Inhibition of the liberation of arachidonic acid by cadmium ions in rabbit alveolar macrophages

The effects of CdCl₂ on the liberation of arachidonic acid (20∶4) from membrane phospholipids of A23187-stimulated rabbit alveolar macrophages and on the activity of phospholipase A₂ (PLA₂) in a cytosolic fraction were studied. Alveolar macrophages were prelabeled with [³H]arachidonic acid (20∶4) and then treated with A23187. This treatment resulted in a remarkable increase in the liberation of [³H]20∶4 from their phospholipids. Exposure of cells to Cd²⁺ inhibited the liberation of [³H]20∶4 in a dose-dependent manner. Liberation of [³H]20∶4 from cell lipids was calcium dependent and the inhibitory effect of Cd²⁺ competed with the stimulatory effect of Ca²⁺. When Ca²⁺ was removed from the incubation medium, Cd²⁺ did not influence the liberation of [³H]20∶4. Entry of⁴⁵Ca²⁺ into cells was enhanced by treatment of A23187. However, Cd²⁺ did not influence the cellular uptake of⁴⁵Ca²⁺. Treatment with A23187 markedly enhanced entry of¹⁰⁹Cd²⁺ into cells. The effect of Cd²⁺ on the activity of phospholipase A₂ was determined with 1-palmitoyl-2-[¹⁴C]arachidonoyl-sn-glycero-3-phosphocholine as substrate. Calcium-dependent activation of PLA₂ was observed and Cd²⁺ inhibited activation in a dose-dependent manner. These results suggest that exposure of alveolar macrophages to Cd²⁺ causes a reduction in the rate of liberation of 20∶4 from cell lipids, as a possible result of the inhibition of PLA₂ activity by Cd²⁺.     

The toxicity of H2O2 on the ionic homeostasis of airway epithelial cells in vitro

Oxygen species may be formed in the air spaces of the respiratory tract in response to environmental pollution such as particulate matter. The mechanisms and target molecules of these oxidants are still mainly unknown but may involve modifications of the ionic homeostasis in epithelial cells. Cytosolic concentrations of Ca²⁺ (Fura2) and Na⁺ (SBFI) and short-circuit current (Isc) were followed in primary cultures of human nasal epithelial cells and in the cell line 16HBE14o⁻ after exposure to H₂O₂ or ·OH (H₂O₂+Fe²⁺). Cells were grown on glass coverslips for ionic imaging or on permeable snapwell inserts for Isc studies. Exposure of the apical as well as the basal side of the cultures to H₂O₂ or ·OH induced a concentration-dependent transient increase in Isc which is due to a transient secretion of Cl⁻. Ca_i also increased transiently with approximately the same kinetics. The response was dependent on the release of calcium from intracellular stores. Na_i on the contrary increased steadily over more than an hour. When the apical membrane was permeabilized with gramicidin, ·OH inhibited the Na⁺ current (a measure of Na⁺-K⁺-ATPase activity in the baso-lateral membrane). The arrest of the pump was significant after 30 min exposure to oxidant. On the other hand no increase in the apical or baso-lateral sodium conductances could be detected. The progressive arrest of the Na⁺/K⁺-pump may contribute to the sustained elevation of Na_i. This strong modification in the cellular ionic homeostasis may participate in the stress response of the respiratory epithelium through alterations in signal transduction pathways.     

Surface binding and uptake of cadmium (Cd2+) by LLC-PK1 cells on permeable membrane supports

Recent studies have shown that Cd²⁺ has relatively specific damaging effects on cell-cell junctions in the renal epithelial cell line, LLC-PK₁. The objective of the present studies was to examine the surface binding and uptake of Cd²⁺ by LLC-PK₁ cells in relation to the disruption of cell-cell junctions. LLC-PK₁ cells on Falcon Cell Culture Inserts were exposed to CdCl₂ containing trace amounts of¹⁰⁹Cd²⁺ from either the apical or the basolateral compartments, and the accumulation of¹⁰⁹Cd²⁺ was monitored for up to 8 h. The integrity of cell-cell junctions was assessed by monitoring the transepithelial electrical resistance. The results showed that the cells accumulated 3–4 times more Cd²⁺ from the basolateral compartment than from the apical compartment. The accumulation of Cd²⁺ from the basolateral compartment occurred in two phases: a rapid, exponential phase that occurred in 1–2 h and coincided with a decrease in transepithelial resistance, and a slower, linear phase that continued for 6–8 h. The Cd²⁺ that accumulated during the rapid phase was easily removed by washing the cells in EGTA, indicating that most of it was bound to sites on the cell surface. By contrast, most of the Cd²⁺ that accumulated during the slower phase could not be removed by EGTA, indicating that it had been taken up by the cells. Additional studies showed that the rapid phase of Cd²⁺ accumulation was enhanced when Ca²⁺ was present at low concentrations (0.1 mM), and was greatly reduced when Ca²⁺ was present at high concentrations (10 mM). These results suggest that Cd²⁺ damages the junctions between LLC-PK₁ cells by interacting with Ca²⁺-sensitive sites on the basolateral cell surface.     

Dual actions of lindane (γ-hexachlorocyclohexane) on calcium homeostasis and exocytosis in rat PC12 cells

The persistent organochlorine pesticide lindane is still abundantly found in the environment and in human and animal tissue samples. Lindane induces a wide range of adverse health effects, which are at least partially mediated via the known inhibition of GABA_A and glycine receptors. Additionally, lindane has been reported to increase the basal intracellular Ca²⁺ concentration ([Ca²⁺]_i). As Ca²⁺ triggers many cellular processes, including cell death and vesicular neurotransmitter release (exocytosis), we investigated whether lindane affects exocytosis, Ca²⁺ homeostasis, production of reactive oxygen species (ROS) and cytotoxicity in neuroendocrine PC12 cells. Amperometric recordings and [Ca²⁺]_i imaging experiments with fura-2 demonstrated that lindane (≥ 10 μM) rapidly increases basal exocytosis and basal [Ca²⁺]_i. Additional imaging and electrophysiological recordings revealed that this increase was largely due to a lindane-induced membrane depolarization and subsequent opening of N- and P/Q-type voltage-gated Ca²⁺ channels (VGCC). On the other hand, lindane (≥ 3 μM) induced a concentration-dependent but non-specific inhibition of VGCCs, thereby limiting the lindane-induced increase in basal [Ca²⁺]_i and exocytosis. Importantly, the non-specific inhibition of VGCCs also reduced stimulation-evoked exocytosis and Ca²⁺ influx. Though lindane exposure concentration-dependently increased ROS production, cell viability was not affected indicating that the used concentrations were not acute cytotoxic.These combined findings indicate that lindane has two, partly counteracting effects. Lindane causes membrane depolarization, thereby increasing basal [Ca²⁺]_i and exocytosis. In parallel, lindane inhibits VGCCs, thereby limiting the basal effects and reducing stimulation-evoked [Ca²⁺]_i and exocytosis. This study further underlines the need to consider presynaptic, non-receptor-mediated effects in human risk assessment.     

Arachidonic acid release mediated by OX1 orexin receptors

Background and purpose:

We have previously shown that lipid mediators, produced by phospholipase D and C, are generated in OX₁ orexin receptor signalling with high potency, and presumably mediate some of the physiological responses to orexin. In this study, we investigated whether the ubiquitous phospholipase A₂ (PLA₂) signalling system is also involved in orexin receptor signalling.

Experimental approach:

Recombinant Chinese hamster ovary-K1 cells, expressing human OX₁ receptors, were used as a model system. Arachidonic acid (AA) release was measured from ³H-AA-labelled cells. Ca²⁺ signalling was assessed using single-cell imaging.

Key results:

Orexins strongly stimulated [³H]-AA release (maximally 4.4-fold). Orexin-A was somewhat more potent than orexin-B (pEC₅₀= 8.90 and 8.38 respectively). The concentration–response curves appeared biphasic. The release was fully inhibited by the potent cPLA₂ and iPLA₂ inhibitor, methyl arachidonyl fluorophosphonate, whereas the iPLA₂ inhibitors, R- and S-bromoenol lactone, caused only a partial inhibition. The response was also fully dependent on Ca²⁺ influx, and the inhibitor studies suggested involvement of the receptor-operated influx pathway. The receptor-operated pathway, on the other hand, was partially dependent on PLA₂ activity. The extracellular signal-regulated kinase, but not protein kinase C, were involved in the PLA₂ activation at low orexin concentrations.

Conclusions and implications:

Activation of OX₁ orexin receptors induced a strong, high-potency AA release, possibly via multiple PLA₂ species, and this response may be important for the receptor-operated Ca²⁺ influx. The response coincided with other high-potency lipid messenger responses, and may interact with these signals.     

Cadmium-induced inflammatory responses in cells relevant for lung toxicity: Expression and release of cytokines in fibroblasts,epithelial cells and macrophages

Inhalation is an important route of cadmium (Cd) exposure, and the lung is considered to be one of the main target organs of Cd toxicity. Pulmonary inflammation seems to be involved in development of many lung diseases. In the present study we show that Cd²⁺ at fairly low concentrations affects gene expression of several different cytokines/chemokines in human M1 fibroblasts. The chemokines CXCL2, CXCL3, IL-8/CXCL8 and CCL26, the pro-inflammatory cytokine IL-6 and the receptor IL-1RL1 were expressed at high levels after exposure to 7 μM Cd²⁺ for 7 h. The expression of some important cytokines was further studied in two different primary cell cultures from rat lungs. Cd²⁺ induced cytokine responses at low concentrations (3–6 μM) and early time-points both in type 2 epithelial cell-enriched cultures and alveolar macrophages. However, the two primary lung cells displayed different patterns of cytokine release. Cd²⁺ induced an increased release of IL-6 and MIP-2/CXCL2 from the epithelial cells and MIP-2, IL-1β and TNF-α from alveolar macrophages. In conclusion, the marked up-regulation of different cytokines in these cell types, that are important in development of lung injury and disease, suggests that inflammation may contribute in Cd-induced lung damage.     

Effects of Cd on AMPA receptor-mediated synaptic transmission in rat hippocampal CA1 area

Cadmium (Cd²⁺) is a common pollutant that causes a wide variety of toxic effects on the central nervous system. However, the mechanism of Cd²⁺ neurotoxicity remains to be elucidated. In the present study, we examined the effects of Cd²⁺ on AMPA receptor-mediated synaptic transmission and short-term synaptic plasticity in hippocampal CA1 area, using whole-cell patch clamp technique. Cd²⁺ significantly inhibited the peak amplitude of evoked EPSCs (eEPSCs) in a concentration-dependent manner and enhanced the short-term synaptic plasticity including paired-pulse facilitation and frequency facilitation. Cd²⁺ also decreased the frequency and amplitude of spontaneous EPSCs (sEPSCs) but had no effect on those of miniature EPSCs (mEPSCs). These effects of Cd²⁺ may involve a presynaptic mechanism of blockade of action potential-sensitive, calcium-dependent release of glutamate. In addition, Cd²⁺ prolonged the decay time of both sEPSCs and mEPSCs, which suggested a postsynaptic action site of Cd²⁺. This study demonstrates that Cd²⁺ impairs the Schaffer collateral-commissural-CA1 glutamatergic synaptic transmission and short-term plasticity in rat hippocampal slices, which may be a possible contributing mechanism for the Cd²⁺-induced neurotoxic effects.     

Comparison of some biochemical effects of teratogenic doses of mercuric mercury and cadmium in the pregnant rat

Mercuric mercury (Hg²⁺), like cadmium (Cd²⁺), interferes with the transport of certain essential metals to the conceptus in the pregnant Wistar rat and, at 48 h after the IV injection of a teratogenic dose (0.79 mg Hg²⁺/kg body weight) on day 12 of gestation, the foetal concentrations of Zn²⁺, Cu²⁺ and Fe³⁺, but not of Mg²⁺, are reduced significantly. Both Hg²⁺ and Cd²⁺, at teratogenic dose levels, inhibit the placental and foetal uptake of ⁶⁵Zn²⁺ and ⁶⁷Cu²⁺, but possibly by different mechanisms. In addition, the effects of Hg²⁺, at different times after dosing, on the uptake of these labelled tracers and of ⁵⁹Fe³⁺, administered as 15-min pulses, do not parallel the changes in the placental and foetal concentrations and contents of the endogenous, stable metallic ions. The teratogenic dose of Hg²⁺ inhibits the placental and foetal uptake of L-[4,5-³H]-leucine, but not the incorporation of the labelled amino acid into foetal protein. In contrast, the corresponding dose of Cd²⁺ inhibits both leucine uptake and protein synthesis in the placenta and foetus. Similarly, Cd²⁺ inhibits the uptake of [2-¹⁴C]-thymidine and its incorporation into foetal DNA, whereas Hg²⁺ reduces the placental and foetal uptake, but has little or no effect on the utilization of the nucleoside. Since both Cd²⁺ and Hg²⁺ reduce the foetal uptake of ⁶⁵Zn and the foetal concentration of Zn, but only Cd²⁺ interferes with DNA synthesis, it is unlikely that the inhibition of the metabolism of thymidine can be attributed to reduction in thymidine kinase activity in consequence of foetal Zn deficiency. It is concluded that the small amount of Cd²⁺ that is taken up by the foetus has a direct effect on the synthesis of DNA and protein, whereas Hg²⁺ primarily affects placental transport processes.     

The insulinotropic effect of fluoroquinolones

Antimicrobial fluoroquinolones induce, with strongly varying frequency, life-threatening hypoglycemias, which is explained by their ability to block K_ATP channels in pancreatic B-cells and thus to initiate insulin secretion. In apparent contradiction to this, we observed that none of the fluoroquinolones in this study (gatifloxacin, moxifloxacin, ciprofloxacin, and a number of fluorophenyl-substituted compounds) initiated insulin secretion of perifused mouse islets when the glucose concentration was basal (5 mM). Only when the glucose concentration was stimulatory by itself (10 mM), the fluoroquinolones enhanced secretion. The fluoroquinolones were ineffective on SUR1 Ko islets, which do not have functional K_ATP channels. All of these fluoroquinolones depolarized the membrane potential of mouse B-cells (patch-clamping in the whole-cell mode). Using metabolically intact B-cells (perforated-patch mode) however, 100 μM of gatifloxacin, ciprofloxacin or moxifloxacin were unable to depolarize when the glucose concentration was 5 mM, whereas other K_ATP channel blockers (tolbutamide and efaroxan) remained effective. Only at a very high concentration (500 μM) gatifloxacin and moxifloxacin, but not ciprofloxacin induced repetitive depolarizations which could be antagonized by diazoxide. In the presence of 10 mM glucose all fluoroquinolones which enhanced secretion markedly elevated cytosolic calcium concentration ([Ca²⁺]_i). In the presence of 5 mM glucose gatifloxacin and moxifloxacin at 500 μM but not at 100 μM elevated [Ca²⁺]_i. It is concluded that fluoroquinolones in the clinically relevant concentration range are not initiators, but rather enhancers of glucose-induced insulin secretion. The block of K_ATP channels appears necessary but not sufficient to explain the hypoglycemic effect of fluoroquinolones.     

Metabolism and transfer of the mycotoxin zearalenone in human intestinal Caco-2 cells

The mycotoxin zearalenone (ZEA) is found worldwide as contaminant in cereals and grains. It is implicated in reproductive disorders and hyperestrogenic syndromes in animals and humans exposed by food. We investigated metabolism and transfer of ZEA using the human Caco-2 cell line as a model of intestinal epithelial barrier. Cells exposed to 10–200 μM ZEA showed efficacious metabolism of the toxin. α-zearalenol and β-zearalenol were the measured preponderant metabolites (respectively 40.7 ± 3.1% and 31.9 ± 4.9% of total metabolites, after a 3 h exposure to 10 μM ZEA), whereas ZEA-glucuronide and α-zearalenol glucuronide were less produced (respectively 8.2 ± 0.9% and 19.1 ± 1.3% of total metabolites, after a 3 h exposure to 10 μM ZEA). Cell production of reduced metabolites was strongly inhibited by α-and β-hydroxysteroid dehydrogenase inhibitors, and Caco-2 cells exhibited α-hydroxysteroid dehydrogenase type II and β-hydroxysteroid dehydrogenase type I mRNA. After cell apical exposure to ZEA, α-zearalenol was preponderantly found at the basal side, whereas β-zearalenol and both glucuronides were preferentially excreted at the apical side. As α-zearalenol shows the strongest estrogenic activity, the preferential production and basal transfer of this metabolite suggests that intestinal cells may contribute to the manifestation of zearalenone adverse effects.     

The signalling role of action potential depolarization in insulin secretion: Metabolism-dependent dissociation between action potential increase and secretion increase by TEA

The K⁺ channel blocker, TEA is known to increase action potential amplitude and insulin secretion of mouse β-cells when added to a nutrient secretagogue. In the presence of a maximally effective sulfonylurea concentration (2.7 μM glipizide) the nutrient secretagogue α-ketoisocaproic acid (KIC, 10 mM) strongly increased insulin secretion (about elevenfold). Instead of enhancing the effect of KIC, TEA reduced the KIC-induced secretion by more than 50%. Also, the secretion rate produced by 2.7 μM glipizide alone was significantly reduced by TEA. In contrast, TEA enhanced the insulinotropic effect of glipizide when a basal glucose concentration (5 mM) was present. In the presence as well as in the absence of glucose glipizide produced a plateau depolarization with superimposed action potentials. Under both conditions, TEA increased the glipizide-induced action potential amplitude and further elevated the cytosolic free calcium concentration ([Ca²⁺]_i) with an oscillatory characteristic. These effects depended on the activity of L-type Ca²⁺ channels, even though the effect of TEA differed from that of 1 μM of the Ca²⁺ channel opener, Bay K8644, which primarily increased action potential duration. TEA did not negatively affect parameters of β-cell energy metabolism (NAD(P)H fluorescence and ATP/ADP ratio), rather, it slightly increased NAD(P)H fluorescence. Apparently, TEA inhibits insulin secretion in the absence of glucose in spite of a persistent ability to block K⁺ ion conductance. Thus, the signalling role of action potential depolarization in insulin secretion may require reconsideration and ion conductance-independent actions of K⁺ channels may be involved in this paradox effect of TEA.     

Congo red modulates ACh-induced Ca2+ oscillations in single pancreatic acinar cells of mice

Aim:

Congo red, a secondary diazo dye, is usually used as an indicator for the presence of amyloid fibrils. Recent studies show that congo red exerts neuroprotective effects in a variety of models of neurodegenerative diseases. However, its pharmacological profile remains unknown. In this study, we investigated the effects of congo red on ACh-induced Ca²⁺ oscillations in mouse pancreatic acinar cells in vitro.

Methods:

Acutely dissociated pancreatic acinar cells of mice were prepared. A U-tube drug application system was used to deliver drugs into the bath. Intracellular Ca²⁺ oscillations were monitored by whole-cell recording of Ca²⁺-activated Cl⁻ currents and by using confocal Ca²⁺ imaging. For intracellular drug application, the drug was added in pipette solution and diffused into cell after the whole-cell configuration was established.

Results:

Bath application of ACh (10 nmol/L) induced typical Ca²⁺ oscillations in dissociated pancreatic acinar cells. Addition of congo red (1, 10, 100 μmol/L) dose-dependently enhanced Ach-induced Ca²⁺ oscillations, but congo red alone did not induce any detectable response. Furthermore, this enhancement depended on the concentrations of ACh: congo red markedly enhanced the Ca²⁺ oscillations induced by ACh (10–30 nmol/L), but did not alter the Ca²⁺ oscillations induced by ACh (100–10000 nmol/L). Congo red also enhanced the Ca²⁺ oscillations induced by bath application of IP₃ (30 μmol/L). Intracellular application of congo red failed to alter ACh-induced Ca²⁺ oscillations.

Conclusion:

Congo red significantly modulates intracellular Ca²⁺ signaling in pancreatic acinar cells, and this pharmacological effect should be fully considered when developing congo red as a novel therapeutic drug.     

Mechanism of cytotoxic action of perfluorinated acids. III. Disturbance in Ca²+ homeostasis

The global distribution of perfluorinated acids (PFAs) in industry and in household is well known. Their increasing environmental occurrence and biomagnification in the living organisms have drawn growing interests in efforts to describe precisely the mechanisms of action in vitro and in vivo.Our previous investigations widely described lipophilicity-dependent cytotoxicity of PFAs as well as the effect of perfluorination of carbon chain on depolarization of plasma membrane potential, acidification or mitochondrial dysfunctions. In this study we presented in dose- and time-dependent manner the impact of PFAs on calcium homeostasis in HCT116 cells. Comparative analysis of cytosolic [Ca²⁺]_c and mitochondrial calcium [Ca²⁺]_m carried out by flow cytometry revealed distinct uptake of calcium into mitochondria in correlation to increasing lipophilicity of PFAs. Massive accumulation of [Ca²⁺]_m was not accompanied by equivalent loss of [Ca²⁺]_c. Indeed, moderate changes of [Ca²⁺]_c were observed after incubation with 400 μM PFDoDA reaching 29.83% and 49.17% decrease at 4th and 72nd hour, respectively. At the same time, mitochondrial calcium uptake increased from 2- to more than 4-fold comparing with non-treated cells. Incubation with non-fluorinated decanoic acid (DA) did not cause any changes in calcium homeostasis.Presented data show that PFAs-induced perturbations in calcium distribution seem to be a missing link related to mitochondria dysfunction playing a crucial role in determination of apoptotic cell death. Complete scheme for the mechanism of cytotoxic action of PFAs has been included.     

Inhibition of store-operated Ca(2+) channels prevent ethanol-induced intracellular Ca(2+) increase and cell injury in a human hepatoma cell line

Elevated intracellular Ca²⁺ content is implicated in ethanol-induced hepatocyte apoptosis and necrosis. Extracellular Ca²⁺ influx has been suggested to play a role in this process. However, the exact Ca²⁺-permeable channel involved in the plasma membrane is still unclear. This study investigated the role of store-operated calcium entry (SOCE) in ethanol-induced cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) increase and hepatotoxicity. Ethanol (25-800 mM) dose-dependently increased [Ca²⁺]_i content and hepatocyte damage in HepG2 cells. 2-aminoethoxydiphenyl borate (2-APB), the proved efficient antagonist of SOCs, dose-dependently suppressed the ethanol (200 nM)-increased [Ca²⁺]_i content and protected against ethanol-induced viability loss and transaminase leakage. Exposure to 200 mM ethanol for 24 h significantly upregulated the mRNA and protein expression of calcium release-activated calcium channel protein 1 (CRACM1, Orai1) and stromal interaction molecule 1 (STIM1), the two main molecular constituents of SOCs, which was sustained for at least 72 h. In addition, small interfering RNA knockdown of STIM1 attenuated the ethanol-increased [Ca²⁺]_i content and hepatotoxicity. Taken together, these data indicate that the Ca²⁺ channel of SOCE may be involved in the pathogenesis of ethanol-induced intracellular Ca²⁺ elevation and consequent hepatocyte damage.     

AMP-activated protein kinase (AMPK)-dependent and -independent pathways regulate hypoxic inhibition of transepithelial Na(+) transport across human airway epithelial cells

BACKGROUND AND PURPOSE

Pulmonary transepithelial Na⁺ transport is reduced by hypoxia, but in the airway the regulatory mechanisms remain unclear. We investigated the role of AMPK and ROS in the hypoxic regulation of apical amiloride-sensitive Na⁺ channels and basolateral Na⁺K⁺ ATPase activity.

EXPERIMENTAL APPROACH

H441 human airway epithelial cells were used to examine the effects of hypoxia on Na⁺ transport, AMP : ATP ratio and AMPK activity. Lentiviral constructs were used to modify cellular AMPK abundance and activity; pharmacological agents were used to modify cellular ROS.

KEY RESULTS

AMPK was activated by exposure to 3% or 0.2% O₂ for 60 min in cells grown in submerged culture or when fluid (0.1 mL·cm⁻²) was added to the apical surface of cells grown at the air–liquid interface. Only 0.2% O₂ activated AMPK in cells grown at the air–liquid interface. AMPK activation was associated with elevation of cellular AMP : ATP ratio and activity of the upstream kinase LKB1. Hypoxia inhibited basolateral ouabain-sensitive I_sc (I_ouabain) and apical amiloride-sensitive Na⁺ conductance (G_Na+). Modification of AMPK activity prevented the effect of hypoxia on I_ouabain (Na⁺K⁺ ATPase) but not apical G_Na+. Scavenging of superoxide and inhibition of NADPH oxidase prevented the effect of hypoxia on apical G_Na+ (epithelial Na⁺ channels).

CONCLUSIONS AND IMPLICATIONS

Hypoxia activates AMPK-dependent and -independent pathways in airway epithelial cells. Importantly, these pathways differentially regulate apical Na⁺ channels and basolateral Na⁺K⁺ ATPase activity to decrease transepithelial Na⁺ transport. Luminal fluid potentiated the effect of hypoxia and activated AMPK, which could have important consequences in lung disease conditions.     

Role of perinuclear mitochondria in the spatiotemporal dynamics of spontaneous Ca2+ waves in interstitial cells of Cajal-like cells of the rabbit urethra

BACKGROUND AND PURPOSE

Although spontaneous Ca²⁺ waves in interstitial cells of Cajal (ICC)-like cells (ICC-LCs) primarily arise from endoplasmic reticulum (ER) Ca²⁺ release, the interactions among mitochondrial Ca²⁺ buffering, cellular energetics and ER Ca²⁺ release in determining the spatiotemporal dynamics of intracellular Ca²⁺ remain to be elucidated.

EXPERIMENTAL APPROACH

Spontaneous Ca²⁺ transients in freshly isolated ICC-LCs of the rabbit urethra were visualized using fluo-4 Ca²⁺ imaging, while the intracellular distribution of mitochondria was viewed with MitoTracker Red.

KEY RESULTS

Spontaneous Ca²⁺ waves invariably originated from the perinuclear region where clusters of mitochondria surround the nucleus. Perinuclear Ca²⁺ dynamics were characterized by a gradual rise in basal Ca²⁺ that preceded each regenerative Ca²⁺ transient. Caffeine evoked oscillatory Ca²⁺ waves originating from anywhere within ICC-LCs. Ryanodine or cyclopiazonic acid prevented Ca²⁺ wave generation with a rise in basal Ca²⁺, and subsequent caffeine evoked a single rudimentary Ca²⁺ transient. Inhibition of glycolysis with 2-deoxy-glucose or carbonyl cyanide 3-chlorophenylhydrazone, a mitochondrial protonophore, increased basal Ca²⁺ and abolished Ca²⁺ waves. However, caffeine still induced oscillatory Ca²⁺ transients. Mitochondrial Ca²⁺ uptake inhibition with RU360 attenuated Ca²⁺ wave amplitudes, while mitochondrial Ca²⁺ efflux inhibition with {"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157 suppressed the initial Ca²⁺ rise to reduce Ca²⁺ wave frequency.

CONCLUSIONS AND IMPLICATIONS

Perinuclear mitochondria in ICC-LCs play a dominant role in the spatial regulation of Ca²⁺ wave generation and may regulate ER Ca²⁺ release frequency by buffering Ca²⁺ within microdomains between both organelles. Glycolysis inhibition reduced mitochondrial Ca²⁺ buffering without critically disrupting ER function. Perinuclear mitochondria may function as sensors of intracellular metabolites.     

Brief low [Mg2+]o-induced Ca2+ spikes inhibit subsequent prolonged exposure-induced excitotoxicity in cultured rat hippocampal neurons

Reducing [Mg²⁺]_o to 0.1 mM can evoke repetitive [Ca²⁺]_i spikes and seizure activity, which induces neuronal cell death in a process called excitotoxicity. We examined the issue of whether cultured rat hippocampal neurons preconditioned by a brief exposure to 0.1 mM [Mg²⁺]_o are rendered resistant to excitotoxicity induced by a subsequent prolonged exposure and whether Ca²⁺ spikes are involved in this process. Preconditioning by an exposure to 0.1 mM [Mg²⁺]_o for 5 min inhibited significantly subsequent 24 h exposure-induced cell death 24 h later (tolerance). Such tolerance was prevented by both the NMDA receptor antagonist D-AP5 and the L-type Ca²⁺ channel antagonist nimodipine, which blocked 0.1 mM [Mg²⁺]_o-induced [Ca²⁺]_i spikes. The AMPA receptor antagonist NBQX significantly inhibited both the tolerance and the [Ca²⁺]_i spikes. The intracellular Ca²⁺ chelator BAPTA-AM significantly prevented the tolerance. The nonspecific PKC inhibitor staurosporin inhibited the tolerance without affecting the [Ca²⁺]_i spikes. While Gö6976, a specific inhibitor of PKCα had no effect on the tolerance, both the PKCε translocation inhibitor and the PKCζ pseudosubstrate inhibitor significantly inhibited the tolerance without affecting the [Ca²⁺]_i spikes. Furthermore, JAK-2 inhibitor AG490, MAPK kinase inhibitor PD98059, and CaMKII inhibitor KN-62 inhibited the tolerance, but PI-3 kinase inhibitor LY294,002 did not. The protein synthesis inhibitor cycloheximide significantly inhibited the tolerance. Collectively, these results suggest that low [Mg²⁺]_o preconditioning induced excitotoxic tolerance was directly or indirectly mediated through the [Ca²⁺]_i spike-induced activation of PKCε and PKCξ, JAK-2, MAPK kinase, CaMKII and the de novo synthesis of proteins.     

Stimulation of calmodulin by cadmium ion

Cd²⁺, a serious environmental pollutant in certain industrial regions, accumulates in mammalian tissues with a very slow turnover. Using various criteria, we studied the ability of Cd²⁺ to substitute for Ca²⁺ in calmodulin (CaM), a ubiquitious Ca²⁺-binding protein that mediates many of the Ca²⁺ effects. CaM bound Cd²⁺ with a Kd of 4.5 M, presumably to the Ca²⁺-binding sites. Binding of Cd²⁺ allowed CaM to bind 2 moles chlorpromazine, or to form a complex with skeletal muscle troponin-I, troponin-T, or phosphodiesterase. Complex formation with phosphodiesterase led to its activation, which was observed even in the presence of glutathione or cysteine, agents known to chelate Cd²⁺. This raises the possibility that one manifestation of Cd²⁺ toxicity may be through its activation of CaM, thus upsetting its normal regulation by a cellular flux of Ca²⁺.This work was supported by grants CA 21765, NS 08059 and GM 28178 from the NIH and by American Lebanese Syrian Associated Charities. J. R. Z. was a recipient of a National Research Service Award, CA 09346, USA     

Adenosine A3 receptor-mediated cardioprotection against doxorubicin-induced mitochondrial damage

Cardiotoxicity associated with doxorubicin (DOX) treatment limits the therapeutic efficiency of this drug against cancer. 2-Chloro-N(6)-(3-iodobenzyl)adenosine-5′-N-methyluronamide (Cl-IB-MECA), a selective agonist of A₃ adenosine receptor (A₃R), reduces DOX toxicity in newborn rat cultured cardiomyocytes. The study's aim was to determine whether the protection demonstrated by Cl-IB-MECA attenuates cardiac depression in vivo. In addition, we wished to examine whether this protective pathway affects the sarcoplasmic reticulum (SR) calcium uptake and release, as well as intramitochondrial Ca²⁺ accumulation induced by DOX.Rats were injected every alternate day (6 times) with (1) saline, (2) 2.5 mg/kg i.p. DOX, (3) 33 μg/kg i.v. Cl-IB-MECA, (4) DOX + Cl-IB-MECA. Left ventricular functions were assessed by invasive (pressure) and non-invasive (echocardiography) techniques at the end of the injection period and 4 weeks later. Cytosolic and intramitochondrial calcium levels were measured with indo-1 and rhod-2 probes. SR Ca²⁺ content was determined by exposing cultured rat cardiomyocytes to caffeine.Echocardiography data demonstrate left ventricular wall thinning (23%), an increase in the end systolic dimension (170%) and decreased fractional shortening (35 ± 5% vs. 54 ± 5%, p < 0.01) in DOX-treated animals, compared to the control group. DOX increased Ca²⁺ levels in the cytosol and in mitochondria by diminishing the SR Ca²⁺ uptake. Pretreatment with Cl-IB-MECA attenuated left ventricular dysfunction, improved SR calcium storage capacity and prevented mitochondrial Ca²⁺ overload.We conclude that the adenosine A₃ receptor agonist is effective in vivo against DOX cardiotoxicity via the restoration of Ca²⁺ homeostasis and prevention of mitochondrial damage that occurs as a result of Ca²⁺ overload.     

Mechanism of primary Cd2+-induced rat liver mitochondria dysfunction: discrete modes of Cd2+ action on calcium and thiol-dependent domains

We attempted to discern discrete sites of Cd²⁺ deleterious action on rat liver mitochondrial function. In particular, EGTA, ADP, and cyclosporin A (potent mitochondrial permeability transition antagonists) affected mainly Cd²⁺ -induced changes in resting state respiration, eliminating its stimulation in KCl medium, while dithiothreitol (DTT, a dithiol reductant) produced its effect both on Cd²⁺ activation of the basal respiration and Cd²⁺ depression of uncoupler-stimulated respiration, evoking its restoration. Substantial differences in DTT influence on mitochondrial respiration at low and high [Cd²⁺] were revealed, namely, an enhanced mitochondrial permeabilization in the presence of saturated [DTT] at high [Cd²⁺] took place. Besides, DTT only partially reversed Cd²⁺ -induced swelling in NH₄NO₃ medium when glutamate plus malate or succinate without rotenone was used. Contrarily, DTT produced complete reversal of the swelling of succinate-energized mitochondria when rotenone was present in the medium. In addition, in the presence of rotenone both Cd²⁺ -produced activation of the resting state respiration in KCl medium and Cd²⁺-induced swelling in sucrose medium of succinate-energized mitochondria were more sensitive to cyclosporin A than the same Cd²⁺ effects obtained on mitochondria oxidizing succinate (without rotenone) or glutamate plus malate. We have concluded that Cd²⁺, producing primary mitochondrial dysfunction, acts both as a thiol and Me²⁺ binding site reagent. Suppositions about possible localization of separate sites of direct Cd²⁺ effects on mitochondrial function were made.