative of the calcium binding subunit of troponin, TnC, has been injected into isolated striated muscle fibres from the barnacle Balanus nubilus. The Ca2+ affinity of isolated TnC is close to that of intact troponin when located in the thin filament. Excitation of the TnCDANZ within the muscle cell (325nm) revealed a marked fluorescence at 510 nm and was similar to that observed in vitro, which was absent at 400 or 600 nm after subtraction of the fibre autofluorescence. High Ca2+ salines increased the fluorescence at 510 nm by roughly 2 times. Single voltage clamp pulses produced a rapid rise in fluorescence at 510 nm after allowing for any non-specific changes at 400 nm, and this signal preceded force development by approx. 55 ms at 22 degrees C. It reached a maximum at the same time as force and subsequently decayed more slowly. The fluorescence signal increased in magnitude with increase in stimulus intensity. These results suggest that Ca2+ attaches rapidly to the contractile filament, but is lost relatively slowly and imply a slow decay of the activation process.

Animals↗