Christopher C. Ashley

Free calcium rise and mitogenesis in glial cells caused by endothelin

The peptide endothelin causes a biphasic rise in intracellular free calcium levels in cultured Type-1 astrocytes and C6 glioma cells, suggesting that glial cells may be the physiological target of endothelin in the brain. Endothelin also causes a calcium-dependent increase [3H]thymidine incorporation in primary cultures of rat cerebellum, indicating that, among other possible roles, this peptide may mediate mitogenesis in brain.

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 KCl at 20°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 × 108 M−1·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+.

Mutations in fast skeletal troponin I, troponin T, and beta-tropomyosin that cause distal arthrogryposis all increase contractile function.

Distal arthrogryposes (DAs) are a group of disorders characterized by congenital contractures of distal limbs without overt neurological or muscle disease. Unexpectedly, mutations in genes encoding the fast skeletal muscle regulatory proteins troponin Τ (TnT), troponin I (Tnl), and β‐tropomyosin (β‐TM) have been shown to cause autosomal dominant DA. We tested how these mutations affect contractile function by comparing wild‐type (WT) and mutant proteins in actomyosin ATPase assays and in troponin‐replaced rabbit psoas fibers. We have analyzed all four reported mutants: Arg63His TnT, Arg91Gly β‐TM, Argl74Gln Tnl, and a Tnl truncation mutant (Argl56ter). Thin filaments, reconstituted using actin and WT troponin and β‐TM, activated myosin subfragment‐1 ATPase in a calcium‐dependent, cooperative manner. Thin filaments containing either a troponin or β‐TM DA mutant produced significantly enhanced ATPase rates at all calcium concentrations without alternating calcium‐sensitivity or cooperativity. In troponin‐exchanged skinned fibers, each mutant caused a significant increase in Ca2+ sensitivity, and Argl56ter Tnl generated significantly higher maximum force. Arg91Gly β‐TM was found to have a lower actin affinity than WT and form a less stable coiled coil. We propose the mutations cause increased contractility of developing fast‐twitch skeletal muscles, thus causing muscle contractures and the development of the observed limb deformities.—Robinson, P., Lipscomb, S., Preston, L. C., Altin, E., Watkins, H., Ashley, C. C., Redwood, C. S. Mutations in fast skeletal troponin I, troponin T, and β‐tropomyosin that cause distal arthrogryposis all increase contractile function. FASEB J. 21, 896–905 (2007)

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 voltageclamped single muscle fibres was examined. Significant buffering of the Ca2+ transient occurred at fura‐2 concentrations above ~ 6 μM.

Troponin I phosphorylation does not increase the rate of relaxation following laser flash photolysis of diazo-2 in guinea-pig skinned trabeculae.

Abstract In vivo, two effects of β-adrenergic stimulation in cardiac muscle are phosphorylation of troponin I and an increase in relaxation rate. In vitro, cardiac TnI can be phosphorylated by protein kinase A (PKA). We have used the technique of laser flash photolysis of the calcium chelator diazo-2 to investigate the effect of phosphorylation of TnI on the relaxation rate of skinned trabeculae from the guinea-pig at 12°C. The fibres were phosphorylated by PKA, and double exponential curve fits of the average relaxation transients showed no significant difference between the rate constants of the phosphorylated and control cases. We conclude that TnI phosphorylation has no effect on the rate of relaxation in skinned trabeculae from the guinea-pig following diazo-2 photolysis.

Two mutations in troponin I that cause hypertrophic cardiomyopathy have contrasting effects on cardiac muscle contractility.

We investigated the effects of two mutations in human cardiac troponin I, Arg(145)-->Gly and Gly(203)-->Ser, that are reported to cause familial hypertrophic cardiomyopathy. Mutant and wild-type troponin I, overexpressed in Escherichia coli, were used to reconstitute troponin complexes in vanadate-treated guinea pig cardiac trabeculae skinned fibres, and thin filaments were reconstituted with human cardiac troponin and tropomyosin along with rabbit skeletal muscle actin for in vitro motility and actomyosin ATPase assays. Troponin containing the Arg(145)-->Gly mutation inhibited force in skinned trabeculae less than did the wild-type, and had almost no inhibitory function in the in vitro motility assay. There was an enhanced inhibitory function with mixtures of 10-30% [Gly(145)]troponin I with the wild-type protein. Skinned trabeculae reconstituted with troponin I containing the Gly(203)-->Ser mutation and troponin C produced less Ca(2+)-activated force (64+/-8% of wild-type) and demonstrated lower Ca(2+) sensitivity [Delta(p)Ca(50) (log of the Ca(2+) concentration that gave 50% of maximal activation) 0.25 unit (P<0.05)] compared with wild-type troponin I, but thin filaments containing [Ser(203)]-troponin I were indistinguishable from those containing the wild-type protein in in vitro motility and ATPase assays. Thus these two mutations each result in hypertrophic cardiomyopathy, but have opposite effects on the overall contractility of the muscle in the systems we investigated, indicating either that we have not yet identified the relevant alteration in contractility for the Gly(203)->Ser mutation, or that the disease does not result directly from any particular alteration in contractility.

An examination of the ability ofinositol 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 μM) 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 impenneant 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 μM 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 (cadmium, quercetin, furosemide), omission of EGTA from the solution and varying the temperature from 4° to 22°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 μM 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.

Cross-bridge attachment and stiffness during isotonic shortening of intact single muscle fibers

Equatorial x-ray diffraction pattern intensities (I10 and I11), fiber stiffness and sarcomere length were measured in single, intact muscle fibers under isometric conditions and during constant velocity (ramp) shortening. At the velocity of unloaded shortening (Vmax) the I10 change accompanying activation was reduced to 50.8% of its isometric value, I11 reduced to 60.7%. If the roughly linear relation between numbers of attached bridges and equatorial signals in the isometric state also applies during shortening, this would predict 51-61% attachment. Stiffness (measured using 4 kHz sinusoidal length oscillations), another putative measure of bridge attachment, was 30% of its isometric value at Vmax. When small step length changes were applied to the preparation (such as used for construction of T1 curves), no equatorial intensity changes could be detected with our present time resolution (5 ms). Therefore, unlike the isometric situation, stiffness and equatorial signals obtained during ramp shortening are not in agreement. This may be a result of a changed crossbridge spatial orientation during shortening, a different average stiffness per attached crossbridge, or a higher proportion of single headed crossbridges during shortening.

Effects of thapsigargin and cyclopiazonic acid on the sarcoplasmic reticulum Ca2+ pump of skinned fibres from frog skeletal muscle

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.

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 (K d changes from 2.2μM to 0.073 μM) 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°C, which is faster than the relaxation observed in intact muscles (half‐time 133 ms at 14°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 1O°C [(1982) J. Physiol. 329, 1–20]).