非酒精性脂肪肝与钙稳态关系的研究进展

钙离子（Ca²⁺）是细胞内重要的第二信使,其主要储存在内质网和线粒体中,参与调控细胞多种生理功能及维持内质网、线粒体功能。内质网和线粒体对钙离子的摄取与释放直接影响胞内钙离子水平的变化,继而影响细胞正常生理功能。病理条件下,细胞内钙离子稳态失衡,可引发细胞生理功能异常并进一步影响内质网、线粒体功能。非酒精性脂肪肝（NAFLD）是临床常见病和多发病,其主要病理特征是：肝脏脂肪样变性、内质网应激、线粒体损伤和非特异性炎性反应等。其中持续的内质网应激及线粒体功能障碍是NAFLD发生发展的重要诱因。而细胞内钙稳态的变化可直接导致内质网及线粒体功能异常,继而影响NAFLD的发生发展。本文主要从钙稳态的主要影响因素以及钙稳态变化与NAFLD的关系两方面进行阐述,为寻求和研发防治NAFLD的药物提供新的方向。     

Heptachlor epoxide induces a non-capacitative type of Ca2+ entry and immediate early gene expression in mouse hepatoma cells

The effects of the organochlorine (OC) liver tumor promoter heptachlor epoxide (HE) and a related non-tumor promoting OC, delta-hexachlorocyclohexane (δ-HCH), on the dynamics of intracellular calcium (Ca²⁺) were investigated in mouse 1c1c7 hepatoma cells. HE induced a non-capacitative, Ca²⁺ entry-like phenomenon, which was transient and concentration-dependent with 10 and 50 μM HE. The plasma membrane Ca²⁺ channel blocker SKF-96365 antagonized this HE-induced Ca²⁺ entry. δ-HCH failed to induce Ca²⁺ entry, rather it antagonized the HE-induced Ca²⁺ entry. Both HE and δ-HCH induced Ca²⁺ release from endoplasmic reticulum (ER) at treatment concentrations as low as 10 μM; at 50 μM, the former induced 5× as much Ca²⁺ release as the latter. The HE-induced Ca²⁺ release from the ER was antagonized using the IP₃ receptor/channel blocker xestospongin C, suggesting that HE induces ER Ca²⁺ release through the IP₃ receptor/channel pore. These results show that the effect of HE on cellular Ca²⁺ mimics that of mitogens such as epidermal and hepatocyte growth factors. They also provide insight into the similarities and differences between tumorigenic and non-tumorigenic OCs, in terms of the mechanisms and the extent of the [Ca²⁺]_i increased by these agents.     

Tetrabromobisphenol A (TBBPA), induces cell death in TM4 Sertoli cells by modulating Ca transport proteins and causing dysregulation of Ca homeostasis

Tetrabromobisphenol A (TBBPA) is a commonly used brominated flame retardant (BFR) utilized to reduce the flammability of a variety of products. Studies have indicated that a number of BFRs are becoming widely distributed within the environment and are bio-accumulating within organisms. There has been much speculation that a variety of phenolic pollutants (including compounds chemically related to TBBPA, such as bisphenol A) may cause endocrine disruption and Ca²⁺ dysregulation in cells involved in spermatogenesis. In this study we therefore investigate the effects of TBBPA on mouse TM4 Sertoli cells (essential for sperm development). Results show that TBBPA increases Ca²⁺ within these cells in the 5–60 μM concentration range (EC₅₀, 21 μM). TBBPA also causes cell death (LC₅₀, 18 μM) partly via apoptosis, involving Ca²⁺-dependent mitochondrial depolarisation. Studies on intracellular Ca²⁺ transporters shows that TBBPA can inhibit sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPases (SERCA) at low concentrations (IC₅₀, 0.4 to 1.2 μM) and also activate the Ryanodine receptor Ca²⁺ channel within the 0.4–4 μM concentration range. Therefore these studies suggest that the cytotoxic effects of TBBPA on cells is partly due to dysregulation of Ca²⁺ signalling, by directly affecting Ca²⁺ transport proteins.     

STIM1 regulates acidic Ca2+ store refilling by interaction with SERCA3 in human platelets

Ca²⁺ mobilization regulates a wide variety of cellular functions. Platelets posses agonist-releasable Ca²⁺ stores in acidic organelles where sarcoendoplasmic reticulum Ca²⁺-ATPase-3 (SERCA) pump is involved in store refilling. Stromal interaction molecule 1 (STIM1), which has been presented as a central regulator of platelet function, is a Ca²⁺ sensor of the intracellular Ca²⁺ stores. Here we present that STIM1 is required for acidic store refilling. Electrotransjection of cells with anti-STIM1 (Y²³¹–K²⁴³) antibody, directed towards a cytoplasmic sequence of STIM1, significantly reduced acidic store refilling, which was tested by remobilizing Ca²⁺ from the acidic stores using 2,5-di-(t-butyl)-1,4-hydroquinone (TBHQ) after a brief refilling period that followed thrombin stimulation. Platelet treatment with thrombin or thapsigargin in combination with ionomycin, to induce extensive Ca²⁺ store depletion, resulted in a transient increase in the interaction between STIM1 and SERCA3, reaching a maximum 30 s after stimulation. The coupling between STIM1 and SERCA3 was abolished by electrotransjection with anti-STIM1 antibody. The interaction between STIM1 and SERCA3 induced by thrombin or by treatment with thapsigargin plus ionomycin is reduced in platelets from type 2 diabetic patients, as well as Ca²⁺ reuptake into the acidic Ca²⁺ stores. These findings provide evidence for a role of STIM1 in acidic store refilling in platelets probably acting as a Ca²⁺ sensor and regulating the activity of SERCA3. This action is impaired in platelets from type 2 diabetics, which might lead to the enhanced cytosolic Ca²⁺ concentration observed and, therefore, in platelet hyperactivity.     

Methapyrilene hepatotoxicity is associated with oxidative stress, mitochondrial disfunction and is prevented by the Ca channel blocker verapamil

Methapyrilene (MP) is an unusual hepatotoxin in that it causes periportal necrosis in rats. The mechanism of acute methapyrilene hepatotoxicity has, therefore, been investigated in cultured male rat hepatocytes. Addition of methapyrilene to rat hepatocytes resulted in a time- and dose-dependent loss in cell viability between 4 and 8 h of incubation as judged by cellular enzyme leakage. The cytochrome P450 (CYP) inhibitor metyrapone protected against methapyrilene-mediated toxicity suggesting that MP is metabolised by CYP for toxicity. The concentration-dependent protection from methapyrilene toxicity afforded by metyrapone correlated with an inhibition of microsomal CYP2C11-associated androstenedione 16 hydroxylase activity, and hepatocytes prepared from hypophysectomised rats (containing reduced levels of microsomal immunodetectable CYP2C11 and associated androstenedione 16 hydroxylase activity) showed resistance to the toxic effects of methapyrilene. These data suggest that the toxicity of methapyrilene is predominantly dependent on the CYP2C11 isoform. Treatment of hepatocytes with a toxic concentration of MP caused oxidative stress as indicated by increases in NADP⁺ levels within 2 h and cellular thiol oxidation as evidenced by a reduction—but not complete loss—in glutathione levels. Methapyrilene hepatotoxicity was associated with an early loss in mitochondrial function, as indicated by mitochondrial swelling and significant losses in cellular ATP within 2 h. Co-incubation of methapyrilene-treated hepatocytes with inhibitors of inner mitochondrial transition permeability pore opening—cyclosporin A or the thiol reductant dithiothreitol—abrogated cell death suggesting that pore opening and loss of mitochondrial Ca²⁺ homeostasis play a significant role in methapyrilene-mediated cell death. Co-incubation of methapyrilene-treated hepatocytes with the phenylalkylamine calcium channel blocker verapamil—but not by treating cells in a nominally calcium-free medium—also abrogated cell death, suggesting that if Ca²⁺ is involved in cell killing then it is dependent on an intracellular Ca²⁺ pool. Pre-treatment of hepatocytes for 1 h with verapamil—to inhibit intracellular Ca²⁺ pool filling—increased the potency of verapamil protection against methapyrilene toxicity by approximately 100-fold. Taken together, these data indicate that methapyrilene intoxication leads to mitochondrial disfunction and suggest a critical role for a loss of mitochondrial Ca²⁺ homeostasis in this model of hepatocyte death.     

Calcium-mobilizing receptors

In a wide variety of cells, activation of certain surface membrane receptors results in a rise in intracellular Ca²⁺ due to release of intracellular Ca²⁺ stores, and to an increased rate of Ca²⁺ entry. James Putney summarizes some current ideas about the mechanisms by which Ca²⁺-mobilizing receptors act. The intracellular Ca²⁺ release is believed to result from the action of inositol 1,4,5-trisphosphate, generated as a result of the activation by receptors of a polyphosphoinositide-specific phospholipase C. Evidence suggests that a guanine nucleotide-dependent regulatory protein may mediate this activation. The regulation of Ca²⁺ entry is less well understood, but may involve a mechanism whereby the emptying of the (1,4,5)IP₃-sensitive pool secondarily activates Ca²⁺ influx across the plasma membrane.     

Inhibition of aftercontractions and phasic calcium release by yohimbine in ferret papillary muscle

Phasic release of calcium from the sarcoplasmic reticulum occurs in all mammalian cardiac preparations when the intracellular calcium concentration is sufficiently high. The phasic calcium release is often sufficient to trigger electrophysiological responses and aftercontractions. These can be detrimental to normal cardiac function. We induced phasic calcium release in ferret papillary muscles loaded with the calcium indicator aequorin. Development of phasic calcium release was associated with an increase in resting and peak [Ca²⁺]_i. Inhibiting sodium channels with yohimbine reduced resting [Ca²⁺]_i and prevented phasic calcium release. We propose a mechasism where by reduced [Na⁺]_i, and the subsequent increased efflux of calcium via sodium/calcium exchange reduced [Ca²⁺]_i.     

Sarcoplasmic reticulum Ca2+ release channel ryanodine receptor (RyR2) plays a crucial role in aconitine-induced arrhythmias

The present study established a model of RyR₂ knockdown cardiomyocytes and elucidated the role of RyR₂ in aconitine-induced arrhythmia. Cardiomyocytes were obtained from hearts of neonatal Sprague–Dawlay rats. siRNAs were used to down-regulate RyR₂ expression. Reduction of RyR₂ expression was documented by RT-PCR, western blot, and immunofluorescence. Ca²⁺ signals were investigated by measuring the relative intracellular Ca²⁺ concentration, spontaneous Ca²⁺ oscillations, caffeine-induced Ca²⁺ release, and L-type Ca²⁺ currents. In normal cardiomyocytes, steady and periodic spontaneous Ca²⁺ oscillations were observed, and the baseline [Ca²⁺]_i remained at the low level. Exposure to 3 μM aconitine increased the frequency and decreased the amplitude of Ca²⁺ oscillations; the baseline [Ca²⁺]_i and the level of caffeine-induced Ca²⁺ release were increased but the L-type Ca²⁺ currents were inhibited after application of 3 μM aconitine for 5 min. In RyR₂ knockdown cardiomyocytes, the steady and periodic spontaneous Ca²⁺ oscillations almost disappeared, but were re-induced by aconitine without affecting the baseline [Ca²⁺]_i level; the level of caffeine-induced Ca²⁺ release was increased but L-type Ca²⁺ currents were inhibited. Alterations of RyR₂ are important consequences of aconitine-stimulation and activation of RyR₂ appear to have a direct relationship with aconitine-induced arrhythmias. The present study demonstrates a potential method for preventing aconitine-induced arrhythmias by inhibiting Ca²⁺ leakage through the sarcoplasmic reticulum RyR₂ channel.     

Mechanisms for resin acid effects on membrane currents and GABA(A) receptors in mammalian CNS

Resin acids from bleached wood pulp are toxic to fish. 12,14-Dichlorodehydroabietic acid (12,14-Cl₂DHA) raises cytoplasmic Ca²⁺ in synaptosomes and blocks neural GABA_A receptors; however, the underlying mechanism remains unclear in these earlier rodent studies. 12,14-Cl₂DHA (50 μM) almost completely blocked native GABA_A currents (rat cortical cultures) but had no significant effect on picrotoxin-sensitive recombinant human receptors in oocytes (1, β2 and γ2L: the most prevalent isoforms in mammalian brain). In oocytes, 12,14-Cl₂DHA failed to produce a calcium-activated chloride current, in contrast to the calcium ionophore ionomycin (10 μM). However, in cultured cortical pyramidal cells, both ionomycin and 12,14-Cl₂DHA produced chloride-selective currents of similar magnitude (presumably secondary to Ca²⁺ release). 12,14-Cl₂DHA was unable to stimulate phosphate labelling of [³H]-inositol in mouse synaptosomes, indicating that the study compound does not cause Ca²⁺ release via an IP₃ mechanism. Calcium pump ATPase inhibition also seems unlikely since thapsigargin did not elevate free calcium in synaptosomes. 12,14-Cl₂DHA clearly blocks GABA_A currents indirectly: we infer that its toxicity may be secondary to the elevations in cytoplasmic Ca²⁺ via an unidentified recognition site (or receptor) found in neuronal cells.     

CGP37157 modulates mitochondrial Ca homeostasis in cultured rat dorsal root ganglion neurons

The effects of 7-chloro-3,5-dihydro-5-phenyl-1H-4,1-benzothiazepine-2-on (CGP37157), an inhibitor of mitochondrial Na⁺/Ca²⁺ exchange, on depolarization-induced intracellular free Ca²⁺ concentration ([Ca²⁺]_i) transients were studied in cultured rat dorsal root ganglion neurons with indo-1-based microfluorimetry. A characteristic plateau in the recovery phase of the [Ca²⁺]_i transient resulted from mitochondrion-mediated [Ca²⁺]_i buffering. It was blocked by metabolic poisons and was not dependent on extracellular Ca²⁺. CGP37157 produced a concentration-dependent decrease in the amplitude of the mitochondrion-mediated plateau phase (IC₅₀=4±1 μM). This decrease in [Ca²⁺]_i was followed by an increase in [Ca²⁺]_i upon removal of the drug, suggesting that Ca²⁺ trapped in the matrix was released when the CGP37157 was removed from the bath. CGP37157 also inhibited depolarization-induced Ca²⁺ influx at the concentrations required to see effects on [Ca²⁺]_i buffering. Thus, CGP37157 inhibits mitochondrial Na⁺/Ca²⁺ exchange and directly inhibits voltage-gated Ca²⁺ channels, suggesting caution in its use to study [Ca²⁺]_i regulation in intact cells.     

Binding domain of oligomycin on Na(+),K(+)-ATPase

Oligomycin inhibits Na⁺,K⁺-ATPase activity by stabilizing the Na⁺ occlusion but not the K⁺ occlusion. To locate the binding domain of oligomycin on Na⁺,K⁺-ATPase, the tryptic-digestion profile of Na⁺,K⁺-ATPase was compared with the profile of Na⁺ occlusion within the digested Na⁺,K⁺-ATPase in the presence of oligomycin. The Na⁺ occlusion profile is responsible for the digestion profile of the -subunit, which is the catalytic subunit of the ATPase. The effect of oligomycin on chimeric Ca²⁺-ATPase activity was examined. The chimera used, in which the 163 N-terminal amino acids of chicken sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase 1 were replaced with the 200 N-terminal amino acids of the chicken Na⁺,K⁺-ATPase 1-subunit, partially retains the Na⁺-dependent characteristics of Na⁺,K⁺-ATPase, because the chimeric Ca²⁺-ATPase activity is activated by Na⁺ but inhibited by ouabain, a specific inhibitor of Na⁺,K⁺-ATPase (Ishii, T., Lemas, M.V., Takeyasu, K., 1994, Proc. Natl. Acad. Sci. U. S. A., 91, 6103–6107). Oligomycin depressed the activation by Na⁺ of the chimeric Ca²⁺-ATPase activity. These findings suggest that the 200 N-terminal amino acids of the Na⁺,K⁺-ATPase -subunit include a binding domain for oligomycin.     

Effects of verapamil, diltiazem, nisoldipine and felodipine on sarcoplasmic reticulum

Verapamil, diltiazem, nisoldipine and felodipine, calcium antagonist drugs with different chemical structures, were studied for their effects on activities of sarcoplasmic reticulum (SR) isolated from dog cardiac and rabbit skeletal muscles. Nisoldipine and felodipine exerted biphasic actions on both cardiac and skeletal SR Ca²⁺-ATPase with maximum activation of 40–60% occurring at 20–40 μM for nisoldipine and 30–40% occurring at 15–30 μM for felodipine. At higher drug concentrations, Ca²⁺-ATPase was inhibited. In the presence of oxalate the maximum activation of the Ca²⁺ uptake rates at 5–20 μM nisoldipine were 30–50% for cardiac SR and 80–100 μM of the drug were 300–500% for skeletal SR. Felodipine inhibited the rate of Ca²⁺ uptake by dog cardiac SR, but activated Ca²⁺ uptake by rabbit skeletal SR with a maximum of 30–50% at 12–25 μM. At higher concentrations of the two drugs the rate of Ca²⁺ uptake was inhibited. In the absence of oxalate, i.e., limited tranport, nisoldipine shortened the duration of time that Ca²⁺ was bound to the cardiac and skeletal SR, while the rate of release of Ca²⁺ from skeletal SR was stimulated. Felodipine at low concentrations similarly caused a premature release of Ca²⁺ from skeletal SR at a rapid rate; at high concentrations both drugs did not alter Ca²⁺ binding but delayed Ca²⁺ release. Unlike nisoldipine and felodipine, verapamil and diltiazem inhibited the rates of Ca²⁺ transport both in cardiac and skeletal SR. The two drugs inhibited Ca²⁺-ATPase in cardiac SR but activated the enzyme in skeletal SR. Thus, these drugs caused complex and different effects on cardiac and skeletal SR, possibly resulting from perturbations of the lipid environment of the SR Ca²⁺-ATPase.     

Force-frequency relationship in intact mammalian ventricular myocardium: physiological and pathophysiological relevance

The force–frequency relationship (FFR) is an important intrinsic regulatory mechanism of cardiac contractility. The FFR in most mammalian ventricular myocardium is positive; that is, an increase in contractile force in association with an increase in the amplitude of Ca²⁺ transients is induced by elevation of the stimulation frequency, which reflects the cardiac contractile reserve. The relationship is different depending on the range of frequency and species of animal. In some species, including rat and mouse, a ‘primary-phase’ negative FFR is induced over the low-frequency range up to approximately 0.5–1 Hz (rat) and 1–2 Hz (mouse). Even in these species, the FFR over the frequency range close to the physiological heart rate is positive and qualitatively similar to that in larger mammalian species, although the positive FFR is less prominent. The integrated dynamic balance of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) is the primary cellular mechanism responsible for the FFR and is determined by sarcoplasmic reticulum (SR) Ca²⁺ load and Ca²⁺ flux through the sarcolemma via L-type Ca²⁺ channels and the Na⁺-Ca²⁺ exchanger. Intracellular Na⁺ concentration is also an important factor in [Ca²⁺]_i regulation. In isolated rabbit papillary muscle, over a lower frequency range (<0.5 Hz), an increase in duration rather than amplitude of Ca²⁺ transients appears to be responsible for the increase in contractile force, while over an intermediate frequency range (0.5–2.0 Hz), the amplitude of Ca²⁺ transients correlates well with the increase in contractile force. Over a higher frequency range (>2.5 Hz), the contractile force is dissociated from the amplitude of Ca²⁺ transients probably due to complex cellular mechanisms, including oxygen limitation in the central fibers of isolated muscle preparations, while the amplitude of Ca²⁺ transients increases further with increasing frequency (‘secondary-phase’ negative FFR). Calmodulin (CaM) may contribute to a positive FFR and the frequency-dependent acceleration of relaxation, although the role of calmodulin has not yet been established unequivocally. In failing ventricular myocardium, the positive FFR disappears or is inverted and becomes negative. The activation and overexpression of cardiac sarcoplasmic reticulum Ca²⁺ ATPase (SERCA2a) is able to reverse these abnormalities. Frequency-dependent alterations of systolic and diastolic force in association with those of Ca²⁺ transients and diastolic [Ca²⁺]_i levels are excellent indicators for analysis of cardiac excitation-contraction coupling, and for evaluating the severity of cardiac contractile dysfunction, cardiac reserve capacity and the effectiveness of therapeutic agents in congestive heart failure.     

Effect of calcium agonists and antagonists on cerebellar granule cells

In cerebellar cultures, comprising predominantly granule neurones, dihydropyridine (DHP) Ca²⁺ agonists were potent stimulators of voltage-sensitive ⁴⁵Ca²⁺ uptake. Their effect was maximal in partially depolarized cells; at 15 mM K_e⁺ half maximal stimulation occurred at about 5 × 10⁻⁸ M BAY K 8644 and 10⁻⁷ M(+)-(S)-202 791. Organic Ca²⁺ antagonists were effective inhibitors of voltage-sensitive calcium entry into granule cells: the order of potency in blocking uptake induced by sub-maximal concentration of K⁺ and BAY K 8644 was nifedipine > (−)-202 791 > D600. BAY K 8644 also stimulated the release of glutamate, the transmitter of the granule cells, from depolarized cells. Granule cells are therefore a class of neurones whose responsiveness to organic Ca²⁺ effectors is similar to that of cardiac and smooth muscle. The discrepant findings on the effect of calcium effectors in various preparations of nervous tissues may thus reflect a differential distribution of voltage-sensitive Ca²⁺ channels in different neuronal cell types.     

Ryanodine as a trigger of tension oscillations in rat ventricular muscle

In rat ventricular muscles, ryanodine (10–30 nM) evoked tension oscillation in the relaxation phase of an isometric twitch (relaxation oscillations) and an increase of tonic tension (ryanodine contracture). Both events were more pronounced in Ca²⁺-loaded rat muscles due to the addition of 0.5 μM adrenaline, an increase in the Ca²⁺ concentration in the solution and high muscle activity. The ryanodine-induced tension oscillations were comparable to those triggered by caffeine in this species. In both cases, blockers of the release of Ca²⁺ from the sarcoplasmic reticulum, namely tetracaine and dantrolene, abolished the relaxation oscillations and the ryanodine contracture. The results suggest that the ability of low concentrations of ryanodine to facilitate the release of Ca²⁺ from the sarcoplasmic reticulum, as shown recently in biochemical experiments, makes a direct contribution to triggering the relaxation oscillations and the ryanodine contracture in intact ventricular muscles. The Ca²⁺ load of the sarcoplasmic reticulum appears to be essential for the manifistation of the ryanodine Ca²⁺-releasing activity in rat ventricular muscles.     

Effect of 3,4,5-Trimethoxybenzoic Acid 8-Diethylamino-octyl ester (TMB-8) on Neuronal Calcium Homeostasis,Protein Synthesis,and Energy Metabolism

Abstract: It has been suggested recently that disturbances of endoplasmic reticulum calcium homeostasis plays a major role in ischaemic cell injury of the brain. Depletion of endoplasmic reticulum calcium stores induces suppression of the initiation process of protein synthesis, a prominent feature of ischaemic cell damage. The benzoic acid derivative 3,4,5-trimethoxybenzoic acid 8-diethylamino-octyl ester (TMB-8), an established inhibitor of calcium release from endoplasmic reticulum, would be an ideal tool for elucidating the role of endoplasmic reticulum dysfunction in this pathological process. The present investigation was performed to study the effects of TMB-8 on neuronal metabolism (cytoplasmic calcium activity, ATP levels and protein synthesis) using hippocampal slices and primary neuronal cell cultures. In addition, we investigated whether the rise in cytoplasmic calcium activity and the suppression of protein synthesis induced by endoplasmic reticulum calcium pool depletion, is reversed by this agent. Exposure of neurones to TMB-8 (100 μM) induced a small transient increase in cytoplasmic calcium activity ([Ca²⁺]_i), whereas a second dose of TMB-8 (200 μM) produced a marked and sustained rise in [Ca²⁺]_i. The increase in [Ca²⁺]_i evoked by blocking endoplasmic reticulum Ca²⁺-ATPase was only transiently suppressed and then aggravated by TMB-8. The dose-dependent suppression of protein synthesis by TMB-8, observed both in neuronal cultures and hippocampal slices, indicates that TMB-8 has a pathological effect on neuronal metabolism. This inhibition was not reversed after washing-off of the drug. TMB-8 did not reverse the inhibition of protein synthesis evoked by caffeine, which depletes endoplasmic reticulum calcium stores by activating the ryanodine receptor. The results indicate that TMB-8 is not a suitable investigative tool for blocking in neuronal cell cultures the depletion of endoplasmic reticulum calcium stores and the suppression of protein synthesis induced by endoplasmic reticulum calcium pool depletion.     

Intracellular calcium and neurotoxic events

Calcium is important in many intracellular regulatory processes. However, the maintenance of low levels of this cation within the cytosol is essential for maintenance of cell viability, in view of the large concentration gradient of ionic calcium across the plasma membrane. The expenditure of energy is needed to maintain intracellular calcium concentration [Ca²⁺]_i at normal levels. In addition, the integrity of the limiting membrane is also vital for this function. Thus, any disruption of membrane characteristics or of mitochondrial anabolic processes may lead to deleterious levels of [Ca²⁺]_i. The toxicity of a wide range of unrelated agents may, therefore, be in part due to elevation of cytosolic calcium. This general event may synergize with the more selective harmful properties of a compound, thus adversely affecting cell metabolism. The capacity now exists to measure levels of [Ca²⁺]_i in isolated cells or organelles such as synaptosomes. The use of such in vitro models can be of value in the evaluation of the neurotoxic potential of compounds. This method, in conjunction with the use of pharmacological agents known to act at specific sites, and with the use of radioactive calcium in translocation studies, also has utility in the delineation of the biochemical mode of action of neurotoxic agents.     

Effect of glutamate and extracellular calcium on uptake of inorganic lead (Pb2+) in immortalized mouse hypothalamic GT1-7 neuronal cells

We have previously shown that although glutamate alone has no effects on viability of mouse hypothalamic GT1–7 cells, it clearly enhances Pb²⁺-induced cytotoxicity. It is likely that Pb²⁺ must enter cells to exert most of its toxic effects. Pb²⁺ is known to substitute for Ca²⁺ in many cellular processes. Therefore, we studied the uptake mechanisms of Pb²⁺ into GT1–7 neuronal cells with a special focus on the role of extracellular calcium (Ca²⁺), voltage-sensitive calcium channels (VSCCs) and glutamate. Basal uptake of Pb²⁺ (1 μM or 10 μM), i.e. without any external stimulus, clearly increased in nominally Ca²⁺-free buffer and was partially abolished by 13 mM Ca²⁺ when compared to uptake in the presence of a physiological concentration of extracellular Ca²⁺ (1.3 mM). Depolarization by 25 mM K⁺, or antagonists of VSCCs, verapamil (10 μM) or flunarizine (10 μM) had no clear effect on basal Pb²⁺ uptake. Glutamate (1 mM) increased Pb²⁺ uptake, but only when cells were treated with 1 μM Pb²⁺ in the presence of 1.3 mM Ca²⁺. Our data suggest that Pb²⁺ competes for the same cellular uptake pathways with Ca²⁺, although not via VSCCs. In addition, enhancement of Pb²⁺-induced neurotoxicity by glutamate may be due to increased neuronal uptake of Pb²⁺.     

The plasma membrane calcium pump, its role and regulation: new complexities and possibilities

Significant progress has been achieved in elucidating the role of the plasma membrane Ca²⁺-ATPase in cellular Ca²⁺ homeostasis and physiology since the enzyme was first purified and physiology since the enzyme was first purified and cloned a number of years ago. The simple notion that the PM Ca²⁺-ATPase controls resting levels of [Ca²⁺]_CYT has been challenged by the complexity arising from the finding of four major isoforms and splice variants of the Ca²⁺ pump, and the finding that these are differentially localized in various organs and subcellular regions. Furthermore, the isoforms exhibit differential sensitivities to Ca²⁺, calmodulin, ATP, and kinase-mediated phosphorylation. The latter pathways of regulation can give rise to activation or inhibition of the Ca²⁺ pump activity, depending on the kinase and the particular Ca²⁺ pump isoform. Significant progress is being made in elucidating subtle and more profound roles of the PM Ca²⁺-ATPase in the control of cellular function. Further understanding of these roles awaits new studies in both transfected cells and intact organelles, a process that will be greatly aided by the development of new and selective Ca²⁺ pump inhibitors.