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1.
Transforming growth factors betas (TGFbetas) are known to have important roles in neuronal survival and can be upregulated in disease. However, unlike many other trophic factors, nothing is known about the rapid neurotransmitter-like actions of TGFbeta in the CNS. We explored this by examining the effects of TGFbeta on calcium influx of large enzymatically dissociated basal forebrain neurons. We show that brief application of TGFbeta2, but not TGFbeta1, to fura-2AM-loaded neurons reversibly and acutely (within seconds) inhibited K(+)-evoked calcium influx. Moreover, using single-cell RT-PCR, we confirmed that the large TGFbeta2-responsive neurons presented a cholinergic phenotype. Investigation of the signaling mechanism underlying TGFbeta2 actions using whole-cell recordings of calcium currents revealed that TGFbeta2-mediated responses were insensitive to the nonhydrolyzable GTP analogue GTPgammaS. However, TGFbeta2-mediated calcium current reductions were prevented by intracellular perfusion of a Smad2/3 peptide antagonist. Together, these results suggest that TGFbeta2 can acutely regulate the excitability of basal forebrain cholinergic neurons through an atypical signaling mechanism.  相似文献   

2.
In the CNS, immune-like competent cells (microglia and astrocytes) were first described as potential sites of chemokine synthesis, but more recent evidence has indicated that neurones might also express chemokines and their receptors. The aim of the present work was to investigate further, both in vivo and in vitro, CC Chemokine Family Receptor 2 (CCR2) expression and functionality in rat spinal cord neurones. First, we demonstrated by RT-PCR and western blot analysis that CCR2 mRNA and protein were present in spinal extracts. Furthermore, we showed by immunolabelling that CCR2 was exclusively expressed by neurones in spinal sections of healthy rat. Finally, to test the functionality of CCR2, we used primary cultures of rat spinal neurones. In this model, similar to what was observed in vivo, CCR2 mRNA and protein were expressed by neurones. Cultured neurones stimulated with Monocyte Chemoattractant Protein-1 (MCP-1)/CCL2, the best characterized CCR2 agonist, showed activation of the Akt pathway. Finally, patch-clamp recording of cultured spinal neurones was used to investigate whether MCP-1/CCL2 could modulate their electrophysiological properties. MCP-1 alone did not affect the electrical properties of spinal neurones, but potently and efficiently inhibited GABA(A)-mediated GABAergic responses in these neurones. These data constitute the first demonstration of a modulatory role of MCP-1 on GABAergic neurotransmission and contribute to our understanding of the roles of CCR2 and MCP-1/CCL2 in spinal cord physiology, in particular with respect to nociceptive transmission, as well as the implication of this chemokine in neuronal adaptation or dysfunction during neuropathy.  相似文献   

3.
Mutations in amyloid precursor protein (APP), and presenilin‐1 and presenilin‐2 (PS1 and PS2) have causally been implicated in Familial Alzheimer’s Disease (FAD), but the mechanistic link between the mutations and the early onset of neurodegeneration is still debated. Although no consensus has yet been reached, most data suggest that both FAD‐linked PS mutants and endogenous PSs are involved in cellular Ca2+ homeostasis. We here investigated subcellular Ca2+ handling in primary neuronal cultures and acute brain slices from wild type and transgenic mice carrying the FAD‐linked PS2‐N141I mutation, either alone or in the presence of the APP Swedish mutation. Compared with wild type, both types of transgenic neurons show a similar reduction in endoplasmic reticulum (ER) Ca2+ content and decreased response to metabotropic agonists, albeit increased Ca2+ release induced by caffeine. In both transgenic neurons, we also observed a higher ER–mitochondria juxtaposition that favors increased mitochondrial Ca2+ uptake upon ER Ca2+ release. A model is described that integrates into a unifying hypothesis the contradictory effects on Ca2+ homeostasis of different PS mutations and points to the relevance of these findings in neurodegeneration and aging.  相似文献   

4.
Disruption of neuronal Ca(2+) homeostasis plays a well-established role in cell death in a number of neurodegenerative disorders. Recent evidence suggests that proteolysis of the type 1 inositol 1,4,5-trisphosphate receptor (InsP(3) R1), a Ca(2+) release channel on the endoplasmic reticulum, generates a dysregulated channel, which may contribute to aberrant Ca(2+) signaling and neurodegeneration in disease states. However, the specific effects of InsP(3) R1 proteolysis on neuronal Ca(2+) homeostasis are unknown, as are the functional contributions of this pathway to neuronal death. This study evaluates the consequences of calpain-mediated InsP(3) R1 proteolysis on neuronal Ca(2+) signaling and survival using adeno-associated viruses to express a recombinant cleaved form of the channel (capn-InsP(3) R1) in rat primary cortical neurons. Here, we demonstrate that expression of capn-InsP(3) R1 in cortical cultures reduced cellular viability. This effect was associated with increased resting cytoplasmic Ca(2+) concentration ([Ca(2+) ](i) ), increased [Ca(2+) ](i) response to glutamate, and enhanced sensitivity to excitotoxic stimuli. Together, our results demonstrate that InsP(3) R1 proteolysis disrupts neuronal Ca(2+) homeostasis, and potentially acts as a feed-forward pathway to initiate or execute neuronal death.  相似文献   

5.
Topiramate (TPM) is a structurally novel broad spectrum anticonvulsant known to have a negative modulatory effect on the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate subtypes of glutamate receptors and some types of voltage-gated Na(+) and Ca(2+) channels, and a positive modulatory effect on some types of gamma-aminobutyric acid(A) (GABA(A)) receptors and at least one type of K(+) channels in neurons. In an earlier work, we showed that the negative modulatory effect of TPM (100 mum) on AMPA/kainate receptors in neurons is dependent on TPM modulation of the phosphorylation state of these receptors. In this work, we investigated the effect of TPM on AMPA-induced intracellular calcium ([Ca(2+)](i)) responses in cultured rat cortical astrocytes, with special interest in intracellular mechanisms. Here, we report that the ability of TPM (1-100 mum) to inhibit AMPA-induced accumulation of Ca(2+) in astrocytes is inversely related to the level of protein kinase A (PKA) -mediated phosphorylation of channels activated by AMPA. The level of receptor phosphorylation was further determined with western blot using phosphorylation specific antibodies that recognize the glutamate receptor 1 (GluR1) subunit phosphorylated on Ser845. These results demonstrated that, even in cultured cortical astrocytes, TPM significantly reduced the phophorylation level of GluR1 subunits. Furthermore, it was shown that TPM binds to AMPA receptors in the dephosphorylated state and thereby exerts an allosteric modulatory effect on the ion channel.  相似文献   

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The neurosteroids pregnenolone (PE) and pregnenolone-sulfate (PS) have been shown to interact with the GABAA receptor in the central nervous system. In contrast, nothing is known of any possible modulation of voltage-gated calcium channels (VGCC). We have examined the interaction of PE, PS and progesterone on VGCC in acutely isolated adult guinea-pig hippocampal CA1 neurons using the whole-cell patch clamp technique. PE and PS depressed the calcium current at low micromolar concentrations (0.001-100 microM). The time to peak of the calcium current was slowed by PE and PS. The blocking action of PE and PS occurs in the presence of 10 microM picrotoxin. In contrast, progesterone had no effect on the Ca2+ current, indicating specificity for PE and PS. These results demonstrate a direct and novel membrane site of action for PE and PS, suggesting a possible role influencing brain excitability.  相似文献   

8.
Neu2000 [NEU, 2-hydroxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethyl-benzylamino)-benzoic acid], a derivative of sulfasalazine, attenuates NMDA-induced neuronal toxicity. Here we investigated the effects of NEU on the NMDA receptor (NMDAR) using whole-cell patch clamp technique to determine the molecular mechanisms underlying its neuroprotective role. NEU reversibly suppressed NMDA responses in an uncompetitive manner with fast binding kinetics. Its inhibition of NMDAR activity depended on both the concentration and the use of agonist but not on the membrane potential. NEU accelerated NMDA desensitization without affecting the binding affinity of NMDAR for its agonists and stabilized the closed state of NMDAR. Therefore, NEU should effectively alleviate disorders that are a result of glutamate excitoxicity with fewer side effects because it is a low-affinity gating modifier that antagonizes NMDAR in an uncompetitive manner. Moreover, in the presence of ifenprodil (an NR2B antagonist) but not NVP-AAM077 [( R )-[( S )-1-(4-bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydro-quinoxalin-5-yl)-methyl]-phosphonic acid, an NR2A antagonist], the extent of NEU block was decreased, suggesting that NEU is an NR2B-specific antagonist.  相似文献   

9.
We investigated the effect of treatment with an angiotensin II receptor blocker, candesartan-cilexetil, on the mechanical and electrophysiological properties of cardiomyocytes isolated from streptozotocin-induced diabetic (STZ) rats. Contractile activity and electrophysiological properties were measured in papillary muscle and ventricular cardiomyocytes from normoglycemic and STZ-induced diabetic rats given vehicle or 5mg/kg/day candesartan-cilexetil for 4 weeks. Alterations in the kinetics of contractile activity and intracellular Ca(2+) transients were observed as well as a typical prolongation of action potential duration and significant decrease of potassium currents in diabetic rat heart preparations. Candesartan-cilexetil treatment recovered significantly prolonged action potential and depressed potassium currents in diabetic rats. It was also shown that treatment with AT(1) blocker restored altered kinetics of both the Ca(2+) transients in cardiomyocytes and the contractile activity in papillary muscle strips of diabetic rats. We also showed that incubation of cardiomyocytes from diabetic rats with a protein kinase C (PKC) inhibitor bisindolylmaleimide I (BIM) had a similar effect to candesartan treatment on the Ca(2+) transients. Thus, angiotensin II receptor blockade protects the heart from the development of cellular alterations typically related with diabetes, and this action of AT(1) receptors seems to be related with the activity of PKC.  相似文献   

10.
Beta-amyloid protein is thought to underlie the neurodegeneration associated with Alzheimer's disease by inducing Ca(2+)-dependent apoptosis. Elevated neuronal expression of the proinflammatory cytokine interleukin-1beta is an additional feature of neurodegeneration, and in this study we demonstrate that interleukin-1beta modulates the effects of beta-amyloid on Ca(2+) homeostasis in the rat cortex. beta-Amyloid-(1-40) (1 microM) caused a significant increase in (45)Ca(2+) influx into rat cortical synaptosomes via activation of L- and N-type voltage-dependent Ca(2+) channels and also increased the amplitude of N- and P-type Ca(2+) channel currents recorded from cultured cortical neurons. In contrast, interleukin-1beta (5 ng/ml) reduced the (45)Ca(2+) influx into cortical synaptosomes and inhibited Ca(2+) channel activity in cultured cortical neurons. Furthermore, the stimulatory effects of beta-amyloid protein on Ca(2+) influx were blocked following exposure to interleukin-1beta, suggesting that interleukin-1beta may govern neuronal responses to beta-amyloid by regulating Ca(2+) homeostasis.  相似文献   

11.
Amplitude, spatial width, and rise time of Ca(2+) sparks were compared in frog fast-twitch muscle, in three conditions that alter activation of release channels by [Ca(2+)]. A total of approximately 17,000 sparks from 30 cells were evaluated. In cells under voltage clamp, caffeine (0.5 or 1 mM) increased average spark width by 28%, rise time by 18%, and amplitude by 7%. Increases in width were significant even among events of the same rise time. Spontaneous events recorded in permeabilized fibers with low internal [Mg(2+)] (0.4 mM), had width and rise times greater than in reference, and not significantly different than those in caffeine. The spark average in reference rides on a continuous fluorescence "ridge" and is continued by an "ember," a prolongation of width approximately 1 microm and amplitude <0.2, vanishing in approximately 100 ms. Ridge and ember were absent in caffeine and in permeabilized cells. Exposure of voltage-clamped cells to high internal [Mg(2+)] (7 mM) had effects opposite to caffeine, reducing spark width by 26% and amplitude by 27%. In high [Mg(2+)], the ember was visible in individual sparks as a prolongation of variable duration and amplitude up to 1.2. Based on simulations and calculation of Ca(2+) release flux from averaged sparks, the increase in spark width caused by caffeine was interpreted as evidence of an increase in radius of the release source-presumably by recruitment of additional channels. Conversely, spark narrowing suggests loss of contributing channels in high Mg(2+). Therefore, these changes in spark width at constant rise times are evidence of a multichannel origin of sparks. Because ridge and ember were reduced by promoters of Ca(2+)-dependent activation (caffeine, low [Mg(2+)]) and became more visible in the presence of its inhibitors, they are probably manifestations of Ca(2+) release directly operated by voltage sensors.  相似文献   

12.
We have investigated the requirement for Ca(2+) in the fusion and content mixing of rat hepatocyte late endosomes and lysosomes in a cell-free system. Fusion to form hybrid organelles was inhibited by 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA), but not by EGTA, and this inhibition was reversed by adding additional Ca(2+). Fusion was also inhibited by methyl ester of EGTA (EGTA-AM), a membrane permeable, hydrolyzable ester of EGTA, and pretreatment of organelles with EGTA-AM showed that the chelation of lumenal Ca(2+) reduced the amount of fusion. The requirement for Ca(2+) for fusion was a later event than the requirement for a rab protein since the system became resistant to inhibition by GDP dissociation inhibitor at earlier times than it became resistant to BAPTA. We have developed a cell-free assay to study the reformation of lysosomes from late endosome-lysosome hybrid organelles that were isolated from the rat liver. The recovery of electron dense lysosomes was shown to require ATP and was inhibited by bafilomycin and EGTA-AM. The data support a model in which endocytosed Ca(2+) plays a role in the fusion of late endosomes and lysosomes, the reformation of lysosomes, and the dynamic equilibrium of organelles in the late endocytic pathway.  相似文献   

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15.
Membrane Ca(2+)-ATPase activity was stimulated in vitro separately by T4 (10(-10) M) and by epinephrine (10(-6) M). In the presence of a fixed concentration of T4, additions of 10(-8) and 10(-6) M epinephrine reduced the T4 effect on the enzyme. beta-Adrenergic blockade with propranolol (10(-6) M) prevented stimulation by epinephrine of Ca(2+)-ATPase activity, but did not prevent the suppressive action of epinephrine on T4-stimulable Ca(2+)-ATPase. In contrast, alpha 1-adrenergic blockade with unlabelled prazosin restored the effect of T4 on Ca(2+)-ATPase activity in the presence of epinephrine. Like propranolol, prazosin prevented enhancement of enzyme activity by epinephrine in the absence of thyroid hormone. Neither prazosin nor propranolol had any effect on the stimulation by T4 of red cell Ca(2+)-ATPase in the absence of epinephrine. Analysis of radiolabelled prazosin binding to human red cell membranes revealed the presence of a single class of high-affinity binding sites (Kd, 1.2 x 10(-8) M; Bmax, 847 fmol/mg membrane protein). Thus, the human erythrocyte membrane contains alpha 1-adrenergic receptor sites that are capable of regulating Ca(2+)-ATPase activity.  相似文献   

16.
Recently, we have isolated a cDNA encoding a muscarinic acetylcholine receptor (mAChR) from Caenorhabditis elegans. To investigate the regulation of phospholipase D (PLD) signaling via a muscarinic receptor, we generated stable transfected Chinese hamster ovary (CHO) cells that overexpress the mAChR of C. elegans (CHO-GAR-3). Carbachol (CCh) induced inositol phosphate formation and a significantly higher Ca(2+) elevation and stimulated PLD activity through the mAChR; this was insensitive to pertussis toxin, but its activity was abolished by the phospholipase C (PLC) inhibitor U73122. Western blot analysis revealed several apparent tyrosine-phosphorylated protein bands after CCh treatment. The CCh-induced PLD activation and tyrosine phosphorylation were significantly reduced by the protein kinase C (PKC) inhibitor calphostin C and down-regulation of PKC and the tyrosine kinase inhibitor genistein. Moreover, the Ca(2+)-calmodulin-dependent protein kinase II (CaM kinase II) inhibitor KN62, in addition to chelation of extracellular or intracellular Ca(2+) by EGTA and BAPTA/AM, abolished CCh-induced PLD activation and protein tyrosine phosphorylation. Taken together, these results suggest that the PLC/PKC-PLD pathway and the CaM kinase II/tyrosine kinase-PLD pathway are involved in the activation of PLD through mAChRs of C. elegans.  相似文献   

17.
Polycystin-1 (PC1) is a large transmembrane protein important in renal differentiation and defective in most cases of autosomal dominant polycystic kidney disease (ADPKD), a common cause of renal failure in adults. Although the genetic basis of ADPKD has been elucidated, molecular and cellular mechanisms responsible for the dysregulation of epithelial cell growth in ADPKD cysts are still not well defined. We approached this issue by investigating the role of the carboxyl cytoplasmic domain of PC1 involved in signal transduction on the control of kidney cell proliferation. Therefore, we generated human HEK293 cells stably expressing the PC1 cytoplasmic tail as a membrane targeted TrkA-PC1 chimeric receptor protein (TrkPC1). We found that TrkPC1 increased cell proliferation through an increase in cytoplasmic Ca2+ levels and activation of PKC alpha, thereby upregulating D1 and D3 cyclin, downregulating p21waf1 and p27kip1 cyclin inhibitors, and thus inducing cell cycle progression from G0/G1 to the S phase. Interestingly, TrkPC1-dependent Ca2+ increase and PKC alpha activation are not constitutive, but require serum factor(s) as parallel component. In agreement with this observation, a significant increase in ERK1/2 phosphorylation was observed. Consistently, inhibitors specifically blocking either PKC alpha or ERK1/2 prevented the TrkPC1-dependent proliferation increase. NGF, the TrkA ligand, blocked this increase. We propose that in kidney epithelial cells the overexpression of PC1 C-terminus upregulates serum-evoked intracellular Ca2+ by counteracting the growth-suppression activity of endogenous PC1 and leading to an increase in cell proliferation.  相似文献   

18.
We report a novel mechanism for dopamine D(1) receptor (D(1) R)-mediated extracellular signal-regulated kinases (Erk) activation in rat striatum. Erk signaling depends on phosphorylation and dephosphorylation events mediated by specific kinases and phosphatases. The tyrosine phosphatase Shp-2, that is required for Erk activation by tyrosine kinase receptors, has been recently shown to regulate signaling downstream of few G protein-coupled receptors. We show that the D(1) R interacts with Shp-2, that D(1) R stimulation results in Shp-2 tyrosine phosphorylation and activation in primary striatal neuronal cultures and that D(1) R/Shp-2 interaction is required for transmitting D(1) R-dependent signaling to Erk1/2 activation. D(1) R-mediated Erk1/2 phosphorylation in cultured striatal neurons is in fact abolished by over-expression of the inactive Shp-2(C/S) mutant and by small interfering RNA-induced Shp-2 silencing. Moreover, by using selective inhibitors we show that both D(1) R-induced Shp-2 activation and Erk1/2 phosphorylation are dependent on the cyclic AMP/protein kinase A pathway and require Src. These results, which were substantiated also in transfected human embryonic kidney 293 cells, provide a novel mechanism by which to converge D(1) R signaling to the Erk pathway and suggest that Shp-2 or the D(1) R/Shp-2 interface could represent a potential drug target for disorders of dopamine transmission involving malfunctioning of D(1) R signaling.  相似文献   

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