Mitochondrial function in intact skeletal muscle fibres of creatine kinase deficient mice

Creatine kinase (CK) has a central role in skeletal muscle, acting as a fast energy buffer and shuttle between sites of energy production (mitochondria) and consumption (cross-bridges and ion pumps). Unexpectedly, isolated fast-twitch skeletal muscle cells of mice deficient in both cytosolic and mitochondrial CK (CK-/-) are highly fatigue resistant during stimulation protocols that stress aerobic metabolism. We have now studied different aspects of mitochondrial function in CK-/- skeletal muscle. Intact, single fibres of flexor digitorum brevis (FDB) muscles were fatigued by repeated tetanic stimulation (70 Hz, 350 ms duration, duty cycle 0.14). Under control conditions, CK-/- FDB fibres were more fatigue resistant than wild-type fibres. However, after mitochondrial inhibition with cyanide, force declined markedly faster in CK-/- fibres than in wild-type fibres. The rapid force decline in CK-/- fibres was not due to decreased myoplasmic [Ca²⁺] during tetani (measured with indo-1), which in these fibres remained virtually constant during fatigue in the presence of cyanide. Intact, single fibres of highly oxidative soleus muscles were fatigued by repeated tetani (50 Hz, 500 ms duration, duty cycle 0.5). All CK-/- soleus fibres tested ( n = 9) produced > 40% force at the end of the fatiguing stimulation period (500 tetani), whereas force fell to < 40% before 500 tetani in two of three wild-type fibres. Mitochondrial [Ca²⁺] (measured with rhod-2 and confocal microscopy) increased during repeated tetanic stimulation in CK-/- but not in wild-type FDB fibres. In conclusion, mitochondria and energy shuttling operate effectively in CK-/- fibres and this is associated with an increase in mitochondrial [Ca²⁺].     

Interpolated twitches in fatiguing single mouse muscle fibres: implications for the assessment of central fatigue

An electrically evoked twitch during a maximal voluntary contraction (twitch interpolation) is frequently used to assess central fatigue. In this study we used intact single muscle fibres to determine if intramuscular mechanisms could affect the force increase with the twitch interpolation technique. Intact single fibres from flexor digitorum brevis of NMRI mice were dissected and mounted in a chamber equipped with a force transducer. Free myoplasmic [Ca²⁺] ([Ca²⁺]_i) was measured with the fluorescent Ca²⁺ indicator indo-1. Seven fibres were fatigued with repeated 70 Hz tetani until 40% initial force with an interpolated pulse evoked every fifth tetanus. Results showed that the force generated by the interpolated twitch increased throughout fatigue, being 9 ± 1% of tetanic force at the start and 19 ± 1% at the end ( P < 0.001). This was not due to a larger increase in [Ca²⁺]_i induced by the interpolated twitch during fatigue but rather to the fact that the force–[Ca²⁺]_i relationship is sigmoidal and fibres entered a steeper part of the relationship during fatigue. In another set of experiments, we observed that repeated tetani evoked at 150 Hz resulted in more rapid fatigue development than at 70 Hz and there was a decrease in force ('sag') during contractions, which was not observed at 70 Hz. In conclusion, the extent of central fatigue is difficult to assess and it may be overestimated when using the twitch interpolation technique.     

Reactive oxygen species reduce myofibrillar Ca2+ sensitivity in fatiguing mouse skeletal muscle at 37°C

The mechanisms of muscle fatigue were studied in small muscle bundles and single fibres isolated from the flexor digitorum brevis of the mouse. Fatigue caused by repeated isometric tetani was accelerated at body temperature (37°C) when compared to room temperature (22°C). The membrane-permeant reactive oxygen species (ROS) scavenger, Tiron (5 m m ), had no effect on the rate of fatigue at 22°C but slowed the rate of fatigue at 37°C to that observed at 22°C. Single fibres were microinjected with indo-1 to measure intracellular calcium. In the accelerated fatigue at 37°C the tetanic [Ca²⁺]_i did not change significantly and the decline of maximum Ca²⁺-activated force was similar to that observed at 22°C. The cause of the greater rate of fatigue at 37°C was a large fall in myofibrillar Ca²⁺ sensitivity. In the presence of Tiron, the large fall in Ca²⁺ sensitivity was abolished and the usual decline in tetanic [Ca²⁺]_i was observed. This study confirms the importance of ROS in fatigue at 37°C and shows that the mechanism of action of ROS is a decline in myofibrillar Ca²⁺ sensitivity.     

Calmodulin kinase modulates Ca2+ release in mouse skeletal muscle

Activation of the contractile machinery in skeletal muscle is initiated by the action-potential-induced release of Ca²⁺ from the sarcoplasmic reticulum (SR). Several proteins involved in SR Ca²⁺ release are affected by calmodulin kinase II (CaMKII)-induced phosphorylation in vitro , but the effect in the intact cell remains uncertain and is the focus of the present study. CaMKII inhibitory peptide or inactive control peptide was injected into single isolated fast-twitch fibres of mouse flexor digitorum brevis muscles, and the effect on free myoplasmic [Ca²⁺] ([Ca²⁺]_i) and force during different patterns of stimulation was measured. Injection of the inactive control peptide had no effect on any of the parameters measured. Conversely, injection of CaMKII inhibitory peptide decreased tetanic [Ca²⁺]_i by ≈25 %, but had no significant effect on the rate of SR Ca²⁺ uptake or the force-[Ca²⁺]_i relationship. Repeated tetanic stimulation resulted in increased tetanic [Ca²⁺]_i, and this increase was smaller after CaMKII inhibition. In conclusion, CaMKII-induced phosphorylation facilitates SR Ca²⁺ release in the basal state and during repeated contractions, providing a positive feedback between [Ca²⁺]_i and SR Ca²⁺ release.     

High temperature does not alter fatigability in intact mouse skeletal muscle fibres

Intense activation of skeletal muscle results in fatigue development, which involves impaired function of the muscle cells resulting in weaker and slower contractions. Intense muscle activity also results in increased heat production and muscle temperature may rise by up to ∼6°C. Hyperthermia is associated with impaired exercise performance in vivo and recent studies have shown contractile dysfunction and premature fatigue development in easily fatigued muscle fibres stimulated at high temperatures and these defects were attributed to oxidative stress. Here we studied whether fatigue-resistant soleus fibres stimulated at increased temperature show premature fatigue development and whether increasing the level of oxidative stress accelerates fatigue development. Intact single fibres or small bundles of soleus fibres were fatigued by 600 ms tetani given at 2 s intervals at 37°C and 43°C, which is the highest temperature the muscle would experience in vivo . Tetanic force in the unfatigued state was not significantly different at the two temperatures. With 100 fatiguing tetani, force decreased by ∼15% at both temperatures; the free cytosolic [Ca²⁺] (assessed with indo-1) showed a similar ∼10% decrease at both temperatures. The oxidative stress during fatigue at 43°C was increased by application of 10 μ m hydrogen peroxide or tert-butyl hydroperoxide and this did not cause premature fatigue development. In summary, fatigue-resistant muscle fibres do not display impaired contractility and fatigue resistance at the highest temperature that mammals, including humans, would experience in vivo . Thus, intrinsic defects in fatigue-resistant muscle fibres cannot explain the decreased physical performance at high temperatures.     

Palmitate increases L-type Ca2+ currents and the size of the readily releasable granule pool in mouse pancreatic β-cells

We have investigated the in vitro effects of the saturated free fatty acid palmitate on mouse pancreatic β-cells by a combination of electrophysiological recordings, intracellular Ca²⁺ ([Ca²⁺]_i) microfluorimetry and insulin release measurements. Addition of palmitate (1 m m , bound to fatty acid-free albumin) to intact islets exposed to 15 m m glucose increased the [Ca²⁺]_i by ∼30% and insulin secretion 2-fold. Palmitate remained capable of increasing [Ca²⁺]_i and insulin release in the presence of tolbutamide and in islets depolarized by high K⁺ in combination with diazoxide, indicating that the stimulation occurs independently of closure of ATP-regulated K⁺ channels (K_ATP channels). Palmitate (0.5 m m ) augmented exocytosis (measured as an increase in cell capacitance) in single β-cells and increased the size of the readily releasable pool (RRP) of granules 2-fold. Whole-cell peak Ca²⁺ currents rose by ∼25% following addition of 0.5 m m palmitate, an effect that was abolished in the presence of 10 μ m isradipine indicating that the free fatty acid specifically acts on L-type Ca²⁺ channels. The actions of palmitate on exocytosis and Ca²⁺ currents were not mimicked by intracellular application of palmitoyl-CoA. We conclude that palmitate increases insulin secretion by a K_ATP channel-independent mechanism exerted at the level of exocytosis and that involves both augmentation of L-type Ca²⁺ currents and an increased size of the RRP.     

Electrophysiological properties of BK channels in Xenopus motor nerve terminals

Single channel properties of Ca²⁺-activated K⁺ (BK or Maxi-K) channels have been investigated in presynaptic membranes in Xenopus motoneurone–muscle cell cultures. The occurrence and density of BK channels increased with maturation/synaptogenesis and was not uniform: highest at the release face of bouton-like synaptic varicosities in contact with muscle cells, and lowest in varicosities that did not contact muscle cells. The Ca²⁺ affinity of the channel ( K _d= 7.7 μ m at a membrane potential of +20 mV) was lower than those of BK channels that have been characterized in other terminals. Hill coefficients varied between 1.5 and 2.8 at different potentials and open probability increased e-fold per 16 mV change in membrane potential over a range of [Ca²⁺]_i from 1 μ m to 1 m m . The maximal activation rate of ensembled single BK channel currents was in the submillisecond range at ≥+20 mV. The activation rate increased ∼10-fold in response to a [Ca²⁺]_i increase from 1 to 100 μ m , but increased only ∼2-fold with a voltage change from +20 to +130 mV. The fastest activation kinetics of BK channels in cell-attached patches resembled that in inside-out patches with [Ca²⁺]_i of 100 μ m or more, suggesting that many BK channels are located very close to calcium channels. Given the low Ca²⁺ affinity and rapid Ca²⁺ binding/unbinding properties, we conclude that BK channels in this preparation are adapted to play an important role in regulation of neurotransmitter release, and they are ideal reporters of local [Ca²⁺] at the inner membrane surface.     

Clathrin-mediated endocytosis in snake motor terminals is directly facilitated by intracellular Ca2+

At the snake neuromuscular junction, low temperature (LT, 5–7°C) blocks clathrin-mediated endocytosis (CME) while exocytosis is largely unaffected. Thus compensatory endocytosis that normally follows transmitter release is inhibited, or 'delayed' until the preparation is warmed to room temperature (RT). This delay was exploited to observe how changes in bulk [Ca²⁺]_i directly affect CME. Motor terminals were loaded with fura-2 to monitor [Ca²⁺]_i. With brief stimulation at LT, [Ca²⁺]_i transiently increased but returned to baseline (∼63 n m ) in < 8 min. After 15 min at LT, [Ca²⁺]_i was altered by incubating preparations in the Ca²⁺ ionophore ionomyocin. Preparations were then warmed to RT to initiate delayed endocytosis, which was quantified as uptake of the fluorescent optical probe sulforhodamine 101. Endocytosis was more rapid when [Ca²⁺]_i increased; the rate at 300 n m Ca²⁺ was ∼double that under basal conditions. Thus the rate of CME – isolated from stimulation, transmitter release, and other forms of endocytosis – is directly influenced by intraterminal Ca²⁺.     

Role of intracellular calcium in fatigue in single skeletal muscle fibers isolated from the rat

The aim of the present study was to demonstrate the role of intracellular calcium ([Ca²⁺]_i) in the performance of fatigued muscle fibers isolated from the skeletal muscle of the rat. We measured developed tension of a single myocyte during short tetanic electrical stimulation of various intensities along with [Ca²⁺]_i dynamics by fura-2. The performance of individual muscle fiber was assessed by developed tension during 100 Hz tetanic stimulation (‘100 Hz force’). We regarded the muscle fiber fatigued when, after repeated tetanic stimulations, the developed tension declined to 50% of the initial level. When fatigue was induced by maximal stimulation (100 Hz tetani), the 100 Hz force measured immediately following completion of fatigue was considerably decreased (48% of control). This change in the muscle performance was associated with significant increase in the resting [Ca²⁺]_i (280% of control) and decrease in Ca²⁺ transient (54% of control). The 50% relaxation time after cessation of tetanic stimulation (RT₅₀) was also prolonged. In contrast, when fatigue was induced by low frequency electrical stimulation (30 Hz tetani), neither the 100 Hz force, RT₅₀, nor Ca²⁺ transient in fatigue were significantly different from the controls, while the resting [Ca²⁺]_i increased only slightly. These findings suggest a tight relationship between [Ca²⁺]_i and the performance of fatigued single isolated skeletal muscles. Also, the results show that performance of the fatigued muscle fiber may in part depend on the protocol used to produce muscle fatigue.     

The effect of mitochondrial inhibitors on membrane currents in isolated neonatal rat carotid body type I cells

Inhibitors of mitochondrial energy metabolism have long been known to be potent stimulants of the carotid body, yet their mechanism of action remains obscure. We have therefore investigated the effects of rotenone, myxothiazol, antimycin A, cyanide (CN⁻) and oligomycin on isolated carotid body type I cells. All five compounds caused a rapid rise in intracellular Ca²⁺, which was inhibited on removal of extracellular Ca²⁺. Under current clamp conditions rotenone and CN⁻ caused a rapid membrane depolarization and elevation of [Ca²⁺]_i. Voltage clamping cells to −70 mV substantially attenuated this rise in [Ca²⁺]_i. Rotenone, cyanide, myxothiazol and oligomycin significantly inhibited resting background K⁺ currents. Thus rotenone, myxothiazol, cyanide and oligomycin mimic the effects of hypoxia in that they all inhibit background K⁺ current leading to membrane depolarization and voltage-gated calcium entry. Hypoxia, however, failed to have any additional effect upon membrane currents in the presence of CN⁻ or rotenone or the mitochondrial uncoupler p -trifluoromethoxyphenyl hydrazone (FCCP). Thus not only do mitochondrial inhibitors mimic the effects of hypoxia, but they also abolish oxygen sensitivity. These observations suggest that there is a close link between oxygen sensing and mitochondrial function in type I cells. Mechanisms that could account for this link and the actions of mitochondrial inhibitors are discussed.     

Role of phosphate and calcium stores in muscle fatigue

Intensive activity of muscles causes a decline in performance, known as fatigue, that is thought to be caused by the effects of metabolic changes on either the contractile machinery or the activation processes. The concentration of inorganic phosphate (P_i) in the myoplasm ([P_i]_myo) increases substantially during fatigue and affects both the myofibrillar proteins and the activation processes. It is known that a failure of sarcoplasmic reticulum (SR) Ca²⁺ release contributes to fatigue and in this review we consider how raised [P_i]_myo contributes to this process. Initial evidence came from the observation that increasing [P_i]_myo causes reduced SR Ca²⁺ release in both skinned and intact fibres. In fatigued muscles the store of releasable Ca²⁺ in the SR declines mirroring the decline in SR Ca²⁺ release. In muscle fibres with inoperative creatine kinase the rise of [P_i]_myo is absent during fatigue and the failure of SR Ca²⁺ release is delayed. These results can all be explained if inorganic phosphate can move from the myoplasm into the SR during fatigue and cause precipitation of CaP_i within the SR. The relevance of this mechanism in different types of fatigue in humans is considered.     

Discrete store-operated calcium influx into an intracellular compartment in rabbit arteriolar smooth muscle

This study tested the hypothesis that store-operated channels (SOCs) exist as a discrete population of Ca²⁺ channels activated by depletion of intracellular Ca²⁺ stores in cerebral arteriolar smooth muscle cells and explored their direct contractile function. Using the Ca²⁺ indicator fura-PE3 it was observed that depletion of sarcoplasmic reticulum (SR) Ca²⁺ by inhibition of SR Ca²⁺-ATPase (SERCA) led to sustained elevation of [Ca²⁺]_i that depended on extracellular Ca²⁺ and slightly enhanced Mn²⁺ entry. Enhanced background Ca²⁺ influx did not explain the raised [Ca²⁺]_i in response to SERCA inhibitors because it had marked gadolinium (Gd³⁺) sensitivity, which background pathways did not. Effects were not secondary to changes in membrane potential. Thus SR Ca²⁺ depletion activated SOCs. Strikingly, SOC-mediated Ca²⁺ influx did not evoke constriction of the arterioles, which were in a resting state. This was despite the fura-PE3-indicated [Ca²⁺]_i rise being greater than that evoked by 20 m m [K⁺]_o (which did cause constriction). Release of endothelial vasodilators did not explain the absence of SOC-mediated constriction, nor did a change in Ca²⁺ sensitivity of the contractile proteins. We suggest SOCs are a discrete subset of Ca²⁺ channels allowing Ca²⁺ influx into a 'non-contractile' compartment in cerebral arteriolar smooth muscle cells.     

External Ca2+-dependent excitation–contraction coupling in a population of ageing mouse skeletal muscle fibres

In the present work, we investigate whether changes in excitation–contraction (EC) coupling mode occur in skeletal muscles from ageing mammals by examining the dependence of EC coupling on extracellular Ca²⁺. Single intact muscle fibres from flexor digitorum brevis muscles from young (2–6 months) and old (23–30 months) mice were subjected to tetanic contractile protocols in the presence and absence of external Ca²⁺. Contractile experiments in the absence of external Ca²⁺ show that about half of muscle fibres from old mice are dependent upon external Ca²⁺ for maintaining maximal tetanic force output, while young fibres are not. Decreased force in the absence of external Ca²⁺ was not due to changes in charge movement as revealed by whole-cell patch-clamp experiments. Ca²⁺ transients, measured by fluo-4 fluorescence, declined in voltage-clamped fibres from old mice in the absence of external Ca²⁺. Similarly, Ca²⁺ transients declined in parallel with tetanic contractile force in single intact fibres. Examination of inward Ca²⁺ current and of mRNA and protein assays suggest that these changes in EC coupling mode are not due to shifts in dihydropyridine receptor (DHPR) and/or ryanodine receptor (RyR) isoforms. These results indicate that a change in EC coupling mode occurs in a population of fibres in ageing skeletal muscle, and is responsible for the age-related dependence on extracellular Ca²⁺.     

Functional characterization of a Ca2+-activated non-selective cation channel in human atrial cardiomyocytes

Cardiac arrhythmias, which occur in a wide variety of conditions where intracellular calcium is increased, have been attributed to the activation of a transient inward current ( I _ti). I _ti is the result of three different [Ca]_i-sensitive currents: the Na⁺–Ca²⁺ exchange current, a Ca²⁺-activated chloride current and a Ca²⁺-activated non-selective cationic current. Using the cell-free configuration of the patch-clamp technique, we have characterized the properties of a Ca²⁺-activated non-selective cation channel (NSC_Ca) in freshly dissociated human atrial cardiomyocytes. In excised inside-out patches, the channel presented a linear I–V relationship with a conductance of 19 ± 0.4 pS. It discriminated poorly among monovalent cations (Na⁺ and K⁺) and was slightly permeable to Ca²⁺ ions. The channel's open probability was increased by depolarization and a rise in internal calcium, for which the K _d for [Ca²⁺]_i was 20.8 μ m . Channel activity was reduced in the presence of 0.5 m m ATP or 10 μ m glibenclamide on the cytoplasmic side to 22.1 ± 16.8 and 28.5 ± 8.6%, respectively, of control. It was also inhibited by 0.1 m m flufenamic acid. The channel shares several properties with TRPM4b and TRPM5, two members of the 'TRP melastatin' subfamily. In conclusion, the NSC_Ca channel is a serious candidate to support the delayed after-depolarizations observed in [Ca²⁺] overload and thus may be implicated in the genesis of arrhythmias.     

Reorganized stores and impaired calcium handling in skeletal muscle of mice lacking calsequestrin-1

Calsequestrin (CS), the major Ca²⁺-binding protein in the sarcoplasmic reticulum (SR), is thought to play a dual role in excitation–contraction coupling: buffering free Ca²⁺ increasing SR capacity, and modulating the activity of the Ca²⁺ release channels (RyRs). In this study, we generated and characterized the first murine model lacking the skeletal CS isoform (CS1). CS1- null mice are viable and fertile, even though skeletal muscles appear slightly atrophic compared to the control mice. No compensatory increase of the cardiac isoform CS2 is detectable in any type of skeletal muscle. CS1- null muscle fibres are characterized by structural and functional changes, which are much more evident in fast-twitch muscles (EDL) in which most fibres express only CS1, than in slow-twitch muscles (soleus), where CS2 is expressed in about 50% of the fibres. In isolated EDL muscle, force development is preserved, but characterized by prolonged time-to-peak and half-relaxation time, probably related to impaired calcium release from and re-uptake by the SR. Ca²⁺-imaging studies show that the amount of Ca²⁺ released from the SR and the amplitude of the Ca²⁺ transient are significantly reduced. The lack of CS1 also causes significant ultrastructural changes, which include: (i) striking proliferation of SR junctional domains; (ii) increased density of Ca²⁺-release channels (confirmed also by ³H-ryanodine binding); (iii) decreased SR terminal cisternae volume; (iv) higher density of mitochondria. Taken together these results demonstrate that CS1 is essential for the normal development of the SR and its calcium release units and for the storage and release of appropriate amounts of SR Ca²⁺.     

Changes in contractile activation characteristics of rat fast and slow skeletal muscle fibres during regeneration

Damaged skeletal muscle fibres are replaced with new contractile units via muscle regeneration. Regenerating muscle fibres synthesize functionally distinct isoforms of contractile and regulatory proteins but little is known of their functional properties during the regeneration process. An advantage of utilizing single muscle fibre preparations is that assessment of their function is based on the overall characteristics of the contractile apparatus and regulatory system and as such, these preparations are sensitive in revealing not only coarse, but also subtle functional differences between muscle fibres. We examined the Ca²⁺- and Sr²⁺-activated contractile characteristics of permeabilized fibres from rat fast-twitch (extensor digitorum longus) and slow-twitch (soleus) muscles at 7, 14 and 21 days following myotoxic injury, to test the hypothesis that fibres from regenerating fast and slow muscles have different functional characteristics to fibres from uninjured muscles. Regenerating muscle fibres had ∼10% of the maximal force producing capacity ( P _o) of control (uninjured) fibres, and an altered sensitivity to Ca²⁺ and Sr²⁺ at 7 days post-injury. Increased force production and a shift in Ca²⁺ sensitivity consistent with fibre maturation were observed during regeneration such that P _o was restored to 36–45% of that in control fibres by 21 days, and sensitivity to Ca²⁺ and Sr²⁺ was similar to that of control (uninjured) fibres. The findings support the hypothesis that regenerating muscle fibres have different contractile activation characteristics compared with mature fibres, and that they adopt properties of mature fast- or slow-twitch muscle fibres in a progressive manner as the regeneration process is completed.     

Effect of ADP on slow-twitch muscle fibres of the rat: implications for muscle fatigue

Slow-twitch mechanically skinned fibres from rat soleus muscle were bathed in solutions mimicking the myoplasmic environment but containing different [ADP] (0.1 μ m to 1.0 m m ). The effect of ADP on sarcoplasmic reticulum (SR) Ca²⁺-content was determined from the magnitude of caffeine-induced force responses, while temporal changes in SR Ca²⁺-content allowed determination of the effective rates of the SR Ca²⁺-pump and of the SR Ca²⁺-leak. The SR Ca²⁺-pump rate, estimated at pCa (−log₁₀[Ca²⁺]) 7.8, was reduced by 20% as the [ADP] was increased from 0.1 to 40 μ m , with no further alteration when the [ADP] was increased to 1.0 m m . The SR Ca²⁺-leak rate constant was not altered by increasing [ADP] from 0.1 to 40 μ m , but was increased by 26% when the [ADP] was elevated to 1.0 m m . This ADP-induced SR Ca²⁺-leak was insensitive to ruthenium red but was abolished by 2,5-di(tert-butyl)-1,4-hydroquinone (TBQ), indicating that the leak pathway is via the SR Ca²⁺-pump and not the SR Ca²⁺-release channel. The decrease in SR Ca²⁺-pump rate and SR Ca²⁺-leak rate when [ADP] was increased led to a 40% decrease in SR Ca²⁺-loading capacity. Elevation of [ADP] had only minor direct effects on the contractile apparatus of slow-twitch fibres. These results suggest that ADP has only limited depressing effects on the contractility of slow-twitch muscle fibres. This is in contrast to the marked effects of ADP on force responses in fast-twitch muscle fibres and may contribute to the fatigue-resistant nature of slow-twitch muscle fibres.     

Hypo-osmotic potentiation of acetylcholine-stimulated ciliary beat frequency through ATP release in rat tracheal ciliary cells

The ciliary beat frequency (CBF) of rat tracheal ciliary cells in a slice preparation was measured using video-enhanced contrast (VEC) microscopy. Acetylcholine (ACh) increased CBF mediated via intracellular Ca²⁺ concentration ([Ca²⁺]_i) in a dose-dependent manner. An adequate hypo-osmotic stress (−40 mos m ) potentiated ACh-stimulated CBF increase in tracheal ciliary cells and shifted the ACh dose–response curve to the left (lower concentration side). This potentiation was independent of hypo-osmotic stresses applied ranging from −20 mosM to −90 mosM. A hypo-osmotic stress induces ATP release in many cell types. The present study demonstrated that suramin (an inhibitor of purinergic receptors) and apyrase (an ATPase/ADPase) eliminate the hypo-osmotic potentiation of ACh-stimulated CBF increase and that ATP increased [Ca²⁺]_i and CBF, as well as potentiating ACh-stimulated rises in [Ca²⁺]_i and CBF increase. Moreover, the apical surface of tracheal ciliary cells were stained immunopositive for the P2X₄ purinergic receptor. A hypo-osmotic stress (−40 mosM) transiently increased [Ca²⁺]_i and potentiated the ACh-stimulated [Ca²⁺]_i increase. The hypo-osmotic potentiation of ACh-stimulated CBF increase was not detected under Ca²⁺-free conditions. These observations suggest that a hypo-osmotic stress stimulates ATP release from the trachea. The released ATP may induce further increases in [Ca²⁺]_i and CBF in ACh-stimulated tracheal ciliary cells, which may be mediated by purinergic receptors, such as P2X₄.     

Calcium-induced calcium release regulates action potential generation in guinea-pig sympathetic neurones

Experiments were done using guinea-pig sympathetic neurones dissociated from the stellate ganglia to establish whether calcium-induced calcium release (CICR) modulated action potential (AP) generation in mammalian neurones. Using measurements of intracellular calcium ([Ca²⁺]_i) with the Ca²⁺-sensitive dye fluo-3, we demonstrated that 10 m m caffeine activated ryanodine receptors and caused a rise in [Ca²⁺]_i in both Ca²⁺-containing and Ca²⁺-deficient solutions. We also demonstrated that combined treatment with caffeine and 1 μ m thapsigargin or caffeine and 20 μ m ryanodine blocked subsequent caffeine-induced elevations of [Ca²⁺]_i. Treatment with thapsigargin, ryanodine or 200 μ m Cd²⁺ to disrupt CICR decreased the latency to AP generation during 400 ms depolarizing current ramps using the perforated patch whole cell patch clamp in current clamp mode. Treatment with 500 μ m tetraethylammonium also decreased the latency to AP generation during depolarizing current ramps in control cells, but not in cells pretreated with thapsigargin to deplete internal Ca²⁺ stores. In summary, we propose that an outward current, carried at least in part through BK channels, is activated by CICR at membrane voltages approaching the threshold for AP initiation and that this current opposed depolarizing current ramps applied to guinea-pig sympathetic stellate neurones.     

Effect of taurine on sarcoplasmic reticulum function and force in skinned fast-twitch skeletal muscle fibres of the rat

We examined the effect of taurine on depolarisation-induced force responses and sarcoplasmic reticulum (SR) function in mechanically skinned skeletal muscle fibres from the extensor digitorum longus (EDL) of the rat. Taurine (20 m m ) produced a small but significant ( P < 0.01) decrease in the sensitivity of the contractile apparatus to Ca²⁺ (increase in the [Ca²⁺] corresponding to 50 % of maximum force of about 7 %; n = 10) and in maximum force (92.0 ± 1.0 % of controls) in the skinned fibres. Taurine had no statistically significant effect on the slope of the force-pCa curve. Depolarisation-induced force responses in the skinned fibres were markedly increased in peak value by 20 m m taurine, to 120.8 ± 5.3 % of control measurements ( P = 0.0006, n = 27). Taurine (20 m m ) significantly increased the SR Ca²⁺ accumulation in the skinned fibres by 34.6 ± 9.3 % compared to control conditions (measured by comparing the integral of caffeine contractures in fibres previously loaded with Ca²⁺ in the absence or presence of taurine; P = 0.0014, n = 10). Taurine (20 m m ) also increased both the peak and rate of rise of caffeine-induced force responses in the fibres by 29.2 ± 9.7 % ( P = 0.0298, n = 6) and 27.6 ± 8.9 % ( P = 0.037), respectively, compared with controls. This study shows that taurine is a modulator of contractile function in mammalian skeletal muscle. Taurine may increase the size of depolarisation-induced force responses by augmenting SR Ca²⁺ accumulation and release.