Rewiring Metabolic Flux in Corynebacterium glutamicum Using a CRISPR/dCpf1-Based Bifunctional Regulation System.

Corynebacterium glutamicum, a microorganism classified as generally recognized as safe for use in the industrial production of food raw materials and additives, has encountered challenges in achieving widespread adoption and popularization as microbial cell factories. These obstacles arise from the intricate nature of manipulating metabolic flux through conventional methods, such as gene knockout and enzyme overexpression. To address this challenge, we developed a CRISPR/dCpf1-based bifunctional regulation system to bidirectionally regulate the expression of multiple genes in C. glutamicum. Specifically, through fusing various transcription factors to the C-terminus of dCpf1, the resulting dCpf1-SoxS exhibited both CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) capabilities in C. glutamicum by altering the binding sites of crRNAs. The bifunctional regulation system was used to fine-tune metabolic flux from shikimic acid (SA) and l-serine biosynthesis, resulting in 27-fold and 10-fold increases in SA and l-serine production, respectively, compared to the original strain. These findings highlight the potential of the CRISPR/dCpf1-based bifunctional regulation system in effectively enhancing the yield of target products in C. glutamicum.

Structural derivatization strategies of natural phenols by semi-synthesis and total-synthesis.

Structural derivatization of natural products has been a continuing and irreplaceable source of novel drug leads. Natural phenols are a broad category of natural products with wide pharmacological activity and have offered plenty of clinical drugs. However, the structural complexity and wide variety of natural phenols leads to the difficulty of structural derivatization. Skeleton analysis indicated most types of natural phenols can be structured by the combination and extension of three common fragments containing phenol, phenylpropanoid and benzoyl. Based on these fragments, the derivatization strategies of natural phenols were unified and comprehensively analyzed in this review. In addition to classical methods, advanced strategies with high selectivity, efficiency and practicality were emphasized. Total synthesis strategies of typical fragments such as stilbenes, chalcones and flavonoids were also covered and analyzed as the supplementary for supporting the diversity-oriented derivatization of natural phenols.

Constructing an Efficient Bacillus subtilis Spore Display by Using Cohesin-Dockerin Interactions.

Bacillus subtilis spore display has become a field of increasing interest in the past two decades. To improve the efficiency of B. subtilis spore display, its directed modification was performed based on the cellulosome architecture by introducing onto them divergent cohesin (Coh) modules that can specifically bind to the target enzyme bearing the matching dockerins (Doc). In this study, five different pairs of cohesins and dockerins, selected from four cellulolytic microbes, were examined for their capabilities in displaying a tetrameric enzyme ß-galactosidase from Bacillus stearothermophilus IAM11001 on the surface of B. subtilis WB600 spores. Immunofluorescence microscopy, western blotting, dot blotting, and enzyme assay was applied to confirm its surface expression. All the resultant five Coh-Doc based spore display can hydrolyze o-nitrophenyl-ß-D-galactopyranoside. Further, the optimized Coh-Doc based spore display exhibited the highest display efficiency. Overall, the results of current study may open new perspectives on the use of Coh-Doc interaction, which will find application in improving the efficiency of B. subtilis spore display.

An appropriate ratio of unsaturated fatty acids is the constituent of hickory nut extract for neurite outgrowth in human SH-SY5Y cells.

Hickory nuts (Carya cathayensis Sarg, CCS), a well-known Chinese medicinal nut, is thought to improve memory in Chinese folks. However, functional constituents have not been scientifically identified. In this study, human SH-SY5Y cells, combined with Q-TOF mass spectrometry (Q-TOF-MS) and standard substances, were used to evaluate the function in neuronal development and to identify constituents of CCS hydrophobic extracts (CCS-HE). Data showed that CCS-HE but not the control induced neurite outgrowth of SH-SY5Y cells in a dose-dependent manner, supported by which CCS-HE induced the expression of nerve growth factor (NGF), neurofilament 160 (NF160), and neuronal peptide Y (NPY) mRNA. Q-TOF-MS analysis with standard substances indicated that linolenic acid (LNA), linoleic acid (LA), and oleic acid (OA) were the main constituents in CCS-HE. Furthermore, mixtures of these unsaturated fatty acids (UFAs) at the natural ratio (1:8:16) significantly induced neurite outgrowth and gene expression of NGF, NF160, and NPY in a dose-dependent manner. However, the individual and alternative ratios were not effective to induce the neurite outgrowth and gene expression of NGF, NF160, and NPY. These data implicate that an appropriate ratio of UFAs is the main constituent for the neurite outgrowth.

Screening of an Alkaline CMCase-Producing Strain and the Optimization of its Fermentation Condition.

OBJECTIVE: Alkaline Carboxymethyl Cellulase (CMCase) is an attractive enzyme for the textile, laundry, pulp, and paper industries; however, commercial preparations with sufficient activity at alkaline conditions are scarce. METHODS: High CMCase-producing bacterial isolate, SX9-4, was screened out from soil bacteria, which was identified as Flavobacterium sp. on the basis of 16S rDNA sequencing. RESULTS: The optimum pH and temperature for CMCase reaction were 8.0 and 55°C, respectively. Alkaline CMCase was stable over wide pH (3.0-10.6) and temperature (25-55°C) ranges. Enzyme activity was significantly inhibited by the bivalent cations Mn2+ and Cu2+, and was activated by Fe2+. To improve the alkaline CMCase production of SX9-4, fermentation parameters were selected through onefactor- at-a-time and further carried out by response surface methodologies based on a central composite design. CONCLUSION: High CMCase production (57.18 U/mL) was achieved under the optimal conditions: 10.53 g/L carboxymethylcellulose sodium, 7.74 g/L glucose, 13.71 g/L peptone, and 5.27 g/L ammonium oxalate.

Identification of angiogenesis-inhibiting peptides from Chan Su.

Chan Su is a traditional medicine prepared from toxic secretions from the auricular and skin glands of Chinese toads. Previous studies show that active components in Chan Su can inhibit the proliferation of tumor cells. To study the effect of Chan Su peptides on angiogenesis, fresh Chan Su was collected and its component peptides were isolated by an extraction and precipitation method. A high-performance liquid chromatography (HPLC) fingerprint of the Chan Su component peptides revealed that there were more than 18 peptide component peaks. We demonstrate that Chan Su peptides inhibit angiogenesis in vitro by inhibiting human umbilical vein endothelial cell (HUVEC) proliferation and tube formation in a dose-dependent manner. Western blots indicated that Chan Su peptides inhibited the protein expression of VEGF165 and Ras, leading us to conclude that Chan Su peptide components exert anti-angiogenic effects by suppressing the VEGF165-VEGFR2-Ras signalling pathway. Finally, we identified the partial amino acid sequences of seven Chan Su peptides using the shotgun proteomics method.

Application of composite dissolving microneedles with high drug loading ratio for rapid local anesthesia.

We report a type of dissolving microneedles (DMNs) which was made of composite matrix materials of hydroxypropyl methyl cellulose (HPMC) and poly(methylvinylether­co­maleic anhydride) (PMVE/MA copolymer, Gantrez S-97), and was successfully loaded with lidocaine hydrochloride. The weight of lidocaine hydrochloride loaded in the microneedles tip was 70% of the weight of the whole tip. The content was 3.43 ±â€¯0.12 mg in weight, which was determined by high performance liquid chromatography (HPLC). The results for mechanical test showed that these microneedles were able to penetrate into the skin of the experimental animals, that was proved using organic staining, texture analyzer and histological examination. The fracture force of the microneedles was 5.442 ±â€¯0.412 N, which was much higher than the one required for the skin penetration. The DMNs with lidocaine hydrochloride could be dissolved inside of the rat skin in 5 min. The onset time would be faster (in <5 min) when it was applied to the guinea pig model, in comparing with a commercially available anesthesia cream that had an onset time for 100 min. However, the efficacy of the DMNs for the local anesthesia only lasted for 16 min. It was shorter than that of the commercially available anesthesia cream with which the efficacy could last for about 130 min. After the DMNs was packaged under the vacuum and dark condition, it was stable for 3 months under the condition of a temperature of 40 ±â€¯2 °C and a humidity of 75 ±â€¯5%. The result of the experiment for the safety evaluation showed that the microneedles were non-irritating and non-allergenic to the skin. In conclusion, the DMNs with lidocaine hydrochloride could be safely administered to the skin with a quick onset time for the local anesthesia.

Identification of anti-tumor components from toad venom.

Secretion of granular glands from the skin of amphibians is a fascinating resource of active substances, particularly for cancer therapy in clinical practice of Traditional Chinese Medicine. A variety of anti-tumor peptides have been isolated from different toads and frogs; however, no anti-tumor peptides are reported in toad venom of Bufo gargarizans. Firstly, soluble fraction from fresh toad venom (FTV) was compared with that from dried toad venom (DTV), using HPLC analysis. It was revealed that FTV has a different HPLC chromatography compared with DTV. Soluble fraction of FTV is more toxic to SH-SY5Y cells than that of DTV, as evaluated by MTT assay. Secondly, it was identified that protein components from soluble fractions of FTV and DTV possess different patterns by SDS-PAGE analysis, and proteins from FTV are also more toxic than that from DTV. Thirdly, an immobilized basic fibroblast growth factor (bFGF) affinity column was used to isolate bFGF-binding components from soluble fraction of FTV, and it was identified that bFGF-binding components prohibited bFGF-dependent neurite growth of SH-SY5Y cells. Finally, it was identified that bFGF-binding components activated apoptosis via upregulation of caspase-3 and caspase-8 expression in SH-SY5Y cells. These data suggest that FTV contains active components that interact with bFGF and activate apoptosis in SH-SY5Y cells.

Expression of recombinant human acetylcholinesterase and its application in screening its inhibitors.

This study is designed to obtain recombinant human acetylcholinesterase (rhAChE) and apply it in screening acetylcholinesterase inhibitors. The rhAChE was overexpressed in HEK293 cells transfected by plasmid of pCMV-AChE with the cationic liposome and rhAChE was found to be secreted into cell culture medium. AChE activity was assayed according to modified Ellman method to obtain kinetic parameters. IC so50 values for donepezil compounds of rhAChE were calculated to determine their activities of inhibition. The results showed that Km value was 151.9 micromol.L-1 donepezil inhibited rhAChE in a mixed competitive-noncompetitive way (Ki= 16.03 nmol.L-1, Ki = 18.36 nmol.L-1) and that most new compounds tested exhibited high activities of inhibition on rhAChE. The study suggests that rhAChE is available to be applied in screening AChE inhibitors in vitro.

HSPA5 forms specific complexes with copper.

Our previous study indicated that Hspa5 directly interacts with copper (Cu) to maintain Cu homeostasis in astrocytes. In this study, we explored the possibility that Cu forms a specific complex with Hspa5 by assaying stoichiometric binding of Cu and other metals to recombinant human HSPA5 (rh-HSPA5) in silico. Spectrophotometric analysis showed that incubation of rh-HSPA5 with Cu but not with Fe, Mn, Zn, or Pb in the presence of ascorbic acid produced an absorbance peak at 470 nm. Furthermore, the absorbance peak was absent when bovine serum albumin was incubated with Cu and when another recombinant protein YWHAZ-14-3-3-Zeta carrying a 6× histidine tag identical to the tag in the rh-HSPA5 was incubated with Cu. The absorbance peak produced by Cu and rh-HSPA5 was abolished by EDTA treatment and was stabilized at pH levels above 6.5. Assay of the stoichiometry of metal binding to the purified rh-HSPA5 showed that one molecule of the rh-HSPA5 could chelate 1 or 2 Cu, 13 iron (Fe), 5 zinc (Zn) and 10 lead (Pb) ions but not manganese (Mn). These data further support our previous finding that HSPA5 specifically forms a complex with Cu to help maintain Cu homeostasis.

Involvement of the molecular chaperone Hspa5 in copper homeostasis in astrocytes.

Copper (Cu) ion availability in tissues and cells must be closely regulated within safe limits by Cu transporters and chaperones. Astrocytes play key roles in metal homeostasis and distribution in the brain that are only partially understood. The purpose of this study was to define the role that the protein chaperone Hspa5, also known as Grp78, plays in Cu homeostasis in astrocytes. First passage cultures of primary astrocytes from neonatal rats and cultures of the C6 rat glioma cells were used as models. We found that the level of Cu accumulation in astrocyte cultures increased with Cu concentrations in the medium, and Cu treatment significantly reduced cellular levels of iron (Fe), manganese (Mn) and zinc (Zn). Cu accumulation specifically induced protein expression of Hspa5 but not metallothioneins (MTs) in astrocytes. In C6 cells, Hspa5 was identified as one component of a Cu-binding complex and shown to directly bind Cu. However, the level of Hspa5 expression was not proportional to Cu accumulation in astrocytes and C6 cells: astrocytes expressed low protein levels of Hspa5 compared to C6 cells but accumulated significantly more Cu than did C6 cells. Consistent with this finding, astrocytes expressed a lower level of the Cu-extruding protein Atp7a than did C6 cells, and depletion of Hspa5 by RNA interference resulted in significantly increased Cu accumulation and induction of MT1/2 expression. These data demonstrate that Hspa5 is involved in Cu homeostasis in astrocytes but not as a Cu storage protein.

ER chaperone-metal interactions: links to protein folding disorders.

Chaperones in the endoplasmic reticulum play vital roles in the folding, assembly, and post-translational modification of secretory proteins and also recycle, refold, or initiate degradation of misfolded proteins. Chaperone deficiencies in either amount or function are implicated in the etiology or pathogenesis of Alzheimer's disease and other protein folding disorders of the central nervous system. In this review, we discuss evidence that chaperones become pathologic through deleterious interactions with metals and then promote protein folding disorders. The "master regulator" chaperone GRP78 in the endoplasmic reticulum is a compelling subject for investigation in this context because it is located at the hub of signaling pathways in a complex chaperone network. It has therefore been studied by several laboratories in conjunction with exposure to toxic metals. The key points of this review are that metals are implicated in the etiology or pathogenesis of Alzheimer's disease and other protein folding disorders, metals induce the expression GRP78, often associated with oxidative stress, some metals bind to GRP78, and lead (Pb) impairs GRP78 function when it binds to GRP78. If certain metals do indeed cause or promote the aggregation of toxic proteins in the central nervous system, as the available evidence indicates, the identification of the mechanisms by which they act would provide valuable leads for the development of therapies to prevent or reverse toxic protein aggregation.

Copper handling by astrocytes: insights into neurodegenerative diseases.

Copper (Cu) is an essential trace element in the brain that can be toxic at elevated levels. Cu accumulation is a suspected etiology in several neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and prion-induced disorders. Astrocytes are a proposed depot in the brain for Cu and other metals, including lead (Pb). This article describes the physiological roles of Cu in the central nervous system and in selected neurodegenerative diseases, and reviews evidence that astrocytes accumulate Cu and protect neurons from Cu toxicity. Findings from murine genetic models of Menkes disease and from cell culture models concerning the molecular mechanisms by which astrocytes take up, store, and buffer Cu intracellularly are discussed, as well as potential mechanistic linkages between astrocyte functions in Cu handling and neurodegenerative diseases.

Determination of metal interactions with the chaperone Hspa5 in human astrocytoma cells and rat astrocyte primary cultures.

Molecular chaperones assist the folding of nascent proteins during translation into their correct conformations. Neurotoxic metals such as copper (Cu) and lead (Pb) may produce a deficiency in chaperone function that compromises protein secretion and exacerbates protein aggregation, potentially promoting neurodegenerative diseases that exhibit protein aggregation. Because astrocytes function as depots in the brain for certain metals, including Cu and Pb, the interaction of metals with chaperones in these cells is of interest. Furthermore, Pb and Cu bind strongly to the molecular chaperone heat shock 70 kDa protein Hspa5, also known as glucose-regulated protein 78 (Grp78) or immunoglobulin-binding protein (BiP). This chapter describes methods for expressing fluorescent chimeric proteins in astrocytes and astrocytoma cells in order to examine the metal-induced cytosolic redistribution of Hspa5, as well as associated effects on the secretion of interleukin-6 (IL-6).

Image analysis of Ca2+ signals as a basis for neurotoxicity assays: promises and challenges.

Free intracellular calcium ([Ca(2+)](i)) controls a wide range of cellular functions such as contraction, neurotransmitter and hormone release, metabolism, cell division and differentiation. Cytosolic Ca(2+) levels are abnormal in cells exposed to toxicants and understanding how these levels become altered may improve our ability to design high-throughput methods for the sensitive detection of cellular responses to a toxic exposure. Because Ca(2+) is involved in multiple aspects of cellular function, its role in signaling is complex. It is therefore necessary to identify the individual pathways targeted during toxicant exposure in order to use them as a tool for predictive measurements of toxicity and as targets for prevention or reversal of injury. This review illustrates several methods available for analysis of Ca(2+) responses in vitro and their applicability for understanding mechanisms of toxicity at the molecular and cellular levels. The review will also consider the usefulness of Ca(2+) imaging for predicting a unique signature for classes of toxicants. Towards this end, two methodological approaches for assessment of Ca(2+) responses to toxicants are examined: steady state measurements and complex spatial and/or temporal measurements. Each of the methods described and appropriately used results in reliable and reproducible measurements which may be applied in a high-throughput fashion to individualize in vitro assessment of cellular responses caused by toxicants.

Valproate reversibly reduces neurite outgrowth by human SY5Y neuroblastoma cells.

Valproate (VPA) is a commonly prescribed mood stabilizer. However, emerging evidence indicates that VPA administration may cause reversible symptoms of Parkinsonism and cognitive decline (P/CD) in some manic patients. The mechanism of this phenomenon is unknown. In this study, we used human SY5Y neuroblastoma cells as a neuronal model to investigate the effects of VPA on neurite outgrowth and neurofilament expression. Data showed that the treatment with VPA at therapeutic plasma levels (0.5 mM) significantly reduced cell proliferation from day 4 through day 6, and neurite outgrowth length from day 1 through day 6. Conversely, VPA had no effect on cell proliferation of human CCF astrocytoma cells but stimulated nerve growth factor (NGF)-induced neurite outgrowth from rat PC12 pheochromocytoma cells. In parallel to these alterations in human SY5Y cells, both mRNA and protein levels of neurofilament 160 (NF160) were significantly reduced, starting at day 2 and day 3, respectively, by the treatment. The inhibition of neurite outgrowth by VPA was completely reversed 2 days after cessation of VPA exposure. Furthermore, NF160 protein levels also rebounded to control levels after VPA removal. NGF application significantly alleviated the inhibition of neurite outgrowth by VPA. These data suggest that VPA-modulated NF160 expression was involved in the inhibition and the reversal of neurite outgrowth in human neuronal cells.

[Lead distribution and 78,000 glucose regulated protein levels in various organs of weaned rats].

OBJECTIVE: To investigate lead distribution and the change of 78 000 glucose regulated protein (GRP78) in various organs of weaned rats challenged with low-level maternal origin lead. METHODS: Male littermates, bred from the female Fisher 344 rats gavaged with lead acetate or sodium acetate (1 ml of 10 mg/ml per day per animal) with male Fisher 344 rats without lead treatment, were divided into 4 groups including control (group A), gestation plus lactation (group B), gestation only (group C), and lactation only (group D). Each group had 6 litters. These littermates were weaned and terminated at postnatal day 21. Lead contents and GRP78 levels in various organs of these littermates were determined by atomic absorbance spectrometry (AAS) and Western blotting analysis, respectively. RESULTS: Maternal lead was observed to transfer to littermates through gestation and lactation. Concentrations of littermate blood lead in groups A to D were (0.0010+/-0.0010), (0.1420+/-0.0190), (0.0250+/-0.0040), and (0.1490+/-0.0160) microg/ml, respectively. Concentrations of littermate brain lead in groups A to D were (0.0005+/-0.0005), (0.1120+/-0.0130), (0.0125+/-0.0042), and (0.0700+/-0.0058) microg/g, respectively. Concentrations of littermate kidney lead in groups A to D were (0.0050+/-0.0050), (1.0400+/-0.1000), (0.1040+/-0.0330), and (0.9920+/-0.0850) microg/g, respectively. Concentrations of littermate liver lead in groups A to D were (0.0030+/-0.0050), (0.3600+/-0.0550), (0.0567+/-0.0126), and (0.3030+/-0.0310) microg/g, respectively. Blood, brain, kidney and liver lead concentrations in groups B and D were significantly higher than those in group C and differences were 5-10 folds. Arbitrary units of littermate leukocytic GRP78 concentration normalized with actin protein in groups A to D were 1.000+/-0.038, 1.180+/-0.060, 0.998+/-0.109, and 1.290+/-0.110, respectively. Arbitrary units of littermate brain GRP78 concentration normalized with actin protein level in groups A to D were 0.996+/-0.128, 0.922+/-0.246, 1.150+/-0.170, and 0.750+/-0.126, respectively. CONCLUSION: Lead in maternal bodies could be transferred to litter bodies through gestation and lactation and distributed in various organs. Lead might also changed GRP78 expression in leukocytes.

Activation profiles of HSPA5 during the glomerular mesangial cell stress response to chemical injury.

Environmental injury has been associated with endoplasmic reticulum (ER) stress, a response characterized by activation of the unfolded protein response, proteasomal degradation of proteins, and induction of HSPA5, also known as GRP78 or BiP. Although HSPA5 has been implicated in the stress response to environmental injury in several cell types, its role in the glomerular ER stress response is unknown. In this study, we evaluated HSPA5 activation profiles in rat glomerular mesangial cells (rGMCs) challenged with heavy metals (HgCl2 or Pb2+ acetate) or polycyclic aromatic hydrocarbons (PAHs, ie, benzo(a)pyrene [BaP]). Challenge of rGMCs with 1 or 10 microM HgCl2 or Pb2+ acetate increased HSPA5 mRNA and protein levels. The induction response was sensitive to transcriptional and translational inhibition by actinomycin D (AD) and cyclohexamide, respectively. HSPA5 mRNA was induced by 3 microM BaP in an AD-sensitive manner, but this response was unaffected by the presence of heavy metals. A promoter construct containing sequences that mediate thapsigargin (TH) inducibility of the HSPA5 promoter was refractory to both heavy metals and BaP. The HSPA5 induction response in rGMCs is conserved because it was reproduced with fidelity in immunolocalization experiments of HSPA5 protein in M15 and HEK293 cells in embryonic lines of murine and human origin, respectively. Collectively, these findings identify HSPA5 in the stress response of rGMCs and implicate regulatory mechanisms that are distinct from those involved in TH inducibility.

A 78-kDa glucose-regulated protein is involved in the decrease of interleukin-6 secretion by lead treatment from astrocytes.

Interleukin (IL)-6 is a cytokine produced mainly by microglia and astrocytes and plays a pleiotropic role in the central nervous system. In this study, we cloned rat IL-6 cDNA into an enhanced green fluorescent protein (EGFP) or a red fluorescent protein (DsRed2) vector and rat 78-kDa glucose-regulated protein (GRP78) cDNA into an EGFP vector to construct IL-6-EGFP, IL-6-DsRed2, and GRP78-EGFP chimeras for the investigation of the mechanism of IL-6 secretion from astrocytes. The data showed that constructed IL-6-EGFP and IL-6-DsRed2 chimeras retained the secretory property, and the secretion of IL-6-EGFP from astrocytes could be attenuated by GRP78 depletion with double-stranded RNA interference. Coexpression of IL-6-DsRed2 and dysfunctional GRP78-EGFP abolished IL-6-DsRed2 secretion, and two chimeric proteins colocalized inside living astrocytes. Coimmunoprecipitation analysis indicated that IL-6 and GRP78 resided in the same complex. The data further revealed that IL-6-EGFP secretion from astrocytes was blocked by the heavy metal lead (Pb) in a concentration-dependent manner. Analysis of the Pb interaction with protein on a Pb-affinity column demonstrated that Pb bound to GRP78 but failed to bind to IL-6. Therefore, these data suggest that IL-6-EGFP or IL-6-DsRed2 chimeras can be used as imaging probes to study IL-6 secretion from living cells, that GRP78 is involved in IL-6 secretion from astrocytes, and that Pb can block IL-6 secretion from astrocytes via targeting GRP78.

Effects of propofol on intracellular Ca2+ homeostasis in human astrocytoma cells.

The effects of propofol, a short-acting general anesthetic, upon cell growth and Ca(2+) signaling in a human astrocytic cell line were examined. Exposure of cells to graded concentrations of propofol resulted in a dose-dependent decrease in cell number with an inhibitory concentration of cell viability (IC50) of 31.7+/-1.2 microM. To evaluate the changes in intracellular Ca(2+) homeostasis induced by propofol, cytoplasmic and mitochondrial Ca(2+) were measured by fluorescence imaging. Mitochondrial Ca(2+) increased while cytoplasmic Ca(2+) decreased significantly at a propofol concentration lower than the IC50 (10 microM for 24 h, 1 microM for 72 h). In addition, propofol diminished the Ca(2+) response induced by fetal bovine serum (FBS). To determine the source of Ca(2+) alterations induced by propofol, pharmacologic agents targeting intracellular Ca(2+) homeostasis mechanisms were used. Nifedipine, an L-type Ca(2+) channel blocker, decreased FBS-induced Ca(2+) response of control cells to a level similar to propofol treated cells. However, diazoxide (a K(+)-ATP channel opener) administered 1 h before FBS addition restored the FBS response in propofol treated cells to a level similar to control. In addition, diazoxide increased mitochondrial Ca(2+) in control cells to a level comparable to propofol treated cells suggesting activation of these channels by propofol treatment. Addition of 1 muM RU-360 (a selective blocker of the mitochondrial Ca(2+) uniporter) for 30 min prior to propofol treatment restored mitochondrial and cytoplasmic Ca(2+) to control levels. These data suggest that voltage operated Ca(2+) channels, mitochondrial Ca(2+) and K(+)-ATP channels may be targets of propofol action in astrocytes.