Basal Intracellular Calcium Levels Research Articles

LDL Promotes Disorders in β-Cell Cholesterol Metabolism, Implications on Insulin Cellular Communication Mediated by EVs.

Dyslipidemia is described as a hallmark of metabolic syndrome, promoting a stage of metabolic inflammation (metainflammation) that could lead to misbalances in energetic metabolism, contributing to insulin resistance, and modifying intracellular cholesterol pathways and the renin–angiotensin system (RAS) in pancreatic islets. Low-density lipoprotein (LDL) hypercholesterolemia could disrupt the tissue communication between Langerhans β-cells and hepatocytes, wherein extracellular vesicles (EVs) are secreted by β-cells, and exposition to LDL can impair these phenomena. β-cells activate compensatory mechanisms to maintain insulin and metabolic homeostasis; therefore, the work aimed to characterize the impact of LDL on β-cell cholesterol metabolism and the implication on insulin secretion, connected with the regulation of cellular communication mediated by EVs on hepatocytes. Our results suggest that β-cells can endocytose LDL, promoting an increase in de novo cholesterol synthesis targets. Notably, LDL treatment increased mRNA levels and insulin secretion; this hyperinsulinism condition was associated with the transcription factor PDX-1. However, a compensatory response that maintains basal levels of intracellular calcium was described, mediated by the overexpression of calcium targets PMCA1/4, SERCA2, and NCX1, together with the upregulation of the unfolded protein response (UPR) through the activation of IRE1 and PERK arms to maintain protein homeostasis. The LDL treatment induced metainflammation by IL-6, NF-κB, and COX-2 overexpression. Furthermore, LDL endocytosis triggered an imbalance of the RAS components. LDL treatment increased the intracellular levels of cholesterol on lipid droplets; the adaptive β-cell response was portrayed by the overexpression of cholesterol transporters ABCA1 and ABCG1. Therefore, lipotoxicity and hyperinsulinism induced by LDL were regulated by the natural compound auraptene, a geranyloxyn coumarin modulator of cholesterol-esterification by ACAT1 enzyme inhibition. EVs isolated from β-cells impaired insulin signaling via mTOR/p70S6Kα in hepatocytes, a phenomenon regulated by auraptene. Our results show that LDL overload plays a novel role in hyperinsulinism, mechanisms associated with a dysregulation of intracellular cholesterol, lipotoxicity, and the adaptive UPR, which may be regulated by coumarin-auraptene; these conditions explain the affectations that occur during the initial stages of insulin resistance.

Elevation of TRPV1 expression on T-cells during experimental immunosuppression

The transient receptor potential vanilloid 1 (TRPV1) channel is a thermo-sensitive, polymodal cation channel. An increase in intracellular calcium (Ca2+) is essential for T-cell responses. Similarly, various immunosuppressive agents are also reported to induce Ca2+ influx. However, the possible involvement of TRPV1 during immunosuppression has not been studied yet. Here, we investigated the possible functional role of TRPV1 in FK506 or B16F10-culture supernatant (B16F10-CS)-driven experimental immunosuppression in T-cells. Intriguingly, it was found that TRPV1 surface expression was further significantly elevated during immunosuppression compared with concanavalin A (ConA) or TCR-activated T-cells. Moreover, in B16F10 tumor-bearing mice, TRPV1 expression was upregulated on splenic T-cells as compared with T-cells derived from control mice. We also observed an immediate increase in intracellular Ca2+ levels in FK506 (marked increase) and B16F10-CS treatment (modest increase) or in combination with T-cell activation as compared with resting and activated T-cells. Likewise, in B16F10 tumor-bearing mice, the basal intracellular calcium level was upregulated in T-cells as compared with controls. The elevated Ca2+ level(s) were found to be significantly downregulated by 5'-iodoresiniferatoxin (50-IRTX) (a TRPV1-specific inhibitor), suggesting an important role of TRPV1 during immune activation and immunosuppression. The current study may have implications for immunosuppressive diseases along with inflammatory disorders associated with the coordinating role of TRPV1 and Ca2+ influx.

Gadolinium chelates inhibit intracellular calcium signaling in rat trigeminal ganglion neurons similar to rat dorsal root ganglion neurons, but less effective

Objectives: Gadolinium (Gd3+) based MR contrast agents (GBMCA) are manufactured with a chelation process, where organic ligand molecules form a stable complex around Gd3+. The aim of the present study was to investigate the possible impact of free gadolinium and GBMCA in ganglia (trigeminal and dorsal root) neuron cell cultures on intracellular calcium (Ca2+) since these agents could dissociate and lead to the release of toxic Gd3+ ions in metabolic conditions. Methods: Ganglia cells were studied for responses to agents and nonspecific depolarization by high KCl+ in Fura 2 AM loaded neurons by monitoring the changes in [Ca2+]i using a microscopic digital image analysis system. GBMCA and molecular gadolinium doses of 0.1 mmol/kg were prepared with the same pH and temperature and applied to the cells. Results: Application of Gd3+, gadobenate dimeglumine, gadobutrol and gadodiamide to rat ganglia neurons led to significant decreases in basal intracellular calcium levels, while gadoterate meglumine administration did not have a significant impact. As expected, Gd3+ was more effective when compared tı GBMCA. While the impact was almost full with gadodiamide, Gd3+ and other GBMCAs had irreversible effects at different levels. The impact of gadoterate meglumine was neglectable when compared to Gd3+ and other GBMCAs, although it exhibited slightly gadodiamide-like effects. Conclusions: In conclusion, gadolinium chelates released gadolinium and irreversibly reduced the intracellular calcium levels in identical TG and DRG cells at different levels.

Characterization of the taste receptor-related G-protein, α-gustducin, in pancreatic β-cells.

Aims/IntroductionTaste receptors, T1rs and T2rs, and the taste‐selective G‐protein, α‐gustducin, are expressed outside the taste‐sensing system, such as enteroendocrine L cells. Here, we examined whether α‐gustducin also affects nutrition sensing and insulin secretion by pancreatic β‐cells.Materials and MethodsThe expression of α‐gustducin and taste receptors was evaluated in β‐cell lines, and in rat and mouse islets either by quantitative polymerase chain reaction or fluorescence immunostaining. The effects of α‐gustducin knockdown on insulin secretion and on cyclic adenosine monophosphate and intracellular Ca2+ levels in rat INS‐1 cells were estimated. Sucralose (taste receptor agonist)‐induced insulin secretion was investigated in INS‐1 cells with α‐gustducin suppression and in islets from mouse disease models.ResultsThe expression of Tas1r3 and α‐gustducin was confirmed in β‐cell lines and pancreatic islets. Basal levels of cyclic adenosine monophosphate, intracellular calcium and insulin secretion were significantly enhanced with α‐gustducin knockdown in INS‐1 cells. The expression of α‐gustducin was decreased in high‐fat diet‐fed mice and in diabetic db/db mice. Sucralose‐induced insulin secretion was not attenuated in INS‐1 cells with α‐gustducin knockdown or in mouse islets with decreased expression of α‐gustducin.Conclusionsα‐Gustducin is involved in the regulation of cyclic adenosine monophosphate, intracellular calcium levels and insulin secretion in pancreatic β‐cells in a manner independent of taste receptor signaling. α‐Gustducin might play a novel role in β‐cell physiology and the development of type 2 diabetes.

Altered calcium dynamics and glutamate receptor properties in iPSC-derived motor neurons from ALS patients with C9orf72, FUS, SOD1 or TDP43 mutations.

The fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS) is characterized by a profound loss of motor neurons (MNs). Until now only riluzole minimally extends life expectancy in ALS, presumably by inhibiting glutamatergic neurotransmission and calcium overload of MNs. Therefore, the aim of this study was to investigate the glutamate receptor properties and key aspects of intracellular calcium dynamics in induced pluripotent stem cell (iPSC)-derived MNs from ALS patients with C9orf72 (n = 4 cell lines), fused in sarcoma (FUS) (n = 9), superoxide dismutase 1 (SOD1) (n = 3) or transactive response DNA-binding protein 43 (TDP43) (n = 3) mutations as well as healthy (n = 7 cell lines) and isogenic controls (n = 3). Using calcium imaging, we most frequently observed spontaneous transients in mutant C9orf72 MNs. Basal intracellular calcium levels and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-induced signal amplitudes were elevated in mutant TDP43 MNs. Besides, a majority of mutant TDP43 MNs responded to 3.5-dihydroxyphenylglycine as metabotropic glutamate receptor agonist. Quantitative real-time PCR demonstrated significantly increased expression levels of AMPA and kainate receptors in mutant FUS cells compared to healthy and isogenic controls. Furthermore, the expression of kainate receptors and voltage gated calcium channels in mutant C9orf72 MNs as well as metabotropic glutamate receptors in mutant SOD1 cells was markedly elevated compared to controls. Our data of iPSC-derived MNs from familial ALS patients revealed several mutation-specific alterations in glutamate receptor properties and calcium dynamics that could play a role in ALS pathogenesis and may lead to future translational strategies with individual stratification of neuroprotective ALS treatments.

The Protective Role of TRPC6 Knockout in the Progression of Diabetic Nephropathy

Diabetic nephropathy (DN) is a chronic kidney pathology that leads to end stage renal disease (ESRD). It was previously found that a gain‐of‐function mutation in the Transient Receptor Potential Canonical 6 (TRPC6) channel, a non‐selective cation channel that is highly permeable for calcium ions, causes Focal Segmental Glomerulosclerosis (FSG) that is characterized with podocyte loss. Previous studies from our laboratory indicate that there is an association between the development of DN and over‐activation of the TRPC6 channel. We have demonstrated that there is an increase in TRPC6 expression and activity in the streptozotocin (STZ)‐induced diabetic Dahl Salt‐sensitive (SS) rat, an established model of type 1 DN. We have created the TRPC6 knock‐out on a SS rat background (SSTRPC6−/−), and test the hypothesis that the deletion of TRPC6 will protect podocytes and glomeruli during the development of DN. Four groups of animals – control SS, SSTRPC6−/−, and STZ‐treated SS and SSTRPC6−/− – were utilized in this study. Diabetes development was monitored for 11 weeks after STZ injection with periodic weight, glucose, and urinary output measurements. Glomerular filtration rate (GFR) was measured and tissue collections were made at the end of the protocol. Freshly isolated glomeruli of perfused kidneys were used from all four groups for either electron microscopy or confocal fluorescence microscopy to assess the foot process effacement and basal level of intracellular calcium ([Ca2+]i), respectively. Histochemical analysis of whole kidney slices was done to assess renal injury. There was a decrease in urinary output (49±29 vs 73±39 mL/day/100g BW) and a trend for blunted increase in albuminuria (97±34 vs 112±19 mg/day x 100g BW) in the STZ‐treated SSTRPC6−/− rats compared to the STZ‐SS animals. There was no significant difference in GFR between either of the 4 experimental groups; however there does appear to be a trend for hyperfiltration in the STZ‐treated animals. Importantly, pathological increases in basal [Ca2+]i levels and foot process damage of podocytes during the development of DN was attenuated in the STZ‐treated SSTRPC6−/− animals. Overall, our data indicates that inhibiting the TRPC6 channel may have renoprotective effects and the potential to lead to the development of new pharmacological tools to treat or prevent the progression of DN and kidney damage.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Pyk2 inhibition promotes contractile differentiation in arterial smooth muscle

Modulation from contractile to synthetic phenotype of vascular smooth muscle cells is a central process in disorders involving compromised integrity of the vascular wall. Phenotype modulation has been shown to include transition from voltage-dependent toward voltage-independent regulation of the intracellular calcium level, and inhibition of non-voltage dependent calcium influx contributes to maintenance of the contractile phenotype. One possible mediator of calcium-dependent signaling is the FAK-family non-receptor protein kinase Pyk2, which is activated by a number of stimuli in a calcium-dependent manner. We used the Pyk2 inhibitor PF-4594755 and Pyk2 siRNA to investigate the role of Pyk2 in phenotype modulation in rat carotid artery smooth muscle cells and in cultured intact arteries. Pyk2 inhibition promoted the expression of smooth muscle markers at the mRNA and protein levels under stimulation by FBS or PDGF-BB and counteracted phenotype shift in cultured intact carotid arteries and balloon injury ex vivo. During long-term (24-96 hr) treatment with PF-4594755, smooth muscle markers increased before cell proliferation was inhibited, correlating with decreased KLF4 expression and differing from effects of MEK inhibition. The Pyk2 inhibitor reduced Orai1 and preserved SERCA2a expression in carotid artery segments in organ culture, and eliminated the inhibitory effect of PDGF stimulation on L-type calcium channel and large-conductance calcium-activated potassium channel expression in carotid cells. Basal intracellular calcium level, calcium wave activity, and store-operated calcium influx were reduced after Pyk2 inhibition of growth-stimulated cells. Pyk2 inhibition may provide an interesting approach for preserving vascular smooth muscle differentiation under pathophysiological conditions.

Accumulation of human full-length tau induces degradation of nicotinic acetylcholine receptor α4 via activating calpain-2.

Cholinergic impairments and tau accumulation are hallmark pathologies in sporadic Alzheimer’s disease (AD), however, the intrinsic link between tau accumulation and cholinergic deficits is missing. Here, we found that overexpression of human wild-type full-length tau (termed hTau) induced a significant reduction of α4 subunit of nicotinic acetylcholine receptors (nAChRs) with an increased cleavage of the receptor producing a ~55kDa fragment in primary hippocampal neurons and in the rat brains, meanwhile, the α4 nAChR currents decreased. Further studies demonstrated that calpains, including calpain-1 and calpain-2, were remarkably activated with no change of caspase-3, while simultaneous suppression of calpain-2 by selective calpain-2 inhibitor but not calpain-1 attenuated the hTau-induced degradation of α4 nAChR. Finally, we demonstrated that hTau accumulation increased the basal intracellular calcium level in primary hippocampal neurons. We conclude that the hTau accumulation inhibits nAChRs α4 by activating calpain-2. To our best knowledge, this is the first evidence showing that the intracellular accumulation of tau causes cholinergic impairments.

Erythropoietin negatively regulates pituitary ACTH secretion

Erythropoietin (Epo) and Epo-receptor (EpoR) signaling, in addition to its classical role in erythropoiesis, exhibit a protective response in non-hematopoietic tissues. Mice with EpoR expression restricted to only hematopoietic tissues (ΔEpoRE), become obese, have low energy expenditure, and are glucose intolerant and insulin resistant. In the arcuate nucleus of the mouse hypothalamus, EpoR expression co-localizes in proopiomelanocortin (POMC) neurons. In vivo high-dose Epo treatment increases hypothalamus POMC, reduces food intake and fat mass accumulation. Here we report that Epo treatment also decreases plasma concentration of the pituitary derived POMC peptide, adrenocorticotropic hormone (ACTH). Conversely, ΔEpoRE mice show reduced hypothalamus POMC and high plasma concentrations of ACTH. In the pituitary, POMC is synthesized in the corticotroph cells, and here we examine Epo effect on pituitary POMC expression using the AtT-20 mouse corticotroph pituitary cell line. In AtT-20 cells, enzyme immunoassay analysis showed that Epo inhibits ACTH secretion. This effect is post-translational, as Epo treatment did not affect POMC mRNA expression but increased intracellular levels of ACTH peptide. Moreover, Epo reduced the basal intracellular calcium (Ca2+) levels, suggesting an effect in the Ca2+-signaling pathway. In summary, our studies suggest a novel regulatory pathway of ACTH secretion in the pituitary via EpoR-signaling. The higher plasma ACTH level in ΔEpoRE mice also suggests a possible mechanism of deregulated pituitary function with loss of Epo-signaling.

Caffeine induces cardiomyocyte hypertrophy via p300 and CaMKII pathways

Caffeine is commonly utilized to trigger intracellular calcium in cardiomyocyte. It is well accepted that caffeine could induce cardiac arrhythmia, but it is not clear with regard of its impacts on the cardiac function. This article presents a recent study concerning the effects of caffeine on the cardiomyocyte hypertrophy and the associated signal pathway. The experimental results showed that the total protein contents, the surface area of cardiomyocyte and β-myosin heavy chain (β-MHC) expression increased in ventricular myocytes of neonatal Sprague–Dawley (SD) rats after 24h caffeine incubation. It is also observed that the basal intracellular calcium (Ca2+) level has increased, while the amplitude of Ca2+ oscillation and Ca2+ content have decreased in sarcoplasmic reticulum (SR). The caffeine-induced myocyte enhancer factor-2 (MEF2) expression and hypertrophy can be completely abolished by the inhibition of cardiac ryanodine receptor (RyR2), as well as KN93 and curcumin treatments. Meanwhile, the amplitude of Ca2+ oscillation and the Ca2+ content of SR in the completely-inhibited group have reached the physiological level. These results suggest that the caffeine-induced cardiomyocyte hypertrophy established the connection between Ca2+ release from SR and cytosol that activates CaMKII and p300, which in turn enhances the expression of MEF2 that promotes cardiomyocyte hypertrophy.

PINK1 deficiency in β-cells increases basal insulin secretion and improves glucose tolerance in mice.

The Parkinson's disease (PD) gene, PARK6, encodes the PTEN-induced putative kinase 1 (PINK1) mitochondrial kinase, which provides protection against oxidative stress-induced apoptosis. Given the link between glucose metabolism, mitochondrial function and insulin secretion in β-cells, and the reported association of PD with type 2 diabetes, we investigated the response of PINK1-deficient β-cells to glucose stimuli to determine whether loss of PINK1 affected their function. We find that loss of PINK1 significantly impairs the ability of mouse pancreatic β-cells (MIN6 cells) and primary intact islets to take up glucose. This was accompanied by higher basal levels of intracellular calcium leading to increased basal levels of insulin secretion under low glucose conditions. Finally, we investigated the effect of PINK1 deficiency in vivo and find that PINK1 knockout mice have improved glucose tolerance. For the first time, these combined results demonstrate that loss of PINK1 function appears to disrupt glucose-sensing leading to enhanced insulin release, which is uncoupled from glucose uptake, and suggest a key role for PINK1 in β-cell function.

Polycystin-1 Mediates Mechanical Strain-Induced Osteoblastic Mechanoresponses via Potentiation of Intracellular Calcium and Akt/β-Catenin Pathway

Mechanical regulation of bone formation involves a complex biophysical process, yet the underlying mechanisms remain poorly understood. Polycystin-1 (PC1) is postulated to function as a mechanosensory molecule mediating mechanical signal transduction in renal epithelial cells. To investigate the involvement of PC1 in mechanical strain-induced signaling cascades controlling osteogenesis, PKD1 gene was stably silenced in osteoblastic cell line MC3T3-E1 by using lentivirus-mediated shRNA technology. Here, our findings showed that mechanical tensile strain sufficiently enhanced osteogenic gene expressions and osteoblastic proliferation. However, PC1 deficiency resulted in the loss of the ability to sense external mechanical stimuli thereby promoting osteoblastic osteogenesis and proliferation. The signal pathways implicated in this process were intracellular calcium and Akt/β-catenin pathway. The basal levels of intracellular calcium, phospho-Akt, phospho-GSK-3β and nuclear accumulation of active β-catenin were significantly attenuated in PC1 deficient osteoblasts. In addition, PC1 deficiency impaired mechanical strain-induced potentiation of intracellular calcium, and activation of Akt-dependent and Wnt/β-catenin pathways, which was able to be partially reversed by calcium ionophore A23187 treatment. Furthermore, applications of LiCl or A23187 in PC1 deficient osteoblasts could promote osteoblastic differentiation and proliferation under mechanical strain conditions. Therefore, our results demonstrated that osteoblasts require mechanosensory molecule PC1 to adapt to external mechanical tensile strain thereby inducing osteoblastic mechanoresponse, partially through the potentiation of intracellular calcium and downstream Akt/β-catenin signaling pathway.

The arrhythmogenic effect of self-assembling nanopeptide hydrogel scaffolds on neonatal mouse cardiomyocytes

The chaotic spatial disarray due to extracellular matrix expansion disrupts cardiomyocytes interaction and causes arrhythmia. We hypothesized that disordered nanopeptide scaffolds can mimic the chaotic spatial disarray related to cardiac fibrosis and have arrhythmogenic effects on cardiomyocytes. Primary mouse cardiomyocytes were cultured in 2D traditional and 3D nanopeptide hydrogel scaffold systems. Cardiomyocytes in 3D scaffolds showed irregular spontaneous contractile activity as compared with 2D culture controls. Calcium fluorimetric imaging revealed that basal intracellular calcium level increased 1.42-fold in cardiomyocytes cultured in the 3D scaffold, in vitro. The mRNA levels of sarcoplasmic reticulum calcium transport ATPase, ryanodine 2 receptor and connexin 43 elevated 2.14-fold, 2.33-fold and 2.62-fold in 3D compared with 2D. Immunofluorescence imaging revealed lateralization of the distribution of connexin 43 in 3D group. These findings suggest that 3D hydrogel culture system provides a model for the development of cardiac dysrhythmia. These limitations should be considered during cardiac tissue engineering. From the Clinical EditorThis team of scientists has established a unique 3D hydrogel culture system as a model for the development of cardiac dysrhythmia.

Activity dependent modulation of synaptic transmission by presynaptic calcium stores: A dichotomy of short-term depression and facilitation

Ongoing electrical activity can initiate a positive feedback loop via Inositol Triphosphate Receptors (IP3Rs) and Ryanodine Receptors (RyRs) on the Endoplasmic Receptors (ER) that can lead to release of calcium from the ER. We investigated how the presence of this additional source of calcium in the presynaptic terminals in addition to the Voltage Dependent Calcium Channels (VDCCs) can regulate synaptic transmission. We carried out 3D Monte Carlo simulations of all the molecular interactions that govern transmitter release in a 1) Canonical CA3-CA1 synapse 2) A synapse reconstructed from serial section Transmission Electron Microscope images. The relatively simple geometry of CA3-CA1 synapses allows activity-dependent local calcium at the active zone and the related transmitter release profiles to be quantitatively analyzed. In paired-pulse stimulation, the presence of the molecular pathways that regulate the calcium stores increased the calcium buffering capacity of the synapse, which decreased release probability and enhanced paired-pulse facilitation. In contrast, a high-frequency stimulus could trigger the activation of presynaptic RyRs and Metabotropic Glutamate Receptors (mGluRs) leading to IP3 production and ultimately to release of calcium from the SER. IP3Rs and RyRs operated at a much slower time scale, on the order of seconds compared to the millisecond timescale of the VDCCs. This led to an increase in the basal level of intracellular calcium and enhanced transmitter release rates. In reconstructions of hippocampal neuropil, the ER appears in a majority of the presynaptic terminals in varying degrees of abundance. We further explored the functional implications of the range of ER geometries and therefore the calcium carrying capacity, observed in the synaptic ultrastructure and the effect of different arrangements between IP3Rs and VDCCs on synaptic plasticity. The synaptic ultrastructure precisely orchestrated the degree of facilitation and depression at the synapse and the existence of presynaptic calcium stores provided the synapse with an additional intrinsic time scale that could be regulated by activity.

Modulation of cell viability, oxidative stress, calcium homeostasis, and voltage- and ligand-gated ion channels as common mechanisms of action of (mixtures of) non-dioxin-like polychlorinated biphenyls and polybrominated diphenyl ethers

Non-dioxin-like polychlorinated biphenyls (NDL-PCBs) and polybrominated diphenyl ethers (PBDEs) are environmental pollutants that exert neurodevelopmental and neurobehavioral effects in vivo in humans and animals. Acute in vitro neurotoxic effects include changes in cell viability, oxidative stress, and basal intracellular calcium levels. Though these acute cellular effects could partly explain the observed in vivo effects, other mechanisms, such as effects on calcium influx and neurotransmitter receptor function, likely contribute to the disturbance in neurotransmission. This concise review combines in vitro data on cell viability, oxidative stress and basal calcium levels with recent data that clearly demonstrate that (hydroxylated) PCBs and (hydroxylated) PBDEs can exert acute effects on voltage-gated Ca(2+) channels as well as on excitatory and inhibitory neurotransmitter receptors in vitro. These novel mechanisms of action are shared by NDL-PCBs, OH-PBDEs, and some other persistent organic pollutants, such as tetrabromobisphenol-A, and could have profound effects on neurodevelopment, neurotransmission, and neurobehavior in vivo.

Malathion/Oxon and Lead Acetate Increase Gene Expression and Protein Levels of Transient Receptor Potential Canonical Channel Subunits TRPC1 and TRPC4 in Rat Endothelial Cells of the Blood–Brain Barrier

This study examined the effects of malathion and lead on transient receptor potential canonical channel TRPC1/TRPC4 channels in rat brain endothelial cells as a mechanism to explain previously noted blood-brain barrier (BBB) permeability induced by these compounds. Lead, malathion, malaoxon and combinations of these were assessed for protein levels and gene expression of TRPC1/C4 at 2, 4, 8, 16, and 24 hours after exposure. Changes in intracellular free calcium dynamics were also assessed. Compounds increased TRPC1 and TRPC4 protein levels as well as gene expression within 4 hours after exposure. Basal levels of intracellular free calcium were also elevated. Increases in gene and protein expression may be associated with an increase in the numbers of TRP channels, and the increases in intracellular calcium may be associated with activation of such channels. Therefore, upregulation and activation of the TRPC1/TRPC4 may be a mechanism by which these neurotoxicants affect BBB permeability.

In vivo experimental stroke and in vitro organ culture induce similar changes in vasoconstrictor receptors and intracellular calcium handling in rat cerebral arteries

Cerebral arteries subjected to different types of experimental stroke upregulate their expression of certain G-protein-coupled vasoconstrictor receptors, a phenomenon that worsens the ischemic brain damage. Upregulation of contractile endothelin B (ET(B)) and 5-hydroxytryptamine 1B (5-HT(1B)) receptors has been demonstrated after subarachnoid hemorrhage and global ischemic stroke, but the situation is less clear after focal ischemic stroke. Changes in smooth muscle calcium handling have been implicated in different vascular diseases but have not hitherto been investigated in cerebral arteries after stroke. Here, we evaluate changes of ET(B) and 5-HT(1B) receptors, intracellular calcium levels, and calcium channel expression in rat middle cerebral artery (MCA) after focal cerebral ischemia and in vitro organ culture, a proposed model of vasoconstrictor receptor changes after stroke. Rats were subjected to 2h MCA occlusion followed by reperfusion for 1 or 24h. Alternatively, MCAs from naïve rats were cultured for 1 or 24h. ET(B) and 5-HT(1B) receptor-mediated contractions were evaluated by wire myography. Receptor and channel expressions were measured by real-time PCR and immunohistochemistry. Intracellular calcium was measured by FURA-2. Expression and contractile functions of ET(B) and 5-HT(1B) receptors were strongly upregulated and slightly downregulated, respectively, 24h after experimental stroke or organ culture. ET(B) receptor-mediated contraction was mediated by calcium from intracellular and extracellular sources, whereas 5-HT(1B) receptor-mediated contraction was solely dependent on extracellular calcium. Organ culture and stroke increased basal intracellular calcium levels in MCA smooth muscle cells and decreased the expression of inositol triphosphate receptor and transient receptor potential canonical calcium channels, but not voltage-operated calcium channels.

Poster #26 DELINEATING THE FUNCTIONAL NEUROANATOMY OF ILLNESS DENIAL OR ANOSOGNOSIA IN SCHIZOPHRENIA

Alternative splicing of the dopamine D2 receptor gene produces two distinct isoforms referred to as D2long (D2L) and D2short (D2S). In mesencephalic dopamine neurons, inhibition of the firing rate through activation of somatodendritic D2 receptors and blockade of neurotransmitter release through stimulation of terminal D2 receptors represent major roles of D2 autoreceptors. Recently, data obtained from D2L-deficient mice suggested that D2S acts as the preferential D2 autoreceptor. In the present study, we investigate whether this D2 isoform-specific autoreceptor function is linked to differences in the subcellular localization and/or signaling properties of the D2S and D2L using mesencephalic neurons transfected with enhanced green fluorescent protein (EGFP)-tagged receptors. Our results show that EGFP-tagged D2S and D2L are localized to the axonal and somatodendritic compartments of mesencephalic neurons. In addition, we demonstrate that EGFP-tagged D2S and D2L regulate cellular excitability, neurotransmitter release and basal levels of intracellular calcium with similar effectiveness. Overall, our morphological and electrophysiological studies suggest that the major D2 autoreceptor function attributed to D2S is likely explained by the predominant expression of this isoform in dopamine neurons rather than by distinct subcellular localization and signaling properties of D2S and D2L.

Effects of Presynaptic Calcium Stores on Short-Term Synaptic Plasticity

In reconstructions of hippocampal neuropil, Smooth Endoplasmic Reticulum (SER) appears in a majority of the presynaptic terminals. In the presence of ongoing electrical activity, Inositol Triphosphate Receptors (IP3Rs) on the SER initiate a positive feedback loop that can lead to release of calcium from the SER via an IP3-mediated pathway. We investigated how the presence of this additional source of calcium in addition to the Voltage Dependent Calcium Channels (VDCCs) can regulate synaptic transmission. We carried out 3D Monte Carlo simulations of the molecular interactions that govern transmitter release in a 1) Canonical CA3-CA1 synapse 2) Synapse reconstructed from serial section Transmission Electron Microscope images. The relatively simple geometry of CA3-CA1 synapses allows activity-dependent local calcium at the active zone and the related transmitter release profiles to be quantitatively analyzed. In paired-pulse stimulation, the presence of molecular pathways that regulate the calcium stores increased the calcium buffering capacity of the synapse, which decreased the initial release probability and enhanced paired-pulse facilitation. In contrast, a high-frequency stimulus could trigger the activation of presynaptic Metabotropic Glutamate Receptors (mGluRs) leading to IP3 production and ultimately to release of calcium from the SER. IP3Rs operated at a much slower time scale, on the order of seconds compared to the millisecond timescale of the VDCCs. This led to an increase in the basal level of intracellular calcium and enhanced transmitter release rates. We further explored the functional implications of the range of SER geometries observed in the synaptic ultrastructure and the effect of different arrangements between IP3Rs and VDCCs on synaptic plasticity. The synaptic ultrastructure precisely orchestrated the degree of facilitation and depression and the existence of presynaptic calcium stores provided the synapse with an additional intrinsic time scale that could be regulated by activity.

Elevated BCR signaling and decreased survival of Lyn‐deficient transitional and follicular B cells

The Src-family tyrosine kinase Lyn negatively regulates BCR signaling and also myeloid cell activity. Mice deficient in Lyn have substantially decreased numbers of peripheral B cells, despite spontaneously producing IgG anti-DNA antibodies. Here, we examine the mechanism underlying the B-cell depletion in these mice. Lyn-deficient B cells were out-competed by WT B cells in mixed BM chimeras at two steps, at the T1 to T2 transitional maturation stage in the spleen and again between the T2 or T3 stage and the mature follicular B-cell population. Lyn-deficient T2 and follicular B cells expressed elevated levels of the pro-apoptotic factor Bim and deletion of Bim restored splenic B cells of Lyn-deficient mice to close to WT numbers. Lyn-deficient T2 and later stage B cells also had changes in cell surface phenotype consistent with increased in vivo BCR signaling. Similarly, an increased proportion of T2 and follicular B cells had elevated basal intracellular free calcium levels. Overall, these observations suggest that increased BCR signaling is responsible for increased death of weakly self-reactive Lyn-deficient B cells both at the T2 stage and additionally as these cells mature to follicular B cells.