Role Of Extracellular Calcium Research Articles

MO092THE ROLE OF CALCIUM IN UROMODULIN EXPRESSION AND SECRETION FROM RENAL MEDULLARY EPITHELIAL CELLS OF HYPERTENSIVE AND NORMOTENSIVE RATS

Abstract Background and Aims Uromodulin (UMOD) is the most abundantly secreted protein found within the urine, primarily produced by medullary thick ascending limb (mTAL) epithelial cells of the kidneys. There is accruing genetic evidence implicating UMOD in blood pressure regulation and consequently hypertension. The molecular signaling induced by calcium in the kidney and its influence on blood pressure are not well understood. The aim of this study was to investigate the potential role of extracellular calcium and the calcium-sensing receptor (CaSR) in mTAL on UMOD production and secretion in TAL cells with the hope of defining novel clinical targets for the treatment of hypertension. Method Kidneys were harvested from normotensive Wistar-Kyoto (WKY) and stroke-prone spontaneously hypertensive (SHRSP) female rats. To determine the effect of extracellular calcium on UMOD secretion, mTAL tubules were incubated in media with and without 1mM calcium, nifedipine (10µM), NPS2143 (1 or 5 µM) and spermine (2mM). Extracellular and intracellular UMOD protein levels were detected by Western blot. Gene expression of Umod was determined by qRT-PCR. Results Calcium increased mTAL tubule UMOD secretion in WKY and SHRSP. Nifedipine slightly decreased UMOD secretion in WKY without calcium. In both strains, NPS2143 increased calcium-induced UMOD secretion, with an enhanced effect in SHRSP. Stimulation of CaSR with spermine decreased UMOD secretion in WKY. Analysis of intracellular UMOD levels in these conditions demonstrated increased accumulation when extracellular secretion was low, and vice versa. Incubation of primary mTAL cells with calcium confirmed increased localisation of UMOD at the membrane compared to the cytosol, without any major differences in cell morphology. The Umod mRNA level changes were not statistically significant among conditions. Conclusion Trafficking of UMOD in the mTAL is influenced by the type of CaSR ligand and the biased nature of G-protein coupled CaSR signalling. Unravelling the signalling events post-calcium will be necessary for identification of key regulators of UMOD secretion and provide new sites for therapeutic intervention in hypertension.

Role of Extracellular Calcium and Calcium Sensitization in the Anti-Contractile Effect of Perivascular Adipose Tissue in Pregnant Rat Aorta

Previous studies have shown that the anti-contractile effect of the perivascular adipose tissue (PVAT) is attenuated in pregnancy. In the present investigation, we have examined the possibility that this loss of anti-contractile effect could be due to changes in calcium mobilization. PVAT exerted anti-contractile effect against 5-hydroxytryptamine (5-HT)-induced contractions of aorta segments from pregnant and non-pregnant rats and this anti-contractile effect was attenuated in segments from pregnant rats. Nifedipine (10^–6 mol/L), an inhibitor of L-type dihydropyridine calcium channels, significantly reduced 5-HT-induced contraction of aorta segments from non-pregnant and pregnant rats with and without PVAT. The inhibitory effect of nifedipine against 5-HT-induced contractions was attenuated in PVAT-free aorta segments from pregnant rats. However, while PVAT reduced the effectiveness of nifedipine in aorta segments from non-pregnant rats, it partially restored the inhibitory effect of nifedipine in aorta segments from pregnant rats. Inhibitors of calcium sensitization, Y-27632 (10^–6 mol/L) and GF 109203X (10^–6 mol/L), significantly reduced 5-HT-induced contractions of PVAT-free aorta segments from non-pregnant and pregnant rats. Both inhibitors, however, were less effective in aorta segments from pregnant rats. The presence of PVAT reduced the effectiveness of Y-27632 and GF 109203X in aorta segments from pregnant and non-pregnant rats. Protein expression of Rho-associated protein kinase (ROCK) I and II was detected in aorta segments and PVAT from pregnant and non-pregnant rats. There was a reduction in the expression of both isoforms in aorta segments but not PVAT from pregnant rats. In addition, there was no significant difference in the expression of ROCK-I and ROCK-II in PVAT from pregnant and non-pregnant rats. We concluded that the loss of anti-contractile effect of PVAT in aorta segments from pregnant rats could be due to increased influx of extracellular calcium through nifedipine-sensitive dihydropyridine channels.

Extra and intracellular calcium signaling pathway(s) differentially regulate histamine-induced myometrial contractions during early and mid-pregnancy stages in buffaloes (Bubalus bubalis)

This study examines the differential role of calcium signaling pathway(s) in histamine-induced uterotonic action during early and mid-pregnancy stages in buffaloes. Compared to mid pregnancy, tonic contraction, amplitude and mean-integral tension were significantly increased by histamine to produce myometrial contraction during early pregnancy with small effects on phasic contraction and frequency. Although uterotonic action of histamine during both stages of pregnancy is sensitive to nifedipine (a L-type Ca2+ channels blocker) and NNC55-0396 (T-type Ca2+ channels blocker), the role of extracellular calcium seems to be more significant during mid-pregnancy as in this stage histamine produced only 9.38±0.96% contraction in Ca2+ free-RLS compared to 21.60±1.45% in uteri of early pregnancy stage. Intracellular calcium plays major role in histamine-induced myometrial contraction during early pregnancy as compared to mid pregnancy, as in the presence of cyclopiazonic acid (CPA) Ca2+-free RLS, histamine produced significantly higher contraction in myometrial strips of early-pregancy in comparison to mid-pregnancy (10.59±1.58% and 3.13±0.46%, respectively). In the presence of U-73122, the DRC of histamine was significantly shifted towards right with decrease in maximal effect (Emax) only in early pregnancy suggesting the predominant role of phospholipase-C (PL-C) in this stage of pregnancy.

Improving and accelerating the differentiation and functional maturation of human stem cell-derived neurons: role of extracellular calcium and GABA.

Neurons differentiated from pluripotent stem cells using established neural culture conditions often exhibit functional deficits. Recently, we have developed enhanced media which both synchronize the neurogenesis of pluripotent stem cell-derived neural progenitors and accelerate their functional maturation; together these media are termed SynaptoJuice. This pair of media are pro-synaptogenic and generate authentic, mature synaptic networks of connected forebrain neurons from a variety of induced pluripotent and embryonic stem cell lines. Such enhanced rate and extent of synchronized maturation of pluripotent stem cell-derived neural progenitor cells generates neurons which are characterized by a relatively hyperpolarized resting membrane potential, higher spontaneous and induced action potential activity, enhanced synaptic activity, more complete development of a mature inhibitory GABAA receptor phenotype and faster production of electrical network activity when compared to standard differentiation media. This entire process - from pre-patterned neural progenitor to active neuron - takes 3weeks or less, making it an ideal platform for drug discovery and disease modelling in the fields of human neurodegenerative and neuropsychiatric disorders, such as Huntington's disease, Parkinson's disease, Alzheimer's disease and Schizophrenia.

The Restraint of Bovine Sperm Cell Motility Increases Survival: Role of Extracellular Calcium in the Phenomena

Sperm cells are complex models for handling in vitro, their viability is limited, and their physiology is complex. The study of their properties is of great application in the animal production industry, to improve the selection of gametes, control pathologies and for the development of cryobiology protocols. It is therefore important to have viable and functional gametes. Consequently, it has been demonstrated that the increase of sperm cell mortality is related to the increase of the Reactive Oxygen Species (ROS), and ROS is secondary to normal metabolic processes of the cell, i.e., special motility. One of the processes where the mature sperm cells’ main activity is the consumption of more energy, it is the flagellar movement through which high ATP consumption generates high quantities of ROS in the seminal plasma. There is evidence of strategies that lead to reduced metabolic activity for different variables (temperature, pH and other), the intention being that seminal plasma protects the sperm cells and reduces the mortality, and thus it is correct to suggest reducing mortality by reducing motility. It has to be considered that flagellar movement is a complex action that involves energy consumption, regulated by calcium. The phenomenon has not been fully characterized, but it is established that in certain mammalian models, the entry of calcium in specific channels such as CATsper or voltage-dependent channels is a signal for flagellar movement to occur. Reduce the motility of bovine spermatozoa using calcium channel blockers can increase cell survival and we hypothesized that: the general blockade of the calcium channel generated reduced the calcium entry into bovine sperm cells, restricting motility and increased survival of these cells. We propose to in the future explore whether the modulation of calcium channels in bovine sperm cells can reduce motility and increase the survival of these cells in experimental conditions, to reduce the mortality of the sample and improve laboratory manipulation.

276 Does TRP channel play a role of extracellular calcium sensing in urethral smooth muscle?

276 Does TRP channel play a role of extracellular calcium sensing in urethral smooth muscle?

Influence of extracellular magnesium on phagocytosis and free cytosolic Mg levels in differentiated U937 and MH-S cells

The influence of extracellular calcium on phagocytosis is limited, and the involvement of extracellular magnesium is unclear. The role of extracellular calcium and magnesium on phagocytosis efficiency was investigated. Extracellular calcium had no influence on the internalization of 1 μm polystyrene particles by primary monocytes as has been shown before for the human lymphoma-derived, differentiated cell line U937 and murine alveolar macrophage-derived MH-S cells. In contrast, the phagocytosis by differentiated U937 cells was positively affected by the presence of extracellular magnesium whereas that of MH-S cells was not. An extracellular increase in the magnesium level did not cause an increase in the free cytosolic magnesium concentration in either cell line. In contrast to magnesium, extracellular calcium caused an increase in intracellular divalent cation levels in differentiated U937 cells. A phagocytosis-enhancing effect in the extracellular space was observed in relation to extracellular magnesium rather than with an intracellular increase in magnesium levels, indicating that murine and human immune cells might be regulated differently.

Role of extracellular calcium and mitochondrial oxygen species in psychosine-induced oligodendrocyte cell death.

Globoid cell leukodystrophy (GLD) is a metabolic disease caused by mutations in the galactocerebrosidase (GALC) gene. GALC is a lysosomal enzyme whose function is to degrade galacto-lipids, including galactosyl-ceramide and galactosyl-sphingosine (psychosine, PSY). GALC loss of function causes progressive intracellular accumulation of PSY. It is widely held that PSY is the main trigger for the degeneration of myelinating cells and progressive white-matter loss. However, still little is known about the molecular mechanisms by which PSY imparts toxicity. Here, we address the role of calcium dynamics during PSY-induced cell death. Using the human oligodendrocyte cell line MO3.13, we report that cell death by PSY is accompanied by robust cytosolic and mitochondrial calcium (Ca2+) elevations, and by mitochondrial reactive oxygen species (ROS) production. Importantly, we demonstrate that the reduction of extracellular calcium content by the chelating agent ethylenediaminetetraacetic acid can decrease intra-mitochondrial ROS production and enhance cell viability. Antioxidant administration also reduces mitochondrial ROS production and cell loss, but this treatment does not synergize with Ca2+ chelation. Our results disclose novel intracellular pathways involved in PSY-induced death that may be exploited for therapeutic purposes to delay GLD onset and/or slow down its progression.

Culture requirements of prostatic epithelial cell lines for acinar morphogenesis and lumen formation in vitro: Role of extracellular calcium

Three-dimensional (3D) culture of benign prostatic epithelial cell lines can recapitulate acinar morphogenesis in vitro, but the broad applicability of this approach has not been described. The present studies examine the culture conditions important for prostatic acinar morphogenesis in vitro and the role of extracellular calcium in this process. With optimized culture conditions, RWPE-1, pRNS-1-1, PZ-HPV-7, PNT1A, BPH-1, and PrEC were analyzed for their ability to undergo acinar morphogenesis in 3D culture and by immunoblotting. RWPE-1 cells were further examined for the effects of calcium on morphology, E-cadherin membrane localization and multicellular layering in 2D culture and for acinar morphogenesis, luminal apoptosis, and luminal filling in 3D. Cell lines grown in low-calcium medium have the ability to form acinar structures with lumens, which correlates with E-cadherin expression, but low calcium is not required for this process. Adding CaCl(2) to the medium strongly inhibits lumen formation, luminal apoptosis and induces luminal filling, and luminal filling is blocked by an interfering antibody. Optimized medium composition allows nearly all seeded RWPE-1 cells to undergo acinar morphogenesis, forming consistent structures representative of normal adult prostate glands. Low-calcium-containing medium appears selective for cells capable of undergoing acinar morphogenesis in vitro, and branching and luminal space within the acini are strongly influenced by extracellular calcium levels, likely through the actions of E-cadherin. These results provide important information about a relevant in vitro model with which to study prostate development and carcinogenesis and highlight the importance of extracellular calcium in regulating 3D morphology.

Effect of glutamate and extracellular calcium on uptake of inorganic lead (Pb 2+) 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 Pb 2+-induced cytotoxicity. It is likely that Pb 2+ must enter cells to exert most of its toxic effects. Pb 2+ is known to substitute for Ca 2+ in many cellular processes. Therefore, we studied the uptake mechanisms of Pb 2+ into GT1–7 neuronal cells with a special focus on the role of extracellular calcium (Ca 2+), voltage-sensitive calcium channels (VSCCs) and glutamate. Basal uptake of Pb 2+ (1 μM or 10 μM), i.e. without any external stimulus, clearly increased in nominally Ca 2+-free buffer and was partially abolished by 13 mM Ca 2+ when compared to uptake in the presence of a physiological concentration of extracellular Ca 2+ (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 Pb 2+ uptake. Glutamate (1 mM) increased Pb 2+ uptake, but only when cells were treated with 1 μM Pb 2+ in the presence of 1.3 mM Ca 2+. Our data suggest that Pb 2+ competes for the same cellular uptake pathways with Ca 2+, although not via VSCCs. In addition, enhancement of Pb 2+-induced neurotoxicity by glutamate may be due to increased neuronal uptake of Pb 2+.

Bothrops venom induces direct renal tubular injury: role for lipid peroxidation and prevention by antivenom

Acute renal failure (ARF) is one of the most serious complications of Bothrops snakebites. Pathogenesis of ARF in snakebite envenomation may involve hemodynamic disturbances, immunologic reactions and direct nephrotoxicity. This study aimed at evaluating Bothrops jararaca venom direct toxicity on isolated rat renal proximal tubules (PT). PT was kept oxygenated and subjected to hypoxia (H, 15 min) and reoxygenation (R, 45 min). Bothropic antivenom effects, role of extracellular calcium and peroxide production were also evaluated. Cell injury was determined by LDH release (%) and peroxide production determined by xylenol-orange method. B. jararaca venom caused tubular injury (LDH 31.6 vs. 17.2%, P<0.05), which was prevented by simultaneous or delayed antivenom administration, but not with low extracellular calcium medium. Venom increased tubules peroxide production: 2.21 vs. 1.27 μM/mg protein ( P<0.05) which was also prevented by antivenom administration. Venom toxic concentration did not enhance H/R injury. In contrast, non-toxic venom concentration afforded protection (LDH 41.3 vs. 51.5%, P<0.05). In conclusion, B. jararaca venom caused direct injury to normoxic renal tubules, but not to hypoxic/reoxygenated tubules. Tubular toxicity is independent of extracellular calcium and mediated in part by lipid peroxidation. Venom induced tubular injury was prevented by simultaneous or delayed antivenom administration.

The role of extracellular calcium in the regulation of intracellular calcium and cell function (II). Some answers and more questions.

The role of extracellular calcium in the regulation of intracellular calcium and cell function (II). Some answers and more questions.

NG-Nitro-L-arginine Methyl Ester-Induced Potentiaton of the Effect of Aminophylline on Rat Diaphragm: the Role of Extracellular Calcium

The role of extracellular calcium in the interaction between intracellular cAMP and nitric oxide (NO)/cGMP on the contractility of rat diaphragm pretreated with cumulative concentrations of aminophylline (0.36 – 3.60 mM) was investigated. In a Ca2+-free medium, NG-nitro-L-arginine methyl ester (L-NAME) (1 and 3 mM) depressed tension developed (Td) and also aminophylline-induced potentiation of Td in a concentration-dependent manner. Verapamil (2.5 μM) or nicardipine (20 μM) significantly antagonized the potentiating effect of L-NAME on Td in a calcium-containing medium. However, in the presence of verapamil or nicardipine, L-NAME still produced statistically significant potentiation of the cumulative concentrations of aminophylline (0.36 – 3.60 mM), given in the second series.

Neurohormone Secretion Persists after Post-Afterdischarge Membrane Depolarization and Cytosolic Calcium Elevation in Peptidergic Neurons in Intact Nervous Tissue

The purpose of this work was to test the hypothesis that an electrical afterdischarge (AD) causes prolonged elevation in cytosolic calcium levels that is associated with prolonged secretion of egg-laying hormone (ELH) from peptidergic neurons in intact nervous tissue of Aplysia. Using a combination of radioimmunoassay measurement of ELH secretion, electrophysiological measurement of membrane potential, and optical imaging of the concentration of intracellular free calcium ions ([Ca2+]i), we verified that there was persistent secretion of ELH after the end of the AD; this was accompanied by prolonged post-AD membrane depolarization and prolonged post-AD elevation in [Ca2+]i. Extracellular treatment with the calcium chelator EGTA had no effect on the pattern or magnitude of ELH secretion or on the post-AD membrane potential (V(m)) and post-AD Ca2+ signal, ruling out a role for extracellular calcium in the post-AD elevation of [Ca2+]i. Both V(m) and [Ca2+]i returned to baseline well before ELH secretion, such that neither prolonged membrane depolarization nor prolonged Ca2+ signaling can fully account for the extent of the persistent secretion of ELH. These findings suggest a unique relationship between membrane excitability, Ca2+ signaling, and prolonged neuropeptide secretion.

Effects of bethanechol on canine urinary bladder smooth muscle function

We studied whether the effects of bethanechol are mediated via a muscarinic receptor, the role of extracellular calcium on bladder contraction, and down-regulation of bladder contraction by bethanechol after activation with potassium chloride (KCl) and acetylcholine (Ach). Smooth muscle strips of normal urinary bladder were studied with standard methods to measure isometric force. Bethanechol caused a dose-dependent increase in bladder contraction. The potency of bethanechol is higher than Ach, as shown by higher peak active isometric stress (Pmax) and lower half-maximal contraction (ED50) (P<0.01). The contractile responses to bethanechol were diminished in the presence of atropine, nifedipine and in calcium-free medium as shown by Pmaxdecreased by 58%, 87% and 65% andED50 increased by 314-, 24- and 16-fold, respectively. When bladder strips were stimulated with KCl and Ach, pre-treatment with bethanechol reduced the responses to KCl by 116–242% (P<0.05), while the contractile responses to Ach were unaltered. Thus, bethanechol induces bladder contraction via muscarinic receptor activation while both intracellular and extracellular calcium play a crucial role on bladder smooth muscle contraction. The mechanisms of down-regulation by bethanechol may be related to interference with calcium influx into the smooth muscle cells, rather than the desensitisation of muscarinic receptors or post-receptor steps of signal transduction following bethanechol binding to the receptor.

Role of extracellular calcium on the regulation of melatonin synthesis in the Syrian hamster pineal gland.

The role of extracellular calcium on the induction of cyclic AMP production, N-acetyltransferase (NAT) activity and melatonin synthesis, induced by forskolin, was investigated in Syrian hamster pineal glands. Pineals were removed from hamsters killed either in the first half of the normal dark period or late in the dark period. Forskolin immediately increased NAT activity and melatonin levels only when the glands were collected late in the dark period, while in the first half of the dark phase, hamster pineals responded to forskolin with an increase of NAT activity and melatonin production only after 6 hr of incubation. The absence of calcium prevented the induction of melatonin synthesis by forskolin only when glands were collected early in the dark phase and incubated for 6 hr. In the second half of the normal dark period removal of calcium markedly decreased NAT activity and melatonin levels in glands incubated with forskolin for either 4 or 6 hr. However, the absence of extracellular calcium had no significant effect on the induction of cyclic AMP production by forskolin in pineals collected either early in the dark period or late in the dark phase. These data indicate that at least part of the action of extracellular calcium is indirect and that it affects steps in the induction of melatonin synthesis beyond the accumulation of cAMP.

Outside-in Signaling Pathway Linked to CD146 Engagement in Human Endothelial Cells

CD146 (S-Endo 1 Ag or MUC18) is a transmembrane glycoprotein expressed on endothelial cells on the whole vascular tree. CD146 is located at the intercellular junction where it plays a role in the cohesion of the endothelial monolayer. CD146 engagement initiates an outside-in signaling pathway involving the protein tyrosine kinases FYN and FAK as well as paxillin. Here we report that CD146 engagement by its specific monoclonal antibody in human umbilical vein endothelial cells induces a Ca(2+) influx that is sensitive to thapsigargin and EGTA treatment, indicating that CD146 engagement initiates a store-operated calcium mobilization. In addition, biochemical and pharmacological analysis revealed that CD146 engagement initiates the tyrosine phosphorylation of phospholipase C-gamma, Pyk2, and p130(Cas). Pharmacological inhibition of Ca(2+) flux with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acetoxymethyl ester and EGTA indicated that an increase in Ca(2+) is required for Pyk2 and p130(Cas) tyrosine phosphorylation. Moreover, a complex association was observed between Pyk2, p130(Cas), and paxillin. These results indicate that CD146 is coupled to a FYN-dependent pathway that triggers Ca(2+) flux via phospholipase C-gamma activation leading subsequently to the tyrosine phosphorylation of downstream targets such as Pyk2, p130(Cas), FAK, and paxillin. In addition to its role in cell-cell adhesion, CD146 is a signaling molecule involved in the dynamics of actin cytoskeleton rearrangement.

Selective Involvement of Calcium and Calcium Channels in Stimulated Mucin Secretion from Rat Colon

Background: The cellular mechanisms involved in the mucin secretion of rat colon are unknown. The objective of the present study was thus to determine the role of extracellular calcium and of L-type calcium channels in rat intestinal mucin discharge. Methods: The experiments were conducted using the isolated vascularly perfused rat colon. Mucin secretion was evaluated using an enzyme-linked immunosorbent assay. Results: Intra-arterial bethanechol (200 μM) or luminal deoxycholate (5 mM) produced a significant mucin discharge (609% and 386% of controls, respectively). The colonic mucin output induced by these two secretagogues was significantly inhibited by arterial administration of EGTA (2 mM), verapamil (100 μM) or nifedipine (50 μM). In contrast, luminal EGTA (2 mM) had no inhibitory effect. Intra-arterial infusion of the calmodulin antagonist trifluoperazine (10 μM) also reduced mucin discharge induced by bethanechol or deoxycholate (304% and 223% of controls, respectively). Colonic mucin secretion was significantly stimulated after intra-arterial infusion of 3-isobutyl-methylxanthine (IBMX, 100 μM) or forskolin (2-20 μM). Stimulation by forskolin was unaffected by arterial EGTA, verapamil, nifedipine or trifluoperazine. Conclusion: In the isolated vascularly perfused rat colon, mucin discharge induced by bethanechol or deoxycholate requires extracellular calcium and the activation of voltage-dependent calcium channels of L-type. In contrast, forskolin does not appear to stimulate mucin release by increasing calcium entry.

Extracellular Ca 2+ regulates the respiratory burst of human neutrophils

The role of extracellular calcium in the activation of respiratory burst in human neutrophils was studied by using the receptor agonist, N-formyl-methionyl-leucyl-phenylalanine (fMLP), and the activator of protein kinase C phorbol myristate acetate (PMA). The level of intracellular free calcium was measured by using both cell suspensions and single cells in the presence and absence of extracellular calcium. The Ca 2+-ATPase inhibitor, thapsigargin, was used to activate higher Ca 2+ influx, while a novel calcium channel blocker, panax notoginseng saponins (PNGS) was used to block the Ca 2+ entry from extracellular space during the responding period of cells. It was found that about two-thirds of the activation of respiratory burst initiated by the receptor agonist were attributed to the Ca 2+ influx under normal physiological conditions. The higher Ca 2+ influx resulted in tremendous enhancement of the intensity of respiratory burst initiated by fMLP and marked acceleration of the onset of the respiratory burst stimulated by PMA. It is evident that both intra- and extracellular Ca 2+ are required for full activation of the respiratory burst of human neutrophils, and the Ca 2+ influx from extracellular space plays an important role either in generation of reactive oxygen metabolites or in activation of protein kinase C.

Modification of Glutamate-Induced Oxidative Stress by Lead: The Role of Extracellular Calcium

The role of extracellular calcium in glutamate-induced oxidative stress, and the role of glutamatergic neuronal stimulation and oxidative stress in lead neurotoxicity were explored in mouse hypothalamic GT1-7 cells. Glutamate increased the production of reactive oxygen species (ROS) whether or not extracellular calcium was present. Glutamate-induced ROS production was amplified by lead acetate (PbAc), but only in the absence of extracellular calcium. However, PbAc on its own did not increase the production of ROS. A PKC inhibitor (Ro 31-8220) and superoxide dismutase (SOD) abolished the amplification of glutamate-induced production of ROS by PbAc, but did not inhibit ROS production induced by glutamate alone. Both glutamate and PbAc decreased the levels of intracellular glutathione (GSH), and amplified each other’s effect on GSH depletion. Glutamate did not decrease cell viability, whereas the cytotoxicity of PbAc was amplified by glutamate. Extracellular calcium, a PKC inhibitor, or SOD did not modify the effects of glutamate, PbAc or their combination on the levels of GSH or cell viability. These data indicate that in GT1-7 cells extracellular calcium is not essential for glutamate-induced ROS production, which is amplified by PbAc, but only without extracellular calcium. The joint cytotoxicity of glutamate and PbAc is mainly induced by PbAc, preferentially through mechanisms other than ROS production.