一氧化氮合酶激活参与烧伤后Ca2+介导的心肌线粒体损伤

目的:探讨线粒体一氧化氮合酶(mtNOS)在严重烧伤早期心肌线粒体损害中的作用。方法:复制30%TBSAⅢ°烧伤大鼠模型,取正常及伤后1、3、6、12、24h大鼠心肌分离线粒体,测其呼吸功能、Ca²⁺浓度(_m)及细胞色素c氧化酶、mtNOS活性。结果:①伤后1h心肌线粒体呼吸控制率(RCR)显著高于正常组,但3、6、12、24h明显低于正常组,Ⅲ态呼吸速率(ST₃)变化与RCR平行,ST₄仅于伤后3h明显升高;②伤后各时点_m均明显高于正常组,尤以3、6h为甚,而mtNOS活性于伤后3、6、12、24h显著高于对照组,细胞色素c氧化酶活性于伤后3、6、12、24h显著低于正常组;③伤后mtNOS活性与_m呈明显正相关,相关系数为0.8945(P＜0.05),RCR与mtNOS活性呈显著负相关,相关系数为-0.9347(P＜0.05)。结论:伤后_m升高激活mtNOS,可能参与严重烧伤早期心肌线粒体损害。     

Cytoplasmic and mitochondrial Ca2+ levels in brown adipocytes

Aim: We elucidated the mitochondrial functions of brown adipocytes in intracellular signalling, paying attention to mitochondrial activity and noradrenaline‐ and forskolin‐induced Ca²⁺ mobilizations in cold‐acclimated rats. Methods: A confocal laser‐scanning microscope of brown adipocytes from warm‐ or cold‐acclimated rats was employed using probes rhodamine 123 which is a mitochondria‐specific cationic dye, and the cytoplasmic and mitochondrial Ca²⁺ probes fluo‐3 and rhod‐2. X‐ray microanalysis was also studied. Results: The signal of rhodamine 123 in the cells was decreased by antimycin A which effect was less in cold‐acclimated cells than warm‐acclimated cells. Cytoplasmic and mitochondrial Ca²⁺ in cold‐acclimated brown adipocytes double‐loaded with fluo‐3 and rhod‐2 were measured. Noradrenaline induced the rise in cytoplasmic Ca²⁺ ([Ca²⁺]_cyto) followed by mitochondrial Ca²⁺ ([Ca²⁺]_mito), the effect being transformed into an increase in [Ca²⁺]_cyto whereas a decrease in [Ca²⁺]_mito by antimycin A or carbonyl cyanide m‐chlorophenylhydrazone (CCCP). Antimycin A induced small Ca²⁺ release from mitochondria. CCCP induced Ca²⁺ release from mitochondria only after the cells were stimulated with noradrenaline. Further, forskolin also elicited an elevation in [Ca²⁺]_cyto followed by [Ca²⁺]_mito in the cells. The Ca measured by X‐ray microanalysis was higher both in the cytoplasm and mitochondria whereas K was higher in the mitochondria of cold‐acclimated cells in comparison to warm‐acclimated cells. Conclusions: These results suggest that noradrenaline and forskolin evoked an elevation in [Ca²⁺]_cyto followed by [Ca²⁺]_mito, in which H⁺ gradient across the inner membrane is responsible for the accumulation of calcium on mitochondria. Moreover, cAMP also plays a role in intracellular and mitochondrial Ca²⁺ signalling in cold‐acclimated brown adipocytes.     

Mechanisms of reduced mitochondrial Ca2+ accumulation in failing hamster heart

Mitochondrial Ca²⁺ plays important roles in the regulation of energy metabolism and cellular Ca²⁺ homeostasis. In this study, we characterized mitochondrial Ca²⁺ accumulation in Syrian hamster hearts with hereditary cardiomyopathy (strain BIO 14.6). Exposure of isolated mitochondria from 70 nM to 30 μM Ca²⁺ ([Ca²⁺]_o) caused a concentration-dependent increase in intramitochondrial Ca²⁺ concentrations ([Ca²⁺]_m). The [Ca²⁺]_m was significantly lower in cardiomyopathic (CMP) hamsters than in healthy hamsters when [Ca²⁺]_o was higher than 1 μM and a decrease of about 52% was detected at [Ca²⁺]_o of 30 μM (916 ± 67 nM vs 1,932 ± 132 nM in control). A possible mechanism responsible for the decreased mitochondrial Ca²⁺ uptake in CMP hamsters is the depolarization of mitochondrial membrane potential (Δψ _m). Using a tetraphenylphosphonium (TPP⁺) electrode, the measured Δψ _m in failing heart mitochondria was −136 ± 1.5 mV compared with −159 ± 1.3 mV in controls. Analyses of mitochondrial respiratory chain demonstrated a significant impairment of complex I and complex IV activities in failing heart mitochondria. In summary, a less negative Δψ _m resulting from defects in the respiratory chain may lead to attenuated mitochondrial Ca²⁺ accumulation, which in turn may contribute to the depressed energy production and myocardial contractility in this model of heart failure. In addition to other known impairments of ion transport in sarcoplasmic reticulum and plasma membrane, results from this paper on mitochondrial dysfunctions expand our understanding of the molecular mechanisms leading to heart failure.     

Mitochondrial calcium accumulation following activation of vanilloid (VR1) receptors by capsaicin in dorsal root ganglion neurons

Stimulation of the vanilloid (capsaicin) receptor (VR1), currently viewed as a molecular integrator of chemical and physical noxious stimuli, evoked intracellular Ca²⁺ transients in a capsaicin-sensitive subpopulation of rat dorsal root ganglion neurons. These were comprised of an initial fast rise (seconds) followed by a long-lasting intracellular Ca²⁺ recovery (tens of minutes). The rate of intracellular Ca²⁺ recovery was dependent on the magnitude of intracellular Ca²⁺ transients. Opening of voltage-operated Ca²⁺ channels in the same neurons by K⁺ depolarization evoked intracellular Ca²⁺ elevation of a similar amplitude and rate of rise; however, the recovery of intracellular Ca²⁺ to the prestimulated level was significantly faster. A mitochondrial uncoupler (10 μM carbonyl cyanide m-chlorophenylhydrasone) was used to reveal the role of mitochondria in intracellular Ca²⁺ buffering. Carbonyl cyanide m-chlorophenylhydrasone-evoked elevation in intracellular Ca²⁺ was greater in neurons previously stimulated with capsaicin compared with KCl. Neither extracellular Ca²⁺ nor ATP depletion influenced significantly the carbonyl cyanide m-chlorophenylhydrasone-sensitive intracellular Ca²⁺ elevation in neurons loaded with Ca²⁺ via vanilloid 1 receptor stimulation.The effects of carbonyl cyanide m-chlorophenylhydrasone suggest that the amount of Ca²⁺ buffered by mitochondria is greater when extracellular Ca²⁺ enters the neuron via the vanilloid 1 receptor channel than via voltage-operated Ca²⁺ channels. The long duration of intracellular Ca²⁺_i decline in neurons stimulated with capsaicin, which depends on the amount of Ca²⁺ buffered by mitochondria, may reflect a specific mechanism of Ca²⁺ buffering following activation the pain receptor VR1.     

Ratiometric high-resolution imaging of JC-1 fluorescence reveals the subcellular heterogeneity of astrocytic mitochondria

Using the mitochondrial potential (ΔΨ_m) marker JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide) and high-resolution imaging, we functionally analyzed mitochondria in cultured rat hippocampal astrocytes. Ratiometric detection of JC-1 fluorescence identified mitochondria with high and low ΔΨ_m. Mitochondrial density was highest in the perinuclear region, whereas ΔΨ_m tended to be higher in peripheral mitochondria. Spontaneous ΔΨ_m fluctuations, representing episodes of increased energization, appeared in individual mitochondria or synchronized in mitochondrial clusters. They continued upon withdrawal of extracellular Ca²⁺, but were antagonized by dantrolene or 2-aminoethoxydiphenylborate (2-APB). Fluo-3 imaging revealed local cytosolic Ca²⁺ transients with similar kinetics that also were depressed by dantrolene and 2-APB. Massive cellular Ca²⁺ load or metabolic impairment abolished ΔΨ_m fluctuations, occasionally evoking heterogeneous mitochondrial depolarizations. The detected diversity and ΔΨ_m heterogeneity of mitochondria confirms that even in less structurally polarized cells, such as astrocytes, specialized mitochondrial subpopulations coexist. We conclude that ΔΨ_m fluctuations are an indication of mitochondrial viability and are triggered by local Ca²⁺ release from the endoplasmic reticulum. This spatially confined organelle crosstalk contributes to the functional heterogeneity of mitochondria and may serve to adapt the metabolism of glial cells to the activity and metabolic demand of complex neuronal networks. The established ratiometric JC-1 imaging—especially combined with two-photon microscopy—enables quantitative functional analyses of individual mitochondria as well as the comparison of mitochondrial heterogeneity in different preparations and/or treatment conditions.     

The higher susceptibility of congenital analbuminemic rats to Ca2+-induced mitochondrial permeability transition is associated with the increased expression of cyclophilin D and nitrosothiol depletion

Congenital analbuminemia is a rare autosomal recessive disorder characterized by a trace level of albumin in blood plasma and mild clinical symptoms. Analbuminemic patients generally present associated abnormalities, among which dyslipidemia is a hallmark. In this study, we show that mitochondria isolated from different tissues (liver, heart and brain) from 3-month-old analbuminemic rats (NAR) present a higher susceptibility to Ca²⁺-induced mitochondrial permeability transition (MPT), as assessed by either Ca²⁺-induced mitochondrial swelling, dissipation of membrane potential or mitochondrial Ca²⁺ release. The Ca²⁺ retention capacity of the liver mitochondria isolated from 3-month-old NAR was about 50% that of the control. Interestingly, the assessment of this variable in 21-day-old NAR indicated that the mitochondrial Ca²⁺ retention capacity was preserved at this age, as compared to age-matched controls, which indicates that a reduced capacity for mitochondrial Ca²⁺ retention is not a constitutive feature. The search for putative mediators of MPT sensitization in NAR revealed a 20% decrease in mitochondrial nitrosothiol content and a 30% increase in cyclophilin D expression. However, the evaluation of other variables related to mitochondrial redox status showed similar results between the controls and NAR, i.e., namely the contents of reduced mitochondrial membrane protein thiol groups and total glutathione, H₂O₂ release rate, and NAD(P)H reduced state. We conclude that the higher expression of cyclophilin D, a major component of the MPT pore, and decreased nitrosothiol content in NAR mitochondria may underlie MPT sensitization in these animals.     

Stimulus-dependent modulation of smooth muscle intracellular calcium and force by altered intracellular pH

Measurements of simultaneous force and intracellular Ca²⁺ concentration ([Ca²⁺]_i) in rat uterine smooth muscle have been made to elucidate the mechanisms involved when force produced spontaneously, by high-K⁺ depolarization or carbachol is altered by a change of intracellular pH (pH_i). Rises in force and [Ca²⁺]_i were closely correlated for all forms of contraction, with the Ca²⁺ transient peaking before force. In spontaneously active preparations, alkalinization significantly increased, and acidification decreased, force and [Ca²⁺]_i. Inhibition of the sarcoplasmic reticulum ATPase (cyclopiazonic acid) did not affect these changes, whereas removal of external Ca²⁺ abolished both responses, suggesting that the effect of pH_i is on Ca²⁺ entry. Alkalinization caused a prolongation of the action potential complex, associated with a potentiation of contractile activity. Acidification produced hyperpolarization and abolition of action potentials and spontaneous activity, but did not prevent brief applications of carbachol or high-K⁺ from producing depolarization and increasing force, suggesting no impairment of the mechanism of generation of the action potential. For depolarized preparations, acidification increased tonic force and [Ca²⁺]_i; the increase in the calcium signal persisted in zero-external calcium. In the presence of carbachol, acidification transiently increased force and [Ca²⁺]_i, followed by a reduction in both. It is concluded that changes in pH_i act at more than one step in excitation-contraction coupling and that changes in [Ca²⁺]_i can account for most of the changes in uterine force. Received: 1 April 1996 /Accepted: 8 May 1996     

Frequency-dependent mitochondrial Ca2+ accumulation regulates ATP synthesis in pancreatic β cells

Pancreatic β cells respond to increases in glucose concentration with enhanced metabolism, the closure of ATP-sensitive K⁺ channels and electrical spiking. The latter results in oscillatory Ca²⁺ influx through voltage-gated Ca²⁺ channels and the activation of insulin release. The relationship between changes in cytosolic and mitochondrial free calcium concentration ([Ca²⁺]_cyt and [Ca²⁺]_mit, respectively) during these cycles is poorly understood. Importantly, the activation of Ca²⁺-sensitive intramitochondrial dehydrogenases, occurring alongside the stimulation of ATP consumption required for Ca²⁺ pumping and other processes, may exert complex effects on cytosolic ATP/ADP ratios and hence insulin secretion. To explore the relationship between these parameters in single primary β cells, we have deployed cytosolic (Fura red, Indo1) or green fluorescent protein-based recombinant-targeted (Pericam, 2mt8RP for mitochondria; D4ER for the ER) probes for Ca²⁺ and cytosolic ATP/ADP (Perceval) alongside patch-clamp electrophysiology. We demonstrate that: (1) blockade of mitochondrial Ca²⁺ uptake by shRNA-mediated silencing of the uniporter MCU attenuates glucose- and essentially blocks tolbutamide-stimulated, insulin secretion; (2) during electrical stimulation, mitochondria decode cytosolic Ca²⁺ oscillation frequency as stable increases in [Ca²⁺]_mit and cytosolic ATP/ADP; (3) mitochondrial Ca²⁺ uptake rates remained constant between individual spikes, arguing against activity-dependent regulation (“plasticity”) and (4) the relationship between [Ca²⁺]_cyt and [Ca²⁺]_mit is essentially unaffected by changes in endoplasmic reticulum Ca²⁺ ([Ca²⁺]_ER). Our findings thus highlight new aspects of Ca²⁺ signalling in β cells of relevance to the actions of both glucose and sulphonylureas.     

严重烧伤早期心肌[Ca2+]m变化及其机制研究

目的:探讨严重烧伤早期心肌线粒体Ca²⁺浓度([Ca²⁺]_m)的动态变化规律及其发生机制。方法:复制30%Ⅲ°烫伤大鼠模型,测定伤后1、3、6、12、24h大鼠心肌[Ca²⁺]_m,同时检测影响[Ca²⁺]_m的相关指标—胞浆Ca^2＋浓度(_c)及线粒体Ca^2＋转运速率。结果:烧伤后1、3、6h[Ca²⁺]_m依次升高,12、24h较6h虽有所下降,但仍高于正常对照组;_c除伤后1h无明显变化外,其余各时相点变化趋势与[Ca²⁺]_m相同,且伤后[Ca²⁺]_m与_c呈显著正相关,相关系数为0.9177(P＜0.01)。伤后1h心肌线粒体Ca^2＋摄取速率明显升高,而Ca^2＋释放速率无明显改变,但3、6、12、24h心肌线粒体Ca^2＋摄取速率与Ca^2＋释放速率均显著降低,且烧伤后3、6、12、24h[Ca²⁺]_m分别与线粒体Ca^2＋释放速率呈明显负相关。结论:烧伤后心肌线粒体存在明显的Ca^2＋超载和转运紊乱。     

Influence of acid-base changes on the intracellular calcium concentration of neurons in primary culture

The influence of changes in intra- and extracellular pH (pH_i and pH_e, respectively) on the cytosolic, free calcium concentration ([Ca²⁺]_i) of neocortical neurons was studied by microspectrofluorometric techniques and the fluorophore fura-2. When, at constant pH_e, pH_i was lowered with the NH₄Cl prepulse technique, or by a transient increase in CO₂ tension, [Ca²⁺]_i invariably increased, the magnitude of the rise being proportional to pH_i. Since similar results were obtained in Ca²⁺-free solutions, the results suggest that the rise in [Ca²⁺]_i was due to calcium release from intracellular stores. The initial alkaline transient during NH₄Cl exposure was associated with a rise in [Ca²⁺]_i. However, this rise seemed to reflect influx of Ca²⁺ from the external solution. Thus, in Ca²⁺-free solution NH₄Cl exposure led to a decrease in [Ca²⁺]_i. This result and others suggest that, at constant pH_e, intracellular alkalosis reduces [Ca²⁺]_i, probably by enhancing sequestration of calcium. When cells were exposed to a CO₂ transient at reduced pH_e, Ca²⁺ rose initially but then fell, often below basal values. Similar results were obtained when extracellular HCO ₃ ^- concentration was reduced at constant CO₂ tension. Unexpectedly, such results were obtained only in Ca²⁺-containing solutions. In Ca²⁺-free solutions, acidosis always raised [Ca²⁺]_i. It is suggested that a lowering of pH_e stimulates extrusion of Ca²⁺ by ATP-driven Ca²⁺/2H⁺ antiport.     

Phasic changes in intracellular pH during action potentials of sheep Purkinje fibres

Regulation of intracellular pH (pH_i) and the relationship between H⁺ and Ca²⁺ may vary during activity. Ion-selective microelectrodes were used to record pH_i during action potentials of sheep Purkinje fibres prolonged by low temperature (21°C) and elevated CO₂ content. Intracellular pH also was measured during changes in extracellular calcium concentration, [Ca²⁺]_o. Cytosolic alkalinization (peak pH_i change, 0.03–0.05) was observed during the long action-potential plateau and transient acidification (0.01–0.02 units) upon repolarization. Potassium-induced depolarization to plateau potentials (i.e. to –15±2 mV) simulated the peak magnitude of the alkalinization. However, compensation for the alkalinization occurred at a faster rate during the action potential (8.9±4.3 nM/min) than during K⁺ depolarization (1.2±0.5 nM/min). In comparison, the cytoplasm acidified in resting fibres (0.06–0.07 log units) during changes of [Ca²⁺]_o thought to increase intracellular calcium concentration. Alterations of pH_i were translated into changes of proton concentration ([H⁺]_i). Ten-to twenty-fold elevation of [Ca²⁺]_o evoked a comparable change in [H⁺]_i (mean increase, 5.7 nM) but oppositely directed from that during the plateau (mean decrease, 8.8 nM). The findings in resting fibres seem consistent with displacement of bound protons by Ca²⁺. In contrast, the initial change in pH_i during the plateau is proposed to be consequent to Ca²⁺-release from sarcoplasmic reticulum and/or phosphocreatine hydrolysis coupled to ATP regeneration.     

Glutamine-mediated protection from neuronal cell death depends on mitochondrial activity

The specific aim of this study was to elucidate the role of mitochondria in a neuronal death caused by different metabolic effectors and possible role of intracellular calcium ions ([Ca²⁺]_i) and glutamine in mitochondria- and non-mitochondria-mediated cell death. Inhibition of mitochondrial complex I by rotenone was found to cause intensive death of cultured cerebellar granule neurons (CGNs) that was preceded by an increase in intracellular calcium concentration ([Ca²⁺]_i). The neuronal death induced by rotenone was significantly potentiated by glutamine. In addition, inhibition of Na/K-ATPase by ouabain also caused [Ca²⁺]_i increase, but it induced neuronal cell death only in the absence of glucose. Treatment with glutamine prevented the toxic effect of ouabain and decreased [Ca²⁺]_i. Blockade of ionotropic glutamate receptors prevented neuronal death and significantly decreased [Ca²⁺]_i, demonstrating that toxicity of rotenone and ouabain was at least partially mediated by activation of these receptors. Activation of glutamate receptors by NMDA increased [Ca²⁺]_i and decreased mitochondrial membrane potential leading to markedly decreased neuronal survival under glucose deprivation. Glutamine treatment under these conditions prevented cell death and significantly decreased the disturbances of [Ca²⁺]_i and changes in mitochondrial membrane potential caused by NMDA during hypoglycemia. Our results indicate that glutamine stimulates glutamate-dependent neuronal damage when mitochondrial respiration is impaired. However, when mitochondria are functionally active, glutamine can be used by mitochondria as an alternative substrate to maintain cellular energy levels and promote cell survival.     

Mitochondria contribute to Ca2+ removal in smooth muscle cells

Recent evidence, from a variety of cell types, suggests that mitochondria play an important role in shaping the change in intracellular calcium concentration ([Ca²⁺]_i,) that occurs during physiological stimulation. In the present study, using a range of inhibitors of mitochondrial Ca²⁺ uptake, we have examined the contribution of mitochondria to Ca²⁺ removal from the cytosol of smooth muscle cells following stimulation. In voltage-clamped single smooth muscle cells, we found that following a 8-s train of depolarizing pulses, the rate of Ca²⁺ extrusion from the cytosol was reduced by more than 50% by inhibitors of cytochrome oxidase or exposure of cells to the protonophore carbonyl cyanideP-trifluoromethoxy-phenylhydrazone. Using the potential-sensitive indicator tetramethyl rhodamine ethyl ester, we confirmed that the effect of these agents was associated with depolarization of the mitochondrial membrane. Since, the primary function of the mitochondria is to provide the cell's ATP, it could be argued that it is the ATP supply to the ion pumps which is limiting the rate of Ca²⁺ removal. However, experiments carried out with the mitochondrial Ca²⁺ uniporter inhibitor ruthenium red produced similar results, while the ATP synthetase inhibitor oligomycin had no effect, suggesting that the effect was not due to ATP insufficiency. These results establish that mitochondria in smooth muscle cells play a significant role in removing Ca²⁺ from the cytosol following stimulation. The uptake of Ca²⁺ into mitochondria is proposed to stimulate mitochondrial ATP production, thereby providing a means for matching increased energy demand, following the cell's rise in [Ca²⁺]_i;, with increased cellular ATP production.     

The use of CalciumOrange-5N as a specific marker of mitochondrial Ca2+ in mouse skeletal muscle fibers

We report the use of the fluorescent dye CalciumOrange-5N (CaOr-5N) as a specific mitochondria Ca²⁺ marker in enzymatically dissociated mouse FBD muscle fibers. Using laser scanning confocal microscopy and the dyes Mitotracker Green (MTG), di-8-ANEPPS and endoplasmic reticulum tracker green (ERTG), we determined the relative position of mitochondria, transverse tubules and sarcoplasmic reticulum in the sarcomere. Comparison with electron micrographies showed that mitochondria are mostly present at both sides of Z lines and near the triads located at the A-I band border. CaOr-5N fluorescence was mainly distributed in mitochondria, highly co-localised with MTG and basically excluded from the A band space. ERTG localised mostly between the two t-tubules present in each sarcomere. We studied the effect of the protonophore FCCP using CaOr-5N to measure mitochondrial Ca²⁺ and JC-1 dye to measure mitochondria inner membrane potential (ΔΨ _m). After FCCP treatment, the CaOr-5N fluorescence diminished by about 33% in 80 s, while JC-1 fluorescence diminished by 36% in 200 s. Our results show the loss of Ca²⁺ from mitochondria when ΔΨ_m is depolarised and demonstrate the usefulness of CaOr-5N to mark mitochondrial [Ca²⁺]_m. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Dependence of intracellular free calcium and tension on membrane potential and intracellular pH in single crayfish muscle fibres

The dependence of intracellular free calcium ([Ca²⁺]_i) and tension on membrane potential and intracellular pH (pH_i) was studied in single isolated fibres of the crayfish claw-opener muscle using ion-selective microelectrodes. Tension (T) was quantified as a percentage of the maximum force, or as force per cross-sectional area (N/cm²). In resting fibres, pH_i had a mean value of 7.06. Contractions evoked by an increase extracellular potassium ([K⁺]₀) produced a fall in pH_i of 0.01–0.05 units. The lowest measured levels of resting [Ca²⁺]_i corresponded to a pCa_i (= –log [Ca²⁺]_i) of 6.8. Intracellular Ca²⁺ transients recorded during K⁺-induced contractions did not reveal any distinct threshold for force development. Both the resting [Ca²⁺]_i and resting tension were decreased by an intracellular alkalosis and increased by an acidosis. The sensitivity of resting tension to a change in pH_i (quantified as –dT/ dpH_i) showed a progressive increase during a fall in pH_i within the range examined (pH_i 6.2–7.5). The pH_i/[Ca²⁺]_i and pH_i/tension relationships were monotonic throughout the multiphasic pH_i change caused by NH₄Cl. A fall of 0.5–0.6 units in pH_i did not produce a detectable shift in the pCa_i/tension relationship at low levels of force development. The results indicate that resting [Ca²⁺]_i is slightly higher than the level required for contractile activation. They also show that the dependence of tension on pH_i in crayfish muscle fibres is attributable to a direct H⁺ and Ca²⁺ interaction at the level of Ca²⁺ sequestration and/or transport. Finally, the results suggest that in situ, the effect of pH on the Ca²⁺ sensitivity of the myofibrillar system is not as large as could be expected on the basis of previous work on skinned crustacean muscle fibres.     

Acid-evoked Ca2+ signalling in rat sensory neurones: effects of anoxia and aglycaemia

Ischaemia excites sensory neurones (generating pain) and promotes calcitonin gene-related peptide release from nerve endings. Acidosis is thought to play a key role in mediating excitation via the activation of proton-sensitive cation channels. In this study, we investigated the effects of acidosis upon Ca²⁺ signalling in sensory neurones from rat dorsal root ganglia. Both hypercapnic (pH_o 6.8) and metabolic–hypercapnic (pH_o 6.2) acidosis caused a biphasic increase in cytosolic calcium concentration ([Ca²⁺] _i ). This comprised a brief Ca²⁺ transient (half-time approximately 30 s) caused by Ca²⁺ influx followed by a sustained rise in [Ca²⁺] _i due to Ca²⁺ release from caffeine and cyclopiazonic acid-sensitive internal stores. Acid-evoked Ca²⁺ influx was unaffected by voltage-gated Ca²⁺-channel inhibition with nickel and acid sensing ion channel (ASIC) inhibition with amiloride but was blocked by inhibition of transient receptor potential vanilloid receptors (TRPV1) with (E)-3-(4-t-butylphenyl)-N-(2,3-dihydrobenzo[b][1,4] dioxin-6-yl)acrylamide (AMG 9810; 1 μM) and N-(4-tertiarybutylphenyl)-4-(3-cholorphyridin-2-yl) tetrahydropryazine-1(2H)-carbox-amide (BCTC; 1 μM). Combining acidosis with anoxia and aglycaemia increased the amplitude of both phases of Ca²⁺ elevation and prolonged the Ca²⁺ transient. The Ca²⁺ transient evoked by combined acidosis, aglycaemia and anoxia was also substantially blocked by AMG 9810 and BCTC and, to a lesser extent, by amiloride. In summary, the principle mechanisms mediating increase in [Ca²⁺] _i in response to acidosis are a brief Ca²⁺ influx through TRPV1 followed by sustained Ca²⁺ release from internal stores. These effects are potentiated by anoxia and aglycaemia, conditions also prevalent in ischaemia. The effects of anoxia and aglycaemia are suggested to be largely due to the inhibition of Ca²⁺-clearance mechanisms and possible increase in the role of ASICs.     

Mitochondrial aldehyde dehydrogenase (ALDH2) protects against streptozotocin-induced diabetic cardiomyopathy: role of GSK3β and mitochondrial function

Background

Mitochondrial aldehyde dehydrogenase (ALDH2) displays some promise in the protection against cardiovascular diseases although its role in diabetes has not been elucidated.

Methods

This study was designed to evaluate the impact of ALDH2 on streptozotocin-induced diabetic cardiomyopathy. Friendly virus B(FVB) and ALDH2 transgenic mice were treated with streptozotocin (intraperitoneal injection of 200 mg/kg) to induce diabetes.

Results

Echocardiographic evaluation revealed reduced fractional shortening, increased end-systolic and -diastolic diameter, and decreased wall thickness in streptozotocin-treated FVB mice. Streptozotocin led to a reduced respiratory exchange ratio; myocardial apoptosis and mitochondrial damage; cardiomyocyte contractile and intracellular Ca²⁺ defects, including depressed peak shortening and maximal velocity of shortening and relengthening; prolonged duration of shortening and relengthening; and dampened intracellular Ca²⁺ rise and clearance. Western blot analysis revealed disrupted phosphorylation of Akt, glycogen synthase kinase-3?? and Foxo3a (but not mammalian target of rapamycin), elevated PTEN phosphorylation and downregulated expression of mitochondrial proteins, peroxisome proliferator-activated receptor ?? coactivator 1?? and UCP-2. Intriguingly, ALDH2 attenuated or ablated streptozotocin-induced echocardiographic, mitochondrial, apoptotic and myocardial contractile and intracellular Ca²⁺ anomalies as well as changes in the phosphorylation of Akt, glycogen synthase kinase-3??, Foxo3a and phosphatase and tensin homologue on chromosome ten, despite persistent hyperglycemia and a low respiratory exchange ratio. In vitro data revealed that the ALDH2 activator Alda-1 and glycogen synthase kinase-3?? inhibition protected against high glucose-induced mitochondrial and mechanical anomalies, the effect of which was cancelled by mitochondrial uncoupling.

Conclusions

In summary, our data revealed that ALDH2 acted against diabetes-induced cardiac contractile and intracellular Ca²⁺ dysregulation, possibly through regulation of apoptosis, glycogen synthase kinase-3?? activation and mitochondrial function independent of the global metabolic profile.     

A comparative study of the calcium accumulation by mitochondria and microsomes isolated from the smooth muscle of the guinea-pig taenia coli

Summary The calcium uptake by mitochondria and microsomes isolated from the guinea-pig taenia coli was studied at physiological Ca²⁺ concentrations, buffered by Ca-EGTA mixtures. The Ca accumulation by the mitochondria was measured from the difference between the amount of Ca taken up in the presence and in the absence of a specific mitochondrial inhibitor. The Ca uptake by the microsomes was determined in a solution containing oxalate and a mitochondrial inhibitor. It was calculated from the difference in Ca uptake measured with and without ATP. By using this procedure, the necessity of extensive purification of the isolated fractions was avoided.The (Ca²⁺) for half-maximal transport in the mitochondria is 7×10^–6 M. At (Ca²⁺) lower than 2×10^–7 M, Ca is taken up in an energy-dependent way.In the microsomes the apparentK _m for Ca is 7×10^–7 M. Accumulation is still stimulated by ATP at a (Ca²⁺) as low as 4×10^–8 M.The results show that the rate of Ca uptake by the cell organelles corresponding to the microsomal vesicles is sufficiently fast to explain the speed of relaxation of the taenia coli. The results also suggest that these cell organelles are more important than the mitochondria in regulating the cytoplasmic Ca concentration.     

L-精氨酸对兔缺血-再灌注心肌线粒体功能及结构的影响

目的:探讨L-精氨酸对心肌缺血-再灌注(MIR)时心肌细胞线粒体功能及结构的影响。方法:实验兔30只,随机分为正常对照组(control组)、心肌缺血-再灌注组(MIR组)和心肌缺血-再灌注+L-精氨酸治疗组(MRI+L-Arg组);分别观察心肌线粒体呼吸功能、Ca²⁺浓度([Ca²⁺]m)、丙二醛(MDA)浓度、超氧化物歧化酶(SOD)活性及超微结构的改变和心肌组织三磷酸腺苷(ATP)、二磷酸腺苷(ADP)、一磷酸腺苷(AMP)含量、总腺苷酸量(TAN)、能荷(EC)的变化。结果:MIR+L-Arg组线粒体呼吸控制率(RCR)、Ⅲ态呼吸速率(V₃)、SOD、面密度(Sv)、比表面(δ)明显高于MIR组,Ⅳ态呼吸速率(V₄)、[Ca²⁺]m、MDA、体密度(Vv)、横径(Hd)显著低于MIR组,心肌组织ATP、ADP、TAN及EC均明显高于MIR组;且与control组比较,V₃、V₄、SOD、MDA、Vv、Sv、δ、数密度(Nv)、纵径(Vd)及AMP、TAN无明显差异。结论:L-精氨酸可通过降低氧自由基水平和减轻钙超载,而改善缺血-再灌注心肌的线粒体功能及结构。