Biophysical mechanisms underlying tefluthrin-induced modulation of gating changes and resurgent current generation in the human Nav1.4 channel.

Human skeletal muscle contraction is triggered by activation of Nav1.4 channels. Nav1.4 channels can generate resurgent currents by channel reopening at hyperpolarized potentials through a gating transition dependent on the intracellular Navß4 peptide in the physiological conditions. Tefluthrin (TEF) is a pyrethroid insecticide that can disrupt electrical signaling in nerves and skeletal muscle, resulting in seizures, muscle spasms, fasciculations, and mental confusion. TEF can also induce tail currents through other voltage-gated sodium channels in the absence of Navß4 peptide, suggesting that muscle spasms may be caused by resurgent currents. Further, intracellular Navß4 peptide and extracellular TEF may show competitive or synergistic effects; however, their binding sites are still unknown. To address these issues, electrophysiological recordings were performed on CHO-K1 cells expressing Nav1.4 channels with intracellular Navß4 peptide, extracellular TEF, or both. TEF and Navß4 peptide induced a hyperpolarizing shift of activation and inactivation curves in the Nav1.4 channel. TEF also substantially prolonged the inactivation time constants, while simultaneous application of Navß4 peptide partially reversed this effect. Resurgent currents were enhanced by TEF and Navß4 peptide at negative potentials, but TEF more potently enhances resurgent currents and dampens decay of resurgent currents. With longer depolarization, peak resurgent currents decay was fastest with the TEF alone. Molecular docking suggested that TEF and Navß4 peptide binding site(s) are not in the narrowest part of the channel pore, but rather in the bundle-crossing regions and in the domain linkers, respectively. TEF can induce resurgent currents independently and synergistically with Navß4 peptide, which may explain the muscle spasms observed in TEF intoxication.

Targeting GPER1 to suppress autophagy as a male-specific therapeutic strategy for iron-induced striatal injury.

The functional outcome of intracerebral hemorrhage (ICH) in young male patients are poor than in premenopausal women. After ICH, ferrous iron accumulation causes a higher level of oxidative injury associated with autophagic cell death in striatum of male mice than in females. In rodent model of ferrous citrate (FC)-infusion that simulates iron accumulation after ICH, female endogenous estradiol (E2) suppresses autophagy via estrogen receptor α (ERα) and contributes to less injury severity. Moreover, E2 implantation diminished the FC-induced autophagic cell death and injury in males, whose ERα in the striatum is less than females. Since, no sex difference of ERß was observed in striatum, we delineated whether ERα and G-protein-coupled estrogen receptor 1 (GPER1) mediate the suppressions of FC-induced autophagy and oxidative injury by E2 in a sex-dimorphic manner. The results showed that the ratio of constitutive GPER1 to ERα in striatum is higher in males than in females. The GPER1 and ERα predominantly mediated suppressive effects of E2 on FC-induced autophagy in males and antioxidant effect of E2 in females, respectively. This finding opens the prospect of a male-specific therapeutic strategy targeting GPER1 for autophagy suppression in patients suffering from iron overload after hemorrhage.

Autophagy is deficient and inversely correlated with COX-2 expression in nasal polyps: a novel insight into the inflammation mechanism.

BACKGROUND: Nasal polyposis is characterised by persistent inflammation of the upper airways. Autophagy has been implicated in many chronic inflammatory diseases. Whether autophagy plays a role in nasal polyp (NP) inflammation is completely unknown and deserves investigation. METHODS: LC3 and COX-2 expression, the common autophagy and inflammation indicators, respectively, was analysed by immunoblotting in fresh tissues of NP and control nasal mucosa (NM). Primary cultures of NP-derived fibroblasts (NPDFs) and NMDFs were established for in vitro studies. Autophagy was induced by amino acid starvation and LC3 ectopic overexpression or inhibited by 3-methyladenine in the fibroblasts. Inflammation was induced by IL1-ß and TNF-α. LC3 and COX-2 expression was confirmed in NP specimens by immunohistochemistry. RESULTS: LC3 expression was decreased while COX-2 expression was significantly increased in fresh NP tissues compared with the NM control. In NMDFs and NPDFs, autophagy induction by starvation and LC3 overexpression downregulated COX-2 expression. Conversely, autophagy inhibition by 3-methyladenine enhanced COX-2 expression. However, IL1-ß and TNF-α had no effect on autophagy. Immunohistochemical studies on the NP specimens showed that most displayed low LC3 expression, whereas COX-2 was highly expressed in >50% of the specimens. Examination of two consecutive NP sections from the same tissue blocks revealed a negative correlation between LC3 and COX-2 expression. CONCLUSION: Autophagy is deficient in NP tissues and COX-2 is negatively regulated by autophagy in NP-derived fibroblasts. Since COX-2 is essential for the production of pro-inflammatory mediators, this study might help interpret persistent mucosal inflammation in NP. Attenuation of inflammation by restoring autophagy might be a therapeutic strategy for treating NP.

Autophagy is deficient in nasal polyps: implications for the pathogenesis of the disease.

BACKGROUND: Nasal polyposis is characterized by persistent inflammation but the pathogenesis is complex and still debatable. Autophagy has been associated with many human health problems including chronic inflammatory airway diseases. Whether autophagy plays a role in nasal polyps and could be a therapeutic target is completely unknown. METHODS: We studied light chain 3 (LC3) protein expression, a common indication of autophagy, in fresh tissue specimens of 5 nasal polyps and 6 normal nasal mucosa by Western blot analysis. The results were also confirmed by immunohistochemistry (IHC) using additional 25 paraffin-embedded nasal tissue sections. Finally the autophagic activity was validated in nasal polyp-derived fibroblasts by evaluating the number of green fluorescent protein (GFP)-labeled LC3 puncta. RESULTS: The expression of LC3 was dramatically decreased in all 5 nasal polyp tissues. In contrast, protein kinase B-mechanistic target of rapamycin (Akt-mTOR) signaling, an established negative regulator of autophagy, was significantly activated in these tissues. Immunohistochemical results further demonstrated a negative correlation between autophagy and nasal polyps (p < 0.05). GFP-LC3 puncta formation, an alternative indicator of autophagy, was also diminished in nasal polyp-derived fibroblasts (p < 0.01). CONCLUSION: Autophagy is deficient presumably due to suppression by high Akt-mTOR activity in nasal polyps, which may provide a molecular basis for future mechanistic study of the disease.

Early inactivation of PKCε associates with late mitochondrial translocation of Bad and apoptosis in ventricle of septic rat.

BACKGROUND: Sepsis is usually accompanied by cardiomyocyte apoptosis and myocardial depression. Protein kinase C (PKC) has been reported to be important in regulating cardiac function and apoptosis; however, which PKC isoform is involved in sepsis-induced myocardial apoptosis remains unknown. MATERIALS AND METHODS: A rat model of sepsis by cecal ligation and puncture was used. Early and late sepsis refers to those rats sacrificed at 9 and 18 h after cecal ligation and puncture, respectively. Ventricular septum (Sep), left ventricle (LV), and right ventricle were fractionated into membrane, mitochondrial, and cytosolic fractions, individually. The protein levels of PKC isoforms (-α, -ß, -δ, -ε, -ζ, -ι, -λ, and -µ) and mitochondrial translocation of Bad were quantified by Western blot analysis. Apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP in situ nick-end labeling. The morphology of mitochondria was examined by electron microscopy. RESULTS: The membrane/cytosol ratio of PKCε was predominantly higher in the Sep, LV, and right ventricle under physiological conditions. At early sepsis, the membrane/cytosol ratio of PKCε was significantly decreased in Sep and LV. At late sepsis, cardiomyocyte apoptosis associated with severe mitochondrial swelling and crista derangement were observed in Sep and LV at late sepsis. Additionally, mitochondria/cytosol ratio of Bad was significantly increased in Sep and LV. CONCLUSIONS: The early inactivation of PKCε in the ventricle may affect the mitochondrial translocation of Bad and subsequent mitochondrial disruption and apoptosis at late sepsis. This finding opens up the prospect for a potential therapeutic strategy targeting PKCε activation to prevent myocardial depression in septic patients.

Identification of minuscule inward currents as precursors to membrane electroporation-induced currents: real-time prediction of pore appearance.

BACKGROUND/AIMS: The objective of this study is to examine the current signals in response to large hyperpolarizations with the aid of principal component analysis (PCA) to search for or even predict current fluctuations related to membrane electroporation-induced current (I(MEP)). METHODS: The characteristics of principal eigenvalues generated for I(MEP) and the current signals at 10 sec prior to the start of initial I(MEP) (I(Pre)) were examined. As membrane hyperpolarizations were applied at 0.1 Hz, the appearance of I(MEP) coincided with the higher principal eigenvalues extracted in PCA. RESULTS: Subsequent addition of LaCl3 (100 µM) greatly reduced I(MEP) and associated principal eigenvalues. In real-time analysis for a single frame (i.e, 300 msec), in response to large hyperpolarization, multiple runs of heralded minuscule inward currents (Imin) occurring before large rise in current amplitudes were detected. With PCA, such heralded Imin was noted to coincide with the extreme principal eigenvalues. The duration of Imin together with large principal eigenvalues was influenced by different levels of membrane hyperpolarization. In GH3 cells, palmitoyl-L-carnitine (PALCAR), a long-chain acylcarnitine, effectively increased the I(MEP) amplitude with an EC50 value of 2.4 µM. However, in PALCAR-treated cells, the Imin together with higher principal eigenvalues disappeared, while in isoflurane-treated cells, Imin occurring before large rise of current amplitude remained intact. Similarly, the PCA analysis from I(Pre) in RAW 264.6 macrophages showed the presence of herald Imin accompanied by the extreme principal eigenvalues. CONCLUSION: It is clear from this study that these large principal eigenvalues are representative of MEP-associated formation of electropores. Therefore, different compositions around the surface membrane of cells may alter the appearance of Imin followed by I(MEP) emergence.

The inhibition of inwardly rectifying K+ channels by memantine in macrophages and microglial cells.

BACKGROUND/AIMS: Memantine (MEM) can block N-methyl-D-aspartate receptors non-competitively and is recognized to exert anti-inflammatory action. Whether MEM and other related compounds produce any effects on K(+) currents in macrophages and in microglial cells is largely unknown. In this study, we investigated the effects of MEM and other related compounds on inwardly rectifying K(+) current (IK(IR)) in RAW 264.7 macrophages and in BV2 microglial cells. METHODS: Patch-clamp recordings under whole-cell, cell-attached or inside-out configuration were performed in standard patch-clamp technique. MEM suppressed the IK(IR) amplitude in a concentration-dependent manner with an IC50 value of 12 µM. RESULTS: This agent significantly slowed the inactivation time rate of IK(IR) evoked with membrane hyperpolarization. In cells dialyzed spermine (10 µM), MEM-mediated inhibition of IK(IR) no longer existed. MEM-suppressed activity is associated with a decrease in the slow component of mean open time and an increase in mean closed time, despite no detectable change in single-channel conductance of inwardly rectifying K(+) (Kir) channels. Under current-clamp conditions, the addition of MEM resulted in membrane depolarization of RAW 264.7 cells. Similarly, in BV2 microglial cells, addition of MEM suppressed IK(IR) as well as depolarized the membrane. However, neither C6 astrocytic cells nor Jurkat T-lymphoces were noted to display IK(IR). CONCLUSION: The block by MEM of Kir2.1 channels is thus one of the important mechanisms underlying its actions on the functional activities of either macrophages or microglial cells, if similar findings occur in vivo.

Indomethacin inhibits cancer cell migration via attenuation of cellular calcium mobilization.

Non-steroidal anti-inflammatory drugs (NSAIDs) were shown to reduce the risk of colorectal cancer recurrence and are widely used to modulate inflammatory responses. Indomethacin is an NSAID. Herein, we reported that indomethacin can suppress cancer cell migration through its influence on the focal complexes formation. Furthermore, endothelial growth factor (EGF)-mediated Ca2+ influx was attenuated by indomethacin in a dose dependent manner. Our results identified a new mechanism of action for indomethacin: inhibition of calcium influx that is a key determinant of cancer cell migration.

Functional regulation of Alu element of human angiotensin-converting enzyme gene in neuron cells.

The angiotensin-converting enzyme (ACE) insertion/deletion (I/D) genotype and its protein activity have been widely implicated to be associated with Alzheimer's disease (AD). However, whether the insertion sequence, Alu element, in intron 16 of the human ACE gene plays a functional role remains uncertain. To investigate the influence of the I/D polymorphism on ACE promoter, we recombined the I and D form fragments with the human ACE promoter sequence before the reporter gene in pSEAP-Basic2 vector. The effect of the Alu element on regulating the transcriptional activity of ACE promoter was examined using transient transfection in SH-SY5Y cells. We found that the I form fragment upregulated the transcriptional activity of ACE promoter by approximately 70% but that the D form fragment did not. Our study first reveals that Alu sequence in human ACE gene possesses a regulatory function on the ACE promoter activity in neuron. This novel finding bridges the gap between the association of ACE I/D genotype with AD, and suggests that Alu sequence is not merely a "junk" DNA in human ACE gene.

Inhibition of autophagy as a therapeutic strategy of iron-induced brain injury after hemorrhage.

Premenopausal women have better survival than men after intracerebral hemorrhage, which is associated with iron overproduction and autophagy induction. To examine the participation of neuronal autophagy and estrogen receptor α (ERα) in the E 2-mediated protection, PC12 neurons treated with Atg7 (autophagy-related protein 7) siRNA, rapamycin (an autophagy inducer), or Erα siRNA were applied. To study whether autophagy involves in ß-estradiol 3-benzoate (E 2)-mediated neuroprotection against iron-induced striatal injury, castration and E 2 capsule implantation were performed at 2 weeks and 24 h, respectively, before ferrous citrate (FC) infusion into the caudate nucleus (CN) of Sprague Dawley male and female rats. Furthermore, the role of neuronal autophagy in the sex difference of FC-induced CN injury was confirmed by using conditional knockout Atg7 in dopamine receptor 2 (DRD2)-containing neurons in mice. The results showed that the suppression of FC-induced autophagy by E 2 was abolished by Erα siRNA preincubation. Atg7 silencing simulates and rapamycin diminishes E 2-mediated neuroprotection against FC-induced neurotoxicity. In vivo, FC induced a lower degree of autophagy, autophagic cell death, injury severity, histological lesion and behavioral deficit in female rats than in males. E 2 implantation decreased the levels of both FC-induced autophagy and injury in ovariectomized rats. Moreover, the sex difference of FC-induced CN injury was diminished in Atg7 knockout mice. Thus, suppression of autophagy by E 2 via ERα contributes to less severity of iron-induced brain injury in females than in male. This finding opens up the prospect for a therapeutic strategy targeting autophagic inhibition for patients suffering from intracerebral iron overload.

The decline of autophagy contributes to proximal tubular dysfunction during sepsis.

Severe sepsis associated with overproduction of tumor necrosis factor α and reactive oxygen species leads to energy depletion and cellular damage. Both reactive oxygen species and damaged organelles induce autophagy for recycling nutrients to combat pathological stress. To study whether autophagy plays a beneficial role in the pathogenesis of renal failure during sepsis, rats were subjected to cecal ligation and puncture (CLP) or sham operation. Temporal relationship of autophagy and renal dysfunction were examined in vivo. The results showed that the level of lipidated microtubule-associated protein light chain 3 (LC3-II), a marker of autophagy, elevated transiently at 3 h but declined at 9 h until 18 h after CLP. Light chain 3 aggregation in renal tissue showed a similar trend to the change of LC3-II protein. High levels of blood urea nitrogen and creatinine as well as low tubular sodium reabsorption occurred at 18 h after CLP. The distribution of autophagy located primarily in angiotensin-converting enzyme-positive, which is concentrated in proximal tubule, but calbindin D28k (calcium-binding protein D28K, a marker of distal tubule)-negative cells in renal cortex. Therefore, NRK-52E (proximal tubule epithelial cell line) cells were used to further examine cell viability and DNA fragmentation after silencing or inducing autophagy. We found that knockdown of Atg7 (autophagy-related gene 7) exaggerates, whereas preincubation of rapamycin (an autophagy inducer) diminishes tumor necrosis factor α-induced cell death. These results suggest that the decline of sepsis-induced autophagy contributes to the proximal tubular dysfunction, and maintenance of sufficient autophagy prevents cell death. These data open prospects for therapies that activate autophagy during sepsis.

Association of ORAI1 haplotypes with the risk of HLA-B27 positive ankylosing spondylitis.

Ankylosing spondylitis (AS) is a chronic inflammation of the sacroiliac joints, spine and peripheral joints. The aetiology of ankylosing spondylitis is still unclear. Previous studies have indicated that genetics factors such as human leukocyte antigen HLA-B27 associates to AS susceptibility. We carried out a case-control study to determine whether the genetic polymorphisms of ORAI1 gene, a major component of store-operated calcium channels that involved the regulation of immune system, is a susceptibility factor to AS in a Taiwanese population. We enrolled 361 AS patients fulfilled the modified New York criteria and 379 controls from community. Five tagging single nucleotides polymorphisms (tSNPs) at ORAI1 were selected from the data of Han Chinese population in HapMap project. Clinical statuses of AS were assessed by the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Bath Ankylosing Spondylitis Functional Index (BASFI), and Bath Ankylosing Spondylitis Global Index (BAS-G). Our results indicated that subjects carrying the minor allele homozygote (CC) of the promoter SNP rs12313273 or TT homozygote of the SNP rs7135617 had an increased risk of HLA-B27 positive AS. The minor allele C of 3'UTR SNP rs712853 exerted a protective effect to HLA-B27 positive AS. Furthermore, the rs12313273/rs7135617 pairwise allele analysis found that C-G (OR 1.69, 95% CI 1.27, 2.25; p = 0.0003) and T-T (OR 1.75, 95% CI 1.36, 2.27; p<0.0001) haplotypes had a significantly association with the risk of HLA-B27-positive AS in comparison with the T-G carriers. This is the first study that indicate haplotypes of ORAI1 (rs12313273 and rs7135617) are associated with the risk of HLA-B27 positive AS.

Differences in left ventricular cardiomyocyte loss induced by chronic intermittent hypoxia between spontaneously hypertensive and Wistar-Kyoto rats.

RATIONALE: Chronic intermittent hypoxia (CIH) is thought to induce several cardiovascular effects in patients with obstructive sleep apnoea (OSA). However, the effects of CIH on patients with long-standing hypertension are unknown. PURPOSE: This prospective study aimed to investigate the influence of combined OSA and hypertension on cardiomyocyte death. METHODS: Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) were exposed to repetitive hypoxia-reoxygenation cycles (30 s of 5% O(2); 45 s of 21% O(2)) or room air for 6 h/day during the light phase (10 a.m.-4 p.m.) for 10, 20, or 30 days, and the levels of necrosis and apoptosis induced in their left ventricular cardiomyocyte were examined. RESULTS: CIH increased the accumulation of reactive oxygen species, which induced cardiomyocyte necrosis in WKY and SHR (both p < 0.05). Cardiomyocyte oxidative stress levels by CIH were higher in SHR than in WKY (p < 0.05); therefore, cardiomyocyte necrosis was amplified (p < 0.05). Notably, if a superoxide-scavenging agent is injected beforehand, cardiomyocyte necrosis can be effectively inhibited (p < 0.05). When WKY and SHR are exposed to CIH, increases in mitochondria-released cytochrome c and activation of caspase-3 are found in the cytosolic fraction only in WKY. CONCLUSIONS: CIH causes cardiomyocyte loss in SHR mainly through cardiomyocyte necrosis. In WKY however, CIH simultaneously induces apoptosis and necrosis of cardiomyocytes.

Sex-specific role of thioredoxin in neuroprotection against iron-induced brain injury conferred by estradiol.

BACKGROUND AND PURPOSE: Accumulation of iron after intracerebral hemorrhage causes free radical formation and oxidative damage resulting in liquefaction. The aim of this study was the investigation of molecular mechanisms underlying estrogen-mediated neuroprotective effect against iron-induced brain injury in vivo. METHODS: Age-matched male and female Sprague-Dawley rats were stereotaxically infused with either ferrous citrate (FC) or saline (10 muL) into the right caudate nucleus. Beta-estradiol 3-benzoate (E(2)) capsule was implanted subcutaneously at 24 hours before infusion of FC. The severity of brain injury and neurological deficits were measured by histological quantification and forelimb asymmetry test, respectively. The role of thioredoxin (Trx) in E(2)-mediated neuroprotective effect was examined by intrastriatal administration of a Trx reductase inhibitor, 5,5-dithiobis-(2-nitrobenzoic acid), and small interfering RNA. RESULTS: FC induced greater brain injury in male rats than females. E(2) treatment reduced FC-induced brain injury in both sexes. E(2) significantly increased protein level and activity of Trx in the caudate nucleus of females but not males. Administration of female rats with 5,5-dithiobis-(2-nitrobenzoic acid) or Trx small interfering RNA to the caudate nucleus decreased the protective effect of E(2) against FC-induced injury. The protein and mRNA levels of estrogen receptor alpha, but not estrogen receptor beta, were more abundant in the caudate nucleus of female rats. CONCLUSIONS: Increase of brain Trx activity might play an important role in the E(2)-mediated neuroprotective effect against FC-induced brain injury in female rats. Understanding of the sex differences in the Trx-mediated neuroprotective effect by E(2) might help in improving treatment of brain dysfunction after hemorrhagic stroke and/or head trauma.

The molecular events occur during MK-801-induced cytochrome oxidase subunit II down-regulation in GT1-7 cells.

Previously, we showed that predominant expression of the N-methyl-D-aspartate (NMDA) receptor in the neurons of the sexually dimorphic nucleus of the preoptic area of male rats plays an important role in preventing neurons from apoptosis during sexual development. Blocking of the NMDA receptor by dizocilpine ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-iminemaleate (MK-801) causes down-regulation of some survival-related genes including cytochrome oxidase subunit II (COII), a mitochondria-encoded complex IV subunit, which in turn induces ATP depletion and the occurrence of apoptosis. The aim of this study is to investigate the molecular events during down-regulation of the COII gene expression induced by blocking of the NMDA receptor. Treatment of the GnRH cell line (GT1-7) with MK-801 caused 1) a decrease of intracellular calcium concentration ([Ca2+]i) after 20 h; 2) significant decreases of the levels of peroxisome proliferator-activated receptor coactivator-1 (PGC-1) mRNA and protein after 24 h; 3) down-regulation of COII mRNA after 36 h; and 4) the occurrence of neuronal apoptosis after 48 h. Accordingly, we hypothesize that blocking of the NMDA receptor may cause a decrease of the [Ca2+]i, which in turn inhibits the expressions of PGC-1 and COII and then leads to subsequent neuronal apoptosis.

Gene regulation by NMDA receptor activation in the SDN-POA neurons of male rats during sexual development.

The present study was designed to identify possible signaling pathways, which may play a role in prevention of neuronal apoptosis in the sexually dimorphic nucleus of the preoptic area (SDN-POA) after physiological activation of the N-methyl-D-aspartate (NMDA) receptor. Gene response to the blockage of the NMDA receptor by an antagonist (dizocilpine hydrogen maleate; MK-801) was screened after suppression subtractive hybridization (SSH). The results showed that differential screening after SSH detected the presence of some neurotrophic genes (RNA binding motif protein 3 (RBM3), alpha-tubulin) as well as apoptosis-related genes (Bcl-2, cytochrome oxidase subunit II, cytochrome oxidase subunit III) in the SDN-POA of male rats, which were down-regulated by blocking the NMDA receptor. The RT-PCR products of the aforementioned genes in MK-801-treated males were significantly less than that in untreated males. In particular, the expression of Bcl-2 mRNA, including Bcl-2 protein, in male rats were significantly suppressed by MK-801 treatment. Moreover, the binding activity of nuclear factor kappaB (NFkappaB) was significantly higher in male rats than in females, but significantly diminished by blocking the NMDA receptor with MK-801 in male rats. No significant difference in cAMP response element-binding protein (CREB) binding activity was observed among untreated male, MK-801-treated male, untreated female and MK-801-treated female groups. These results suggest that genes regulated by NMDA receptor activation might participate in neuronal growth and/or anti-apoptosis, and support an important signaling pathway of NFkappaB activation and its target gene, Bcl-2, in preventing neuronal apoptosis in the SDN-POA of male rats during sexual development.

Arachidonic acid inhibits capacitative Ca2+ entry and activates non-capacitative Ca2+ entry in cultured astrocytes.

Arachidonic acid (AA) plays important physiological or pathophysiological roles. Here, we show in cultured rat astrocytes that: (i) endothelin-1 or thapsigargin (Tg) induces store-depleted activated Ca(2+) entry (CCE), which is inhibited by 2-aminoethoxydiphenyl borane (2-APB) or La(3+); (ii) AA (10 microM) and other unsaturated fatty acids (8,11,14-eicosatrienoic acid and gamma-linoleic acid) have an initial inhibitory effect on the CCE, due to AA- or fatty acid-induced internal acid load; (iii) after full activation of CCE, AA induces a further Ca(2+) influx, which is not inhibited by 2-APB or La(3+), indicating that AA activates a second Ca(2+) entry pathway, which coexists with CCE; and (iv) Tg or AA activates two independent and co-existing non-selective cation channels and the Tg-induced currents are initially inhibited by addition of AA or weak acids. A possible pathophysiological effect of the AA-induced [Ca](i) overload is to cause delayed cell death in astrocytes.