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C C Ashley

Publications and source records attributed to C C Ashley.

At least 55 records · Page 3Linked to original sources
PubMed1993

Hyperparathyroidism, platelet intracellular free calcium and hypertension in chronic renal failure.

To investigate possible relationships between hyperparathyroidism, alterations in intracellular free calcium concentration ([Ca2+]i) and hypertension in chronic renal failure, serum concentrations of intact parathyroid hormone (PTH) were measured by two-site immunometric assay, and platelet ([Ca2+]i) was assessed using the fluorescent indicator fura-2. Thirty-six patients with chronic renal failure were studied, 10 with normal serum PTH concentrations (mean 8.0 +/- 0.6 pmol/liter), 17 with elevated serum PTH (35.0 +/- 7.2 pmol/liter) and 9 patients with elevated PTH (36.2 +/- 5.9 pmol/liter) who were receiving nifedipine. Platelet [Ca2+]i was increased in patients with elevated PTH, compared with those in whom PTH was normal (138 +/- 16 vs. 83 +/- 7 nmol/liter, P < 0.01). A linear relation was observed between serum PTH and platelet [Ca2+]i in these patients (r = 0.818, P < 0.001). In contrast, platelet [Ca2+]i was not elevated (84 +/- 9 nmol/liter) in the patients with elevated PTH who were receiving nifedipine. A linear relation was also present between both serum PTH (r = 0.616, P < 0.001) and platelet [Ca2+]i (r = 0.576, P < 0.005) and mean blood pressure. Nine patients with hyperparathyroidism were restudied after treatment with the vitamin D analogue alfacalcidol. This resulted in significant decreases in serum PTH (P < 0.01), platelet [Ca2+]i (P < 0.02), and mean blood pressure (P < 0.05). These studies indicate that [Ca2+]i may be increased early in renal failure, and that this increase occurs in association with both hyperparathyroidism and hypertension. Furthermore, treatment of hyperparathyroidism with alfacalcidol may result in reductions in both [Ca2+]i and blood pressure.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1991

Skeletal muscle relaxation with diazo-2: the effect of altered pH.

In a fatigued muscle fibre, the concentrations of ADP, Pi and H+ are all increased and relaxation is slowed. We have used the technique of laser flash photolysis of the caged calcium-chelator, diazo-2, to investigate the direct effect of changes in pH (pH 6.5, 7.0, 7.5) upon tension during relaxation of single chemically skinned fibres, when the effects of the sarcoplasmic reticulum are absent. The relaxation transients were closely fitted with 2 exponentials, a fast (42.3 +/- 1.4; pH 7.0) and a slow process (12.0 +/- 0.7; pH 7.0). The fast phase of relaxation was pH sensitive; lowering pH leading to a slowing of the rate of force decline and raising pH leading to an increase of the rate. The rate of the slow phase was unaltered by changing pH over the range investigated. Thus the slowing of relaxation in fatigued muscle may be due, in part, to the direct action of protons on the myofilaments independent of any effects upon the sarcoplasmic reticulum.

Animals↗
PubMed1991

Amino acid sequences and Ca2(+)-binding properties of two isoforms of barnacle troponin C.

Much of our knowledge of the relationship between elevation of free sarcoplasmic [Ca2+] and skeletal muscle contraction has come from physiological studies on barnacle muscle fibers. Little is known, however, about the biochemical properties of the barnacle proteins responsible for Ca2+ regulation. In order to help rectify this unfortunate situation, we purified the two major isoforms (BTnC1 and BTnC2) of troponin C (TnC) from the giant barnacle, Balanus nubilis, and determined their amino acid sequences. BTnC2, the more abundant isoform, contains 151 amino acid residues and has a calculated molecular weight of 16,838. Due to an elongated N-terminus, BTnC1 contains 158 amino acid residues and has a calculated molecular weight of 17,984. The two sequences can be aligned from their C-termini, with no insertions or deletions, but they are only 61% identical. Sequence differences are twice as frequent in the N-terminal halves as in the C-terminal halves but occur in all regions of the polypeptides. This indicates that BTnC1 and BTnC2 are products of different genes, rather than alternative transcripts of a single gene. Both isoforms contain the usual four ancestral Ca2(+)-binding regions, numbered I-IV from the N-terminus. Analysis of the sequences predicts that functional Ca2(+)-binding sites are present only in regions II and IV and that these sites are low-affinity Ca2(+)-specific type sites. Direct Ca2(+)-binding measurements using fluorescent Ca2+ indicators show that both isoforms bind 2 Ca2+/mol with equal affinity (KCa = 1.3 x 10(5) M-1) and the sites appear to be Ca2(+)-specific.

Amino Acid Sequence↗
PubMed1991

Functional characterization of the two isoforms of troponin C from the arthropod Balanus nubilus.

Two isoforms of troponin C (BTnC1 and BTnC2) from the striated muscle of the arthropod Balanus nubilus Darwin (giant barnacle) have been purified (Potter et al., 1987; Collins et al., 1991). Both isoforms were present in all of the white striated muscle fibres studied but not in the red fibres. The ratio of BTnC2 to BTnC1 in different fibre types varied between 3:1 and 1:1. Both forms of TnC could be readily extracted from myofibrillar bundles of barnacle muscle in low ionic strength EDTA solutions, reducing force activation to less than 10%. Both forms either separately or together reassociated with the TnC-depleted fibres in a relaxing (LR) solution (pCa greater than 8.0, [Mg2+] free = 1 mM, I = 0.15 M), and the reconstituted fibres could be subsequently activated in contraction (LA) solution (pCa = less than 3.8, [Mg2+] free = 1 mM, I = 0.15 M). The dissociation of BTnC 1 + 2 is blocked in low ionic strength solutions containing Mg2+ (greater than or equal to 10 mM). The two isoforms of crayfish TnC (CrTnC1 and CrTnC2) were also found to be equivalent to the barnacle TnCs in their ability to reactivate TnC-depleted barnacle myofibrillar bundles. Similar experiments using rabbit skeletal muscle TnC (STnC) (I = 0.15 M) in BTnC-depleted myofibrillar bundles of barnacle showed considerable variability. STnC could associate, although weakly, with the depleted bundles in either LR or LA, and force could be partially restored. In neither situation was it as effective as either BTnC or CrTnC. Interestingly, bovine cardiac TnC (CTnC), although it did not associate at pCa greater than 7.0, did associate and effectively activate force at pCa less than 3.8, but dissociated on return to pCa greater than 7.0 (LR). Neither barnacle TnC isoform associated with TnC-depleted skinned fibres from rabbit skeletal muscle at pCa greater than 7.0, but did associate and activate these fibres at pCa less than 3.8. Once these fibres were returned to LR and then placed in LA at pCa 3.8 all BTnC-restored force was lost, indicating a dissociation of BTnC once the Ca2+ is lowered, as observed with CTnC in barnacle myofibrillar bundles. Finally, the inhibitory effect of BTnI on force and the absence of an effect of calmodulin, trifluoperazine or ATP-gamma-S on force were all taken as evidence for a thin filament regulated Ca2+ control system.

Animals↗
PubMed1991

Time-resolved changes in equatorial x-ray diffraction and stiffness during rise of tetanic tension in intact length-clamped single muscle fibers.

We report the first time-resolved x-ray diffraction studies on tetanized intact single muscle fibers of the frog. The 10, 11, 20, 21, 30, and Z equatorial reflections were clearly resolved in the relaxed fiber. The preparation readily withstood 100 1-s duration (0.4-s beam exposure) tetani at 4 degrees C (less than 4% decline of force and no deterioration in the 10, 11 equatorial intensity ratio at rest or during activation). Equatorial intensity changes (10 and 11) and fiber stiffness led tension (t1/2 lead 20 ms at 4 degrees C) during the tetanus rise and lagged during the isometric phase of relaxation. These findings support the existence of a low force cross-bridge state during the rise of tetanic tension and isometric relaxation that is not evident at the tetanus plateau. In "fixed end" tetani lattice expansion occurred with a time course similar to stiffness during the tetanus rise. During relaxation, lattice spacing increased slightly, while the sarcomere length remained isometric, but underwent large changes after the "shoulder" of tension. Under length clamp control, lattice expansion during the tetanus rise was reduced or abolished, and compression (2%) of the lattice was observed. A lattice compression is predicted by certain cross-bridge models of force generation (Schoenberg, M. 1980. Biophys. J. 30:51-68; Schoenberg, M. 1980. Biophys. J. 30:69-78).

Animals↗
PubMed1990

Detection of radial crossbridge force by lattice spacing changes in intact single muscle fibers.

Time-resolved lattice spacing changes were measured (10-millisecond time resolution) by x-ray diffraction of synchrotron radiation in single intact muscle fibers of the frog Rana temporaria undergoing electrically stimulated tension development during application of stretches and releases. Ramp releases, which decreased fiber length at constant speed, caused a lattice expansion. After the ramp, increasing tension during recovery was accompanied by lattice compression. Ramp stretches caused a compression of the lattice. While the fiber was held at a constant length after the stretch, tension decreased and lattice spacing increased. These observations demonstrate the existence of a previously undetected radial component of the force generated by a cycling crossbridge. At sarcomere lengths of 2.05 to 2.2 micrometers, the radial force compresses the myofilament lattice. Hence, the myofilament lattice does not maintain a constant volume during changes in force.

Animals↗
PubMed1990

Rapid activation by photolysis of nitr-5 in skinned fibres of the striated adductor muscle from the scallop.

Photolysis of nitr-5, a caged calcium molecule, has been used for rapid activation of skinned fibre bundles of a myosin-regulated muscle, the striated adductor of the scallop, Pecten maximus. Chemically skinned fibre bundles (diameter 70-200 microns) were equilibrated in solutions containing 1-3 mM nitr-5 (pCa 6.1) and then activated by ultraviolet laser pulse (25 ns). Pulse energies of 60-95 mJ gave contractions of over 90% maximum tension and a mean half-time for tension rise of 43 ms (n = 4) at 12 degrees C. Electrically stimulated bundles of intact fibres develop a tetanus with a rise half-time of 60.2 ms at 10 degrees C (n = 5) (Rall, J.A. (1981) J. Physiol. 321, 287-295, and personal communication). At lower pulse energies the skinned fibres gave smaller amplitude contractions with slower rates of rise (up to 260 ms half-time). In addition, a slower component of tension development (mean rise half-time 13.3 s) was often observed. In ATP-free solutions containing hexokinase and glucose, rigour tension developed with a delayed onset. Rapid release of ATP (0.47-0.59 mM) from photolysis of caged ATP (2 mM) at pCa 4.5 then caused a rapid contraction with a mean half-time for tension development of 17 ms (n = 4). The fast activation rates obtained by the photorelease of Ca2+ from nitr-5 are similar to those obtained with skinned skeletal fibres of actin-regulated muscle. The results imply that the rate-limiting step in excitation-contraction coupling of the scallop muscle is not the increase in sarcoplasmic Ca2+, but rather the activation of the muscle in response to this increase. The half-times of ATP-induced contractions at pCa 4.5 suggest that in a contraction activated by a rapid Ca2+ jump the process comprising ATP hydrolysis and cross-bridge cycling occurs at a somewhat faster rate than the Ca2(+)-dependent activation process which precedes it.

Adenosine Triphosphate↗
PubMed1990

Striated scallop muscle relaxation: fast force transients produced by photolysis of Diazo-2.

Relaxation of the myosin regulated striated adductor muscles of Pecten maximus was initiated by the photolysis of the caged Ca2+ chelator, Diazo-2. The fibres relaxed to approximately 30% of the maximum tension with a mean half-time of 17.9 +/- 1.6 ms (n = 7, temp 12 degrees C), much faster than the rates observed in intact muscle at the same temperature. This indicates that in the intact adductor muscle the slower relaxation rate is determined by the speed of Ca2+ removal from the sarcoplasm. The faster rate of relaxation of scallop muscle in vitro, compared with frog skeletal muscle may reflect different mechanisms of regulation of the crossbridge cycle.

Animals↗
PubMed1990

Evidence for receptor-mediated bivalent-cation entry in A10 vascular smooth-muscle cells.

In fura-2-loaded A10 vascular smooth-muscle cells, 1 nM-vasopressin and 200 nM-endothelin evoked a rapid transient rise in intracellular free Ca2+ concentration [( Ca2+]i), which was then followed by a maintained elevation of [Ca2+]i. The maintained elevation of [Ca2+]i was only partially inhibited by 5 microM-nifedipine, but completely abolished in the presence of 1 mM-EGTA. When extracellular Ca2+ was replaced with 1 mM-Mn2+ (Mn2+ quenches fura-2 fluorescence), both endothelin and vasopressin evoked an Mn2+ quench of the fluorescence from the intracellularly trapped fura-2, even in the presence of 5 microM-nifedipine. These data suggest that both vasopressin and endothelin promote a bivalent-cation influx and provide further evidence for receptor-mediated Ca2+ entry in vascular smooth muscle.

Calcium↗
PubMed1990

Mechanical characteristics of skinned and intact muscle fibres from the giant barnacle, Balanus nubilus.

Intact muscle fibres from Balanus nubilus develop tensions of up to 600 kN.m-2 during electrical stimulation. The rise of tension occurs with a half-time (177 ms at 12 degrees C) about fivefold longer than that of tetanised frog muscle at the same temperature. The response of myofibrillar bundles to a rapid stretch resembles that of frog muscle but has a yo value (i.e. the size of an instantaneous release necessary to just discharge tension) which is ca. 2.5 times smaller, and phase 2 of the tension transient (the "quick phase") occurs at a rate comparable to that of frog muscle. In contrast, the ATPase activity (0.018 mmoles.kg wet weight-1.s-1) of this preparation and its maximum shortening velocity (0.15-0.16 muscle lengths.s-1) are both at least fivefold slower than frog muscle. These findings can be accounted for by a cross-bridge cycle in barnacle muscle in which events prior and subsequent to the tension generating step(s) occur at a rate at least fivefold slower than comparable steps in frog muscle, but the step(s) associated with tension development occur at similar rates in the two preparations. Since the rate of mechanical relaxation in barnacle muscle is modified in the presence of intracellular calcium buffers and by depolarisation-induced elevation of the free calcium during the relaxation phase, it is proposed that the time course of relaxation is not determined exclusively by the kinetics of the cross-bridge cycle, but is also dependent on the free calcium concentration during relaxation.

Adenosine Triphosphatases↗
PubMed1990

Ca2+ release from the sarcoplasmic reticulum of barnacle myofibrillar bundles initiated by photolysis of caged Ca2+.

1. Ca2(+)-induced Ca2+ release (CICR) from the sarcoplasmic reticulum was measured by isometric tension recording from barnacle myofibrillar bundles. Laser-induced photolysis of the caged calcium molecule, nitr-5, was used to generate a rapid jump in free Ca2+ (within 1 ms) at the site of the sarcoplasmic reticulum, thus overcoming delays due to Ca2+ diffusion from the bathing solution. 2. The method consisted of equilibrating a myofibrillar bundle (100 micrograms diameter) in a solution containing 0.1 mM-nitr-5 (initial pCa 6.8-6.6) and then exposing it to a UV laser pulse. The resulting phasic contraction had an amplitude of up to 100% maximum tension (P0) in some preparations and a mean half-time for the rise of tension of 2.3 s at 12 degrees C. Longer half-times were obtained at low pulse energies. 3. Pre-treatment of the myofibrillar bundles with ryanodine (10(-4) M) or the detergent Triton X-100 abolished a large part of the phasic contraction, confirming its dependence on SR Ca2+ release. The small tonic response which remained had a shorter rise half-time than the Ca2(+)-induced Ca2+ release response and was attributed to direct activation of the myofibrils by Ca2+ released from the nitr-5. 4. The size of the photolytic Ca2+ jump was estimated from the amplitude of the fast tension component. By increasing the laser pulse energy or the initial Ca2+ loading of the nitr-5, the post-photolysis pCa was varied from 6.7 to 6.0; the CICR response increased in size over this pCa range. 5. Direct activation of Triton-treated myofibrils by photolysis of 2.0 mM-nitr-5 (initial pCa 6.4) gave contractions of up to 100% P0 and a mean rise half-time of 164 ms at 12 degrees C (n = 9 for contractions greater than 40% P0). Both the amplitude and the rate of these contractions were dependent on the laser pulse energy. 6. The Ca2(+)-induced Ca2+ release responses obtained with nitr-5 photolysis were significantly slower than the fastest rate of tetanus development which has been recorded from intact fibres of barnacle muscle (mean half-time = 177 ms at 12 degrees C). This could mean that either Ca2(+)-induced Ca2+ release is less efficient in isolated myofibrillar bundles than in intact fibres or that Ca2(+)-induced Ca2+ release is not the primary Ca2+ releasing mechanism in excitation-contraction coupling in barnacle muscle.

Animals↗
PubMed1989

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.

Animals↗
PubMed1989

Endothelin evoked Ca2+ transients and oscillations in A10 vascular smooth muscle cells.

Endothelin (200 nM) evoked a rapid rise in [Ca2+]i which was then followed by a maintained elevation of [Ca2+]i. The initial transient can be explained by the release of stored Ca2+ whilst the maintained plateau is likely to be an influx of Ca2+ as it was partially inhibited by nifedipine (5 microM) and the remaining component abolished by the removal of extracellular Ca2+. Vasopressin (1 nM) evoked a similar response which also showed a nifedipine insensitive component to it's plateau phase. Endothelin also evoked oscillations in [Ca2+]i; these where characterised by a rapid rising phase followed by a slower decline, with no 'pacemaker' rise in [Ca2+]i preceding the rising phase. The oscillations were inhibited by the addition of 5 microM nifedipine or the removal of extracellular Ca2+ suggesting they are at least in part dependent on voltage gated Ca2+ entry.

Animals↗