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Biomedical subjects

D G Allen

Publications and source records attributed to D G Allen.

At least 37 records · Page 2Linked to original sources
PubMed2001

Dopaminergic therapy with carbidopa L-dopa for left neglect after stroke: a case series.

OBJECTIVE: To evaluate the efficacy of carbidopa L-dopa (Sinemet) in reducing left spatial neglect after stroke. DESIGN: Case series. SETTING: Inpatient neurorehabilitation unit in a regional rehabilitation center. PARTICIPANTS: A convenience sample of 4 women with right brain strokes and left neglect. INTERVENTION: A trial of carbidopa L-dopa to treat left neglect, if indicated by selected subtests of the Behavioral Inattention Test (BIT). MAIN OUTCOME MEASURES: Baseline and posttreatment evaluation with the modified BIT and the FIM instrument. RESULTS: Three of 4 subjects had significant improvements in their modified BIT scores (8%, 12%, 27%, respectively) and their functional status on the FIM. CONCLUSION: With further study, carbidopa L-dopa may be shown to reduce unilateral spatial neglect and thereby improve rehabilitation outcomes.

Activities of Daily Living↗
PubMed2000

The mechanisms of sarcoplasmic reticulum Ca2+ release in toad pacemaker cells.

The mechanisms of sarcoplasmic reticulum (SR) Ca2+ release in pacemaker cells from the sinus venosus of the cane toad (Bufo marinus) were studied. Single, isolated cells were voltage clamped using a nystatin-perforated patch. Ionic currents and intracellular Ca2+ concentration ([Ca2+]i) were recorded simultaneously. Depolarizations of 300 ms duration from a holding potential of -55 mV produced an inward current which had a bell-shaped relationship with voltage. Inward current first appeared at about -45 mV, reached a maximum of -343 +/- 46 pA at -15 mV and reversed at +45 mV. In contrast the amplitude of the increase in [Ca2+]i caused by depolarization (Ca2+ transient) increased monotonically with the increasing depolarization. At -15 mV the amplitude of the Ca2+ transient was 243 +/- 33 nM and at +45 mV it was 411 +/- 43 nM. The inward current produced by depolarizations to -5 mV was largely eliminated by the L-type Ca2+ channel blocker nifedipine (10 microM) while 37 +/- 7 % of the Ca2+ transient persisted. A significantly larger proportion of the Ca2+ transient (56 +/- 5 %) remained at +85 mV in the presence of nifedipine. The SR Ca2+ pump inhibitor 2, 5-di(tert-butyl)-1,4-hydroquinone (10 microM), which causes depletion of the SR Ca2+, reduced the amplitude of the Ca2+ transient to 34 +/- 1 % of control, irrespective of the voltage. Brief exposure to extracellular Ca2+-free solution abolished the Ca2+ transients caused by depolarization while the caffeine-induced Ca2+ release persisted. Tetrodotoxin (1 microM) had no effect on the amplitude of the depolarization-induced Ca2+ transient, although it reduced the fast component of the inward current. In contrast, Ni2+ (5 mM) abolished the Ca2+ transients at any given voltage. Ni2+ also abolished spontaneous Ca2+ transients. In conclusion, in toad pacemaker cells Ca2+ release from SR contributes approximately 66 % of the Ca2+ involved in the Ca2+ transient and requires extracellular Ca2+ influx to trigger its release. The L-type Ca2+ channels and Na+-Ca2+ exchange are major sources of Ca2+ influx under physiological conditions.

Action Potentials↗
PubMed2000

Identification of early biomarkers of inflammation produced by keratinocytes exposed to jet fuels jet A, JP-8, and JP-8(100).

The purpose of this study was to identify biomarkers of inflammation in normal human epidermal keratinocytes (NHEK) exposed to three jet fuel mixtures, Jet A, JP8, and JP8(100). NHEK were treated over 24 hours with 0.1% jet fuels, and mRNA production and protein release of two proinflammatory cytokines, IL-8 and TNF-alpha, were determined. Using an enzyme-linked immunosorbent assay (ELISA), NHEK were found to release both TNF-alpha and IL-8 in response to exposure to all three jet fuels. IL-8 release was noted within 8 hours and continued to rise through 24 hours compared to controls. Maximal levels of TNF-alpha release were seen at 4 hours and decreased in a time-dependent manner, although these levels remained above control levels at all time points assayed. mRNA for IL-8 was elevated 4 hours following exposure to the fuels, which was detected via a quantitative competitive reverse transcriptase-polymerase chain reaction (RT-PCR). mRNA for TNF-alpha was detected at all time points assayed but was not quantified. These results demonstrate that jet fuels induce the production and release of proinflammatory cytokines in NHEK and thus create the potential for chronic inflammation, which may contribute to the development or progression of disease states in the skin.

Biomarkers↗
PubMed2000

Functional significance of Ca2+ in long-lasting fatigue of skeletal muscle.

Repeated activation of skeletal muscle causes fatigue, which involves a reduced ability to produce force and slowed contraction regarding both the speed of shortening and relaxation. One important component in skeletal muscle fatigue is a reduced sarcoplasmic reticulum (SR) Ca2+ release. In the present review we will describe different types of fatigue-induced inhibition of SR Ca2+ release. We will focus on a type of long-lasting failure of SR Ca2+ release which is called low-frequency fatigue, because this type of fatigue may be involved in the muscle dysfunction and chronic pain experienced by computer workers. Paradoxically it appears that the Ca2+ released from the SR, which is required for contraction, may actually be responsible for the failure of SR Ca2+ release during low-frequency fatigue. We will also discuss the relationship between gross morphological changes in muscle fibres and long-lasting failure of SR Ca2+ release. Finally, a model linking muscle cell dysfunction and muscle pain is proposed.

Calcium↗
PubMed2000

The distribution of calcium in toad cardiac pacemaker cells during spontaneous firing.

Isolated, spontaneously active pacemaker cells from the sinus venosus region of the toad heart were loaded with the calcium indicator fluo-3. The cells were examined with a confocal microscope to investigate the distribution of calcium during spontaneous activity. Three classes of calcium-related signals were present. First, intense, localised, time-invariant signals were detected from structures distributed across the cell interior. Based on the insensitivity to saponin and the distribution in the cell, these signals appear to arise from fluo-3 located in the sarcoplasmic reticulum and the nuclear envelope. Second, spatially uniform signals from the cytoplasm were present at rest and showed spontaneous increases in [Ca2+]i which propagated along the cell. These Ca2+ transients were uniform in intensity across the diameter of the cell and we could detect no significant delay in the middle of the cell compared to the edges. However, within the nucleus the Ca2+ transient showed a clear delay compared to the cytoplasm. Third, localised, transient increases in [Ca2+]i (Ca2+ sparks) which did not propagate were also detectable. These could be detected both near the surface membrane and in the interior of the cell and reduced in magnitude and increased in duration in the presence of ryanodine. The frequency of firing of Ca2+ sparks significantly increased in the 200-ms period preceding a spontaneous Ca2+ transient. These results suggest that pacemaker cells contain sarcoplasmic reticulum which is distributed across the cell. The Ca2+ transient is uniform across the cell indicating that near-synchronous release of Ca2+ from the sarcoplasmic reticulum is achieved. Ca2+ sparks occur in pacemaker cells though their role in pacemaker function remains to be elucidated.

Action Potentials↗
PubMed2000

Activity of the Na(+)/H(+) exchanger is critical to reperfusion damage and preconditioning in the isolated rat heart.

OBJECTIVE: Removal of protons from the heart during ischemia and/or reperfusion by the cardiac Na(+)/H(+) exchanger (NHE1) leads to Na(+) entry; this causes Ca(2+) influx and is thought to contribute to ischemic and/or reperfusion damage. The extent to which Na(+) enters during ischemia as opposed to reperfusion is disputed and has important implications for the therapeutic use of NHE1 inhibitors as protection against ischemic damage. Preconditioning has recently been proposed to inhibit NHE1 during reperfusion. The objective of the present study was to determine the activity of NHE1 during ischemia, reperfusion and following preconditioning. METHODS: The experiments were on isolated perfused rat hearts in which left ventricular developed pressure (LVDP) and intracellular sodium and pH were measured. RESULTS: LVDP following 30 min of ischemia recovered to 14+/-3% of pre-ischemic level. Application of the NHE1 inhibitor HOE 642 during ischemia and reperfusion improved recovery of LVDP to 77+/-9%. When HOE was applied at the moment of reperfusion the recovery of LVDP was reduced to 54+/-6%. To overcome possible delays in the delivery of HOE, the drug was applied at 28 min of ischemia; under these conditions recovery of LVDP (71+/-7%) was not significantly different to HOE throughout ischemia and reperfusion. HOE had no effect on the recovery of preconditioned hearts. NHE1 activity was assessed by the [Na(+)](i) and pH(i) changes in response to brief exposure to Na(+) lactate (NaL). In control hearts, activity of NHE1 caused a pH(i) recovery of 0. 034+/-0.007 pH units and was associated with a [Na(+)](i) rise of 7. 5+/-0.5 mmol/l. After 5-min reperfusion following ischemia, NaL application caused a pH(i) recovery (0.046+/-0.007) and a larger [Na(+)](i) rise (15.8+/-0.6 mmol/l). After 5-min reperfusion of preconditioned hearts, NaL application caused a smaller recovery pH recovery (0.013+/-0.002) and a smaller [Na(+)](i) rise (4.2+/-0.5 mmol/l). CONCLUSIONS: These findings suggest that NHE1 is activated in early reperfusion after ischemia but inhibited during early reperfusion in preconditioned hearts. Overall our results point to a critical period of activity of NHE1 in early reperfusion which is inhibited by preconditioning.

Analysis of Variance↗
PubMed2000

Intracellular calcium during fatigue of cane toad skeletal muscle in the absence of glucose.

Mechanisms of fatigue were studied in single muscle fibres of the cane toad (Bufo marinus) in which force, intracellular calcium ([Ca2+]i), [Mg2+]i, glycogen and the rapidly releasable Ca2+ from the sarcoplasmic reticulum (SR) were measured. Fatigue was produced by repeated tetani continued until force had fallen to 50%. Two patterns of fatigue in the absence of glucose were studied. In the first fatigue run force fell to 50% in 8-10 min. Fatigue runs were then repeated until force fell to 50% in < 3 min in the final fatigue run. Addition of extracellular glucose after the final fatigue run prolonged a subsequent fatigue run. In the first fatigue run peak tetanic [Ca2+]i initially increased and then declined and at the time when force had fallen to 50% tetanic [Ca2+]i was 54+/-5% of initial value. In the final fatigue run force and peak tetanic [Ca2+]i declined more rapidly but to the same level as in first fatigue runs. At the end of the first fatigue run, the rapidly releasable SR Ca2+ store fell to 46+/-6% of the pre-fatigue value. At the end of the final fatigue run the rapidly releasable SR Ca2+ store was 109+/-16% of the pre-fatigue value. In unstimulated fibres the nonwashable glycogen content was 176+/-30 mmol glycosyl units/l fibre. After one fatigue run the glycogen content was 117+/-17 mmol glycosyl units/l fibre; at the end of the final fatigue run the glycogen content was reduced to 85+/-9 mmol glycosyl units/l fibre. [Mg2+]i did not change significantly at the end of fatigue in either the first or the final fatigue run suggesting that globally-averaged ATP does not decline substantially in either pattern of fatigue. These results suggest that different mechanisms are involved in the decline of tetanic [Ca2+]i in first compared to final fatigue runs. The SR Ca2+ store is reduced in first fatigue runs; this is not the case for the final fatigue run which is associated with a decline in glycogen and possibly related to either a non-metabolic effect of glycogen or a spatially-localised metabolic decline.

Animals↗
PubMed2000

Activity of the Na+/H+ exchanger contributes to cardiac damage following ischaemia and reperfusion.

1. The present review considers the evidence that Na+-H+ exchange activity contributes to cardiac damage following ischaemia and reperfusion. The basic mechanism involved is that protons are produced during ischaemia and leave the myocytes on the Na+/H+ exchanger during either ischaemia and/or reperfusion. The resulting elevation of [Na+]i causes Ca2+ loading through the Na+/Ca2+ exchanger and the elevated [Ca2+]i is thought to lead to myocardial damage. 2. Inhibition of the Na+/H+ exchanger during ischaemia and/or reperfusion produces a substantial cardioprotective effect by blocking the damage caused by the coupled exchanger mechanism described above. Preconditioning also produces a cardioprotective effect and the evidence that this also involves the Na+/H+ exchanger is reviewed. 3. The intracellular mechanisms associated with ischaemic damage and preconditioning are of great interest because they may provide targets for potential therapeutic interventions. The intracellular regulation of the Na+/H+ exchanger appears to be an important component of these pathways and may become a focus for therapeutic approaches.

Animals↗
PubMed2000

Progressive genetic aberrations detected by comparative genomic hybridization in squamous cell cervical cancer.

Genetic changes orchestrated by human papillomaviruses are the most important known factors in carcinogenesis of the uterine cervix. However, it is clear that additional genetic events are necessary for tumour progression. We have used comparative genomic hybridization to document non-random chromosomal gains and losses within a subset of 37 cervical carcinomas matched for clinical stage Ib, but with different lymph node status. There were significantly more chromosomal changes in the primary tumours when the lymph nodes were positive for metastases. The most frequent copy number alterations were loss of 3p, 11q, 6q and 10q and gain of 3q. The smallest areas of loss and gain on chromosome 3 were 3p14-22 and 3q24-26. The study identifies progressive DNA copy number changes associated with early-stage invasive cervical cancers with and without lymph node metastases, a factor of potential prognostic and therapeutic value.

Adult↗
PubMed2000

Paraxanthine, a caffeine metabolite, dose dependently increases [Ca(2+)](i) in skeletal muscle.

It was hypothesized that the caffeine derivative paraxanthine results in subcontracture increases in intracellular calcium concentration ([Ca(2+)](i)) in resting skeletal muscle. Single fibers obtained from mouse flexor digitorum brevis were loaded with a fluorescent Ca(2+) indicator, indo 1-acetoxymethyl ester. After a stable baseline was recorded, the fiber was superfused with physiological salt solution (Tyrode) containing 0.5, 1.0, 2.5, or 5 mM paraxanthine, resulting in [Ca(2+)](i) increases of 6.4 +/- 2.5, 9.7 +/- 3.6, 26.8 +/- 11.7, and 39.6 +/- 9.6 nM, respectively. The increases in [Ca(2+)](i) were transient and were also observed with exposure to 5 mM theophylline and theobromine. Six fibers were exposed to 5 mM paraxanthine followed by 5 mM paraxanthine in the presence of 10 mM procaine (sarcoplasmic reticulum Ca(2+) release channel blocker). There was no increase from baseline [Ca(2+)](i) when fibers were superfused with paraxanthine and procaine, suggesting that the sarcoplasmic reticulum is the primary Ca(2+) source in the paraxanthine-induced response. In separate experiments, intact flexor digitorum brevis (n = 13) loaded with indo 1-acetoxymethyl ester had a significant increase in [Ca(2+)](i) with exposure to 0.01 mM paraxanthine. It is concluded that physiological and low pharmacological concentrations of paraxanthine result in transient, subcontracture increases in [Ca(2+)](i) in resting skeletal muscle, the magnitude of which is related to paraxanthine concentration.

Animals↗
PubMed1999

Role of Na(+)/H(+) exchanger during ischemia and preconditioning in the isolated rat heart.

The role of the Na(+)/H(+) exchanger in ischemia, reperfusion, and preconditioning was investigated in isolated perfused rat hearts. Contractile function, [Na(+)](i), and pH(i) were measured; ischemic damage was assessed by the recovery of developed pressure (DP) on reperfusion. After 30 minutes of ischemia, DP recovered to only 14+/-4% of preischemic control. In contrast, after preconditioning (3x5-minute periods of ischemia) followed by 30 minutes of ischemia, DP recovered to 75+/-4%. Hearts treated with the Na(+)/H(+) exchange inhibitor 5-(N-methyl-N-isobutyl)amiloride (MIA) also showed an enhanced recovery after ischemia (DP 62+/-9%). Treatment with a low concentration of tetrodotoxin (TTX, 100 nmol/L), which blocks the persistent component of the Na(+) current, had a small beneficial effect on recovery (DP 37+/-8%). Thirty minutes of ischemia caused a small [Na(+)](i) rise (3.2+/-0.9 mmol/L); reperfusion resulted in a further [Na(+)](i) increase (+11.9+/-2.5 mmol/L), which partially recovered over 30 minutes. Preconditioning did not change the [Na(+)](i) rise during ischemia but abolished the large [Na(+)](i) rise on reperfusion, and [Na(+)](i) instead fell (-3.6+/-1.3 mmol/L). In the presence of MIA, the [Na(+)](i) rise was unchanged from ischemia only; on reperfusion, [Na(+)](i) fell (-3.7+/-0.9 mmol/L), similar to the preconditioned hearts. TTX abolished the [Na(+)](i) rise during ischemia (+0.3+/-0.7 mmol/L), and the increase on reperfusion was similar to ischemia only. We conclude that the rise of [Na(+)](i) during ischemia is caused by Na(+) entry through persistent Na(+) channels. The rise of [Na(+)](i) on reperfusion is caused by activation of the Na(+)/H(+) exchanger and is blocked by MIA and by preconditioning. It is known that the Na(+)/H(+) exchanger is inhibited during ischemia; the present result suggests that this inhibition is prolonged into the early part of reperfusion by preconditioning. To test this hypothesis, we measured the time course of pH(i) recovery after ischemia and preconditioning. Preconditioning slowed the rate of pH(i) recovery after ischemia, providing further support for the hypothesis that preconditioning inhibits the Na(+)/H(+) exchanger during early reperfusion. This inhibition of the Na(+)/H(+) exchanger during reperfusion prevents Na(+) entry, and therefore Ca(2+) loading, and is part of the protective pathway involved in preconditioning.

Animals↗
PubMed1999

The role of calcium stores in fatigue of isolated single muscle fibres from the cane toad.

1. Intracellular calcium ([Ca2+]i) and tension were measured from single muscle fibres dissected from the cane toad (Bufo marinus). The amount of Ca2+ which could be released from the sarcoplasmic reticulum (SR) was estimated by brief (approximately 20 s) exposures to 4-chloro-m-cresol (4-CmC) or caffeine. 2. Muscle fatigue was produced by repeated tetani at 4 s or shorter intervals and continued until tension had fallen to 50% of the control. The intracellular free calcium concentration during a tetanus (tetanic [Ca2+]i) first increased and then steadily declined to 43+/-2% of control by the time tension had fallen to 50%. Over the period of fatigue the rapidly releasable Ca2+ from the SR fell to 46+/-6% of control. Tension and tetanic [Ca2+]i recovered to 93+/-3% and 100+/-4% of the control values after 20 min of rest. Over the same period rapidly releasable SR Ca2+ recovered to 98+/-12%. 3. When a similar number of tetani (200) were repeated at longer intervals (10 s), fibres showed only a small reduction in tension (to 85+/-1%) and tetanic [Ca2+]i did not change significantly. Under these conditions the rapidly releasable SR Ca2+ did not change significantly. 4. The recovery of rapidly releasable SR Ca2+ after fatigue was unaffected by removal of extracellular calcium but did not occur when oxidative phosphorylation was inhibited with cyanide. 5. These results suggest that an important cause of the decline of tetanic [Ca2+]i during fatigue is an equivalent decline in the amount of rapidly releasable SR Ca2+. The results show that the decline of rapidly releasable SR Ca2+ is related to a metabolic consequence of fatigue and are consistent with the hypothesis that Ca2+ precipitates with phosphate in the SR during fatigue.

Animals↗
PubMed1999

Skeletal muscle hypertrophy is mediated by a Ca2+-dependent calcineurin signalling pathway.

Skeletal muscle hypertrophy and regeneration are important adaptive responses to both physical activity and pathological stimuli. Failure to maintain these processes underlies the loss of skeletal muscle mass and strength that occurs with ageing and in myopathies. Here we show that stable expression of a gene encoding insulin-like growth factor 1 (IGF-1) in C2C12 skeletal muscle cells, or treatment of these cells with recombinant IGF-1 or with insulin and dexamethasone, results in hypertrophy of differentiated myotubes and a switch to glycolytic metabolism. Treatment with IGF-1 or insulin and dexamethasone mobilizes intracellular calcium, activates the Ca2+/calmodulin-dependent phosphatase calcineurin, and induces the nuclear translocation of the transcription factor NF-ATc1. Hypertrophy is suppressed by the calcineurin inhibitors cyclosporin A or FK506, but not by inhibitors of the MAP-kinase or phosphatidylinositol-3-OH kinase pathways. Injecting rat latissimus dorsi muscle with a plasmid encoding IGF-1 also activates calcineurin, mobilizes satellite cells and causes a switch to glycolytic metabolism. We propose that growth-factor-induced skeletal-muscle hypertrophy and changes in myofibre phenotype are mediated by calcium mobilization and are critically regulated by the calcineurin/NF-ATc1 signalling pathway.

3T3 Cells↗
PubMed1999

How does beta-adrenergic stimulation increase the heart rate? The role of intracellular Ca2+ release in amphibian pacemaker cells.

1. The mechanism by which sympathetic transmitters increase the firing rate of pacemaker cells was explored in isolated cells from the sinus venosus of the cane toad Bufo marinus. Intracellular calcium concentration ([Ca2+]i) was measured with indo-1 and membrane potential and currents were recorded with the nystatin perforated-patch technique. 2. Adrenaline or isoprenaline (2 microM) increased the transient rise in [Ca2+]i and increased the firing rate; these effects were blocked by propranolol (2 microM). 3. To determine whether the changes in [Ca2+]i might influence the firing rate we studied agents which affect either the loading or the release of Ca2+ from the sarcoplasmic reticulum (SR). Rapid application of caffeine (10 mM) to spontaneously firing cells caused a large Ca2+ release from the SR and the cells were then quiescent for 24 s. In the presence of beta-adrenergic stimulation the caffeine-induced [Ca2+]i was 14 % larger but the period of quiescence after application was reduced to 12 s. 4. Ryanodine, at either low (1 microM) or high (> 10 microM) concentration, stopped firing. However, when the SR store content of Ca2+ was tested with caffeine, at low ryanodine concentration the SR Ca2+ store was empty whereas at the high concentration the SR store was still loaded with Ca2+. beta-Adrenergic stimulation was not able to restore firing at the low concentration of ryanodine but did restore firing at the high ryanodine concentration. 5. An SR Ca2+ pump blocker, 2, 5-di(tert-butyl)-1,4-hydroquinone (TBQ) which depletes the SR store of Ca2+, also rapidly and reversibly stopped spontaneous firing. 6. The relation between the amplitude of the [Ca2+]i transient and firing rate established in the presence of ryanodine was similar when firing was restored by beta-stimulation. 7. In both spontaneously firing and voltage-clamped cells, depleting the SR store with either ryanodine or TBQ suggested that about half of the Ca2+ which contributes to the calcium transient is released from the SR. 8. These results show that the amplitude of the [Ca2+]i transient is an important factor in the firing rate of toad pacemaker cells and consequently agents which modify SR Ca2+ release influence firing rate. The effects of beta-stimulation on firing rate seem to be largely mediated by changes in amplitude of the [Ca2+]i transient.

Adrenergic beta-Agonists↗
PubMed1999

Does adrenaline modulate the Na+-Ca2+ exchanger in isolated toad pacemaker cells?

beta-Adrenergic stimulation of pacemaker cells from the sinus venosus of the cane toad (Bufo marinus) increases intracellular calcium ([Ca2+]i) and firing rate. The increase in [Ca2+]i could contribute to the increased firing rate by increasing the inward Na+-Ca2+ exchange current (INa-Ca) during diastole. In this study we measured [Ca2+]i and membrane currents in single, isolated, voltage-clamped pacemaker cells. We show that INa-Ca increases during beta-adrenergic stimulation. To test whether this increase in INa-Ca is caused by elevated [Ca2+]i or by changes in the properties of the Na+-Ca2+ exchanger, we made rapid applications of caffeine and plotted the INa-Ca against [Ca2+]i. This relationship was linear during the declining phase of the [Ca2+]i signal caused by caffeine and was not significantly different in the presence or absence of beta stimulation. These results show that INa-Ca is increased during beta-adrenergic stimulation and will contribute to the increased firing rate. However the increase in INa-Ca appears to be a consequence of the increase in [Ca2+]i and is not caused by changes in the intrinsic properties of the Na+-Ca2+ exchanger.

Adrenergic beta-Agonists↗
PubMed1999

Measurement of sarcoplasmic reticulum Ca2+ content in intact amphibian skeletal muscle fibres with 4-chloro-m-cresol.

Single skeletal muscle fibres were isolated from the toad (Bufo marinus) and isometric force and myoplasmic free calcium concentration ([Ca2+]i) were measured. Brief applications of 4-chloro- m-cresol (4-CmC, 0.2-5 mM) elevated [Ca2+]i reversibly in a dose-dependent manner. The lowest concentration of 4-CmC which reliably gave maximal [Ca2+]i was 2 mM and it was, therefore, used for measurement of sarcoplasmic reticulum (SR) Ca2+ content. Tetanic stimulations (100 Hz) increased [Ca2+]i from a resting level of 105 +/- 47 nM (n = 10) to 1370 +/- 220 nM (n = 6). Application of 2 mM 4-CmC produced a contracture that was 54 +/- 16% (n = 6) of the tetanic force and elevated [Ca2+]i to a peak of 3520 +/- 540 nM (n = 8). Both force and [Ca2+]i levels (resting and tetanic) were restored after 10 min of washout of 4-CmC. In skinned muscle fibres, the myofibrillar Ca(2+)-sensitivity was not changed by 4-CmC, but maximal force was reduced to 74 +/- 10% (n = 4). The magnitude of the peak of the 4-CmC-induced Ca2+ transient was not significantly changed by removal of extracellular Ca2+ nor by inhibiting the SR Ca2+ pump with 2,5-di-tert-butylhydroquinone. Treatment of intact fibres with 30 mM caffeine produced a peak Ca2+ level that was indistinguishable from 2 mM 4-CmC. These results indicate that it is possible to measure the SR Ca2+ content in the same fibre with 4-CmC without loss of normal muscle function.

Animals↗