Increase In Intracellular Calcium Concentration Research Articles

Co-expression of endothelial and neuronal nitric oxide synthases in the developing vasculatures of the human fetal eye

Nitric oxide (NO) is a multifunctional gaseous molecule that regulates various physiological functions in both neuronal and non-neuronal cells. NO is synthesized by nitric oxide synthases (NOSs), of which three isoforms have been identified. Neuronal NOS (nNOS) and endothelial NOS (eNOS) constitutively produce low levels of NO as a cell-signaling molecule in response to an increase in intracellular calcium concentration. Recent data have revealed a predominant role of eNOS in both angiogenesis and vasculogenesis. The immunohistochemical localization of nNOS and eNOS was investigated during embryonic and fetal ocular vascular development from 7 to 21 weeks gestation (WG) on sections of cryopreserved tissue. eNOS was confined to endothelial cells of developing vessels at all ages studied. nNOS was prominent in nuclei of vascular endothelial and smooth muscle cells in the fetal vasculature of vitreous and choriocapillaris. nNOS was also prominent in the nuclei of CXCR4(+) progenitors in the inner retina and inner neuroblastic layer. These findings demonstrate co-expression of n- and eNOS isoforms in different compartments of vasoformative cells during development. Nuclear nNOS was present in vascular and nonvascular progenitors as well as endothelial cells and pericytes. This suggests that nNOS may play a role in the transcription regulatory systems in endothelial cells and pericytes during ocular hemo-vasculogenesis, vasculogenesis, and angiogenesis.

Regulator of calcineurin 1 (Rcan1) has a protective role in brain ischemia/reperfusion injury

BackgroundAn increase in intracellular calcium concentration [Ca2+]i is one of the first events to take place after brain ischemia. A key [Ca2+]i-regulated signaling molecule is the phosphatase calcineurin (CN), which plays important roles in the modulation of inflammatory cascades. Here, we have analyzed the role of endogenous regulator of CN 1 (Rcan1) in response to experimental ischemic stroke induced by middle cerebral artery occlusion.MethodsAnimals were subjected to focal cerebral ischemia with reperfusion. To assess the role of Rcan1 after stroke, we measured infarct volume after 48 h of reperfusion in Rcan1 knockout (KO) and wild-type (WT) mice. In vitro studies were performed in astrocyte-enriched cortical primary cultures subjected to 3% oxygen (hypoxia) and glucose deprivation (HGD). Adenoviral vectors were used to analyze the effect of overexpression of Rcan1-4 protein. Protein expression was examined by immunohistochemistry and immunoblotting and expression of mRNA by quantitative real-time Reverse-Transcription Polymerase Chain Reaction (real time qRT-PCR).ResultsBrain ischemia/reperfusion (I/R) injury in vivo increased mRNA and protein expression of the calcium-inducible Rcan1 isoform (Rcan1-4). I/R-inducible expression of Rcan1 protein occurred mainly in astroglial cells, and in an in vitro model of ischemia, HGD treatment of primary murine astrocyte cultures induced Rcan1-4 mRNA and protein expression. Exogenous Rcan1-4 overexpression inhibited production of the inflammatory marker cyclo-oxygenase 2. Mice lacking Rcan1 had higher expression of inflammation associated genes, resulting in larger infarct volumes.ConclusionsOur results support a protective role for Rcan1 during the inflammatory response to stroke, and underline the importance of the glial compartment in the inflammatory reaction that takes place after ischemia. Improved understanding of non-neuronal mechanisms in ischemic injury promises novel approaches to the treatment of acute ischemic stroke.

Enteroaggregative Escherichia coli induced increase in intracellular calcium concentration modulates cytoskeletal F-actin rearrangement and bacterial entry in INT-407 cells

BackgroundEnteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen, associated with cases of acute and persistent diarrhoea worldwide. The pathogenesis of EAEC is yet to be understood. In intestinal epithelium, an increase in [Ca2+]i has been attributed due to the action of different enteric pathogens. EAEC was shown to increase [Ca2+]i in HEp-2 cells.The present study was undertaken to investigate the effect of EAEC induced increase in [Ca2+]i oncultured human intestinal epithelial cells. MethodsINT-407 cells were infected with EAEC (T8 strain) in the absence and presence of dantrolene (inhibitor of release of Ca2+ from intracellular stores)/verapamil (L-type Ca2+ channel blocker)/BAPTA-AM (Ca2+ chelator)/U73122 (PLC inhibitor)/Cytochalasin-D (inhibitor of actin polymerization). [Ca2+]i was estimated using Fura-2/AM. Cytoskeletal rearrangement was assessed by F-actin staining using TRITC-phalloidin. The invasiveness of EAEC-T8 to INT-407 cells was checked by electron microscopy and invasion assay. ResultsA significant increase in [Ca2+]i was observed in EAEC-T8 infected INT-407 cells, which was reduced in presence of dantrolene/verapamil/U73122. EAEC-T8 could induce cytoskeletal F-actin polymerization in INT-407 cells and was found to be invasive in nature. The cytoskeletal rearrangement as well as invasion of EAEC-T8 was attenuated in presence of U73122/dantrolene/BAPTA-AM/verapamil/cytochalasin D. ConclusionsEAEC induced increase in [Ca2+]i seems to play a major role in host cytoskeletal F-actin rearrangements leading to invasion of the organism. General significanceOur study undoubtedly will lead to an improved understanding of EAEC-pathogenesis.

Induction of Intracellular Calcium Concentration by Environmental Benzo(a)pyrene Involves a β2-Adrenergic Receptor/Adenylyl Cyclase/Epac-1/Inositol 1,4,5-Trisphosphate Pathway in Endothelial Cells

Polycyclic aromatic hydrocarbons (PAHs) such as benzo(a)pyrene (B(a)P) are widely distributed environmental contaminants, known as potent ligands of the aryl hydrocarbon receptor (AhR). These chemicals trigger an early and transient increase of intracellular calcium concentration ([Ca(2+)](i)), required for AhR-related effects of PAHs. The mechanisms involved in this calcium mobilization were investigated in the present study. We demonstrated that B(a)P-mediated [Ca(2+)](i) induction was prevented in endothelial HMEC-1 cells by counteracting β2-adrenoreceptor (β2ADR) activity using pharmacological antagonists, anti-β2ADR antibodies, or siRNA-mediated knockdown of β2ADR expression; by contrast, it was strongly potentiated by β2ADR overexpression in human kidney HEK293 cells. B(a)P was shown, moreover, to directly bind to β2ADR, as assessed by in vitro binding assays and molecular modeling. Pharmacological inhibition and/or siRNA-mediated silencing of various signaling actors acting downstream of β2ADR in a sequential manner, such as G protein, adenylyl cyclase, Epac-1 protein, and inositol 1,4,5-trisphosphate (IP(3))/IP(3) receptor, were next demonstrated to prevent B(a)P-induced calcium signal. Inhibition or knockdown of these signaling elements, as well as the use of chemical β-blockers, were finally shown to counteract B(a)P-mediated induction of cytochrome P-450 1B1, a prototypical AhR target gene. Taken together, our results show that B(a)P binds directly to β2ADR and consequently utilizes β2ADR machinery to mobilize [Ca(2+)](i), through activation of a G protein/adenylyl cyclase/cAMP/Epac-1/IP(3) pathway. This β2ADR-dependent signaling pathway activated by PAHs may likely be crucial for PAH-mediated up-regulation of AhR target genes, thus suggesting a contribution of β2ADR to the health-threatening effects of these environmental pollutants.

Functional interaction between AQP2 and TRPV4 in renal cells

We have previously demonstrated that renal cortical collecting duct cells (RCCD(1)), responded to hypotonic stress with a rapid activation of regulatory volume decrease (RVD) mechanisms. This process requires the presence of the water channel AQP2 and calcium influx, opening the question about the molecular identity of this calcium entry path. Since the calcium permeable nonselective cation channel TRPV4 plays a crucial role in the response to mechanical and osmotic perturbations in a wide range of cell types, the aim of this work was to test the hypothesis that the increase in intracellular calcium concentration and the subsequent rapid RVD, only observed in the presence of AQP2, could be due to a specific activation of TRPV4. We evaluated the expression and function of TRPV4 channels and their contribution to RVD in WT-RCCD(1) (not expressing aquaporins) and in AQP2-RCCD(1) (transfected with AQP2) cells. Our results demonstrated that both cell lines endogenously express functional TRPV4, however, a large activation of the channel by hypotonicity only occurs in cells that express AQP2. Blocking of TRPV4 by ruthenium red abolished calcium influx as well as RVD, identifying TRPV4 as a necessary component in volume regulation. Even more, this process is dependent on the translocation of TRPV4 to the plasma membrane. Our data provide evidence of a novel association between TRPV4 and AQP2 that is involved in the activation of TRPV4 by hypotonicity and regulation of cellular response to the osmotic stress, suggesting that both proteins are assembled in a signaling complex that responds to anisosmotic conditions.

Intracellular Dynamics of HIV-Gag: The Role of Calcium and the Activation of Phospholipase C

The assembly of the human immunodeficiency virus type 1 occurs at the plasma membrane or within late endosomes/multivesicular bodies, and is determined by the polyprotein Gag. This structural protein is also the only viral component necessary and sufficient for the assembly and the release of virus-like particles (VLPs). It is well known that participation of host cell components is particularly required for any of the many Gag-encoded functions. In particular, as already shown by L.S. Ehrlich et al. 2010, the activation of the phospholipase C and the inositol-(1,4,5)-triphosphate receptor, resulting in an increase of intracellular calcium concentration, are both required for efficient Gag trafficking and VLPs release.So far, it is still unclear whether the Gag protein itself or a cellular factor specifically activates this signaling pathway for the virus release process.We are interested to investigate the activation and the role of the PLC signaling pathway in Gag-expressing cells, and how the intracellular calcium concentration can influence the dynamics of Gag and the VLPs release-process.HeLa cells transfected with Gag fused with a fluorescent protein (e.g. EGFP, EYFP) are our principal tool to investigate the intracellular localization of the viral protein upon activation or inhibition of the PLC signaling pathway. Acetoxymethylester-derivates of fluorescent indicators are used to study variations of the calcium concentration in live cells. Chemical inhibitors and RNAi technology are utilized to turn off the PLC pathway at different steps and time points. Total Internal Reflection Fluorescence microscopy, Forster Resonance Energy Transfer methods and co-immunoprecipitation are applied to study the localization of Gag at the plasma membrane or in the cell-interior, and to identify interactions with specific binding partners of the host cell.

Endocannabinoid-binding CB1 and TRPV1 receptors as modulators of sperm capacitation

Mammalian spermatozoa reach the ability to fertilize only after they complete a complex series of physical-chemical modification, the capacitation. Recently, the endocannabinoid-binding type-1cannabinoid receptor (CB1) and transient receptor potential vanilloid 1 (TRPV1) channel have been proposed to play a key role in the control of capacitation. In particular CB1, acting via a Gi protein/cAMP/PKA pathway, maintains low cAMP levels in early stages of post ejaculatory life of male gametes. By this way it promotes the maintenance of membrane stability, thus avoiding the premature fusion of plasma membrane (PM) and outer acrosome membrane (OAM), which is mandatory for the exocytosis of acrosome content. TRPV1, on the contrary, becomes active during the latest stages of capacitation, and allows the rapid increase in intracellular calcium concentration that leads to the removal of the F-actin network interposed between PM and OAM, leading to their fusion and, ultimately, to the acrosome reaction.

Toll-like receptor 3 activation promotes desensitization of histamine response in human gingival fibroblasts: Poly (I:C) induces histamine receptor desensitization in human gingival fibroblasts

Viruses are associated with the development of periodontal disease, particularly during periods of suppressed cellular immunity. For this reason, we evaluated the hypothesis that viral components regulate the actions of histamine, an important mediator of immune responses. We assessed the effect of Poly (I:C) on histamine-mediated intracellular calcium mobilization in human gingival fibroblasts. Our results show that histamine induces an increase in intracellular calcium concentrations in a dose-dependent manner. This response was blocked when cells were incubated in the presence of Poly (I:C). In addition, phorbol esters, a diacylglycerol analog, mimics the inhibitory actions of Poly (I:C) in response to histamine. The effect of Poly (I:C) was reversed by Stuarosporine (1μM), GÖ6983 (7μM), Bisindolylmaleimide (1μM) [a protein inhibitor (PKC)], and SB 203580 (3μM) (a p38-MAPK inhibitor). These findings suggest that Poly (I:C) regulates histamine-induced calcium mobilization through activation of PKC and p38.

Calcium-activated chloride current expression in axotomized sensory neurons: what for?

Calcium-activated chloride currents (CaCCs) are activated by an increase in intracellular calcium concentration. Peripheral nerve injury induces the expression of CaCCs in a subset of adult sensory neurons in primary culture including mechano- and proprioceptors, though not nociceptors. Functional screenings of potential candidate genes established that Best1 is a molecular determinant for CaCC expression among axotomized sensory neurons, while Tmem16a is acutely activated by inflammatory mediators in nociceptors. In nociceptors, such CaCCs are preferentially activated under receptor-induced calcium mobilization contributing to cell excitability and pain. In axotomized mechano- and proprioceptors, CaCC activation does not promote electrical activity and prevents firing, a finding consistent with electrical silencing for growth competence of adult sensory neurons. In favor of a role in the process of neurite growth, CaCC expression is temporally correlated to neurons displaying a regenerative mode of growth. This perspective focuses on the molecular identity and role of CaCC in axotomized sensory neurons and the future directions to decipher the cellular mechanisms regulating CaCC during neurite (re)growth.

Comparison of Cytotoxicity of Pristine and Covalently Functionalized Multi-Walled Carbon Nanotubes in RAW 264.7 Macrophages

Carbon nanotubes may be applied in different fields including biomedicine and mechanical engineering. It is important to understand the potential hazards of carbon nanotubes. In the present study, the toxicological effects of the pristine multi-walled carbon nanotubes (p-MWCNTs) and taurine functionalized multi-walled carbon nanotubes (tau-MWCNTs) were assessed on RAW 264.7 macrophages. We tested cell viability, GSH/GSSG ratio, apoptosis, intracellular calcium concentration, ultrastructural changes of cell morphology, and the release of IL-8. We observed the loss of cell viability, decline in the cellular GSH/GSSG ratio, increase of IL-8, and the increase of intracellular calcium concentration in RAW 264.7 macrophages when exposed to p-MWCNTs at high dosage. Additionally, exposure to p-MWCNTs resulted in ultrastructural and morphological changes in RAW 264.7 macrophages. In contrast, the RAW 264.7 macrophages exposed to the tau-MWCNTs did not exhibit altered morphology. Our results conclude that the tau-MWCNTs show lower toxicity than that of p-MWCNTs.

Calcium signaling as a regulator of hematopoiesis

Different extracellular signaling molecules that bind to receptors on the cell membrane use calcium ions for signal transduction. Due to the opening of receptor-operated calcium channels, some cytokine receptors and G-protein coupled receptors induce an increase of intracellular calcium concentration upon activation. Calcium ion is a versatile intracellular secondary messenger that control many different cellular functions by changing its cytoplasmic concentration. A specific and complex network of signaling proteins recognizes intracellular calcium alterations to modulate cellular processes. Some reports have previously demonstrated that calcium also regulates hematopoiesis. This review examines the participation of intracellular calcium in hematopoiesis after the stimulus of various myeloid cytokines such as interleukin-3 and granulocyte-macrophage colony-stimulating factor. In addition, the role of adenosine triphosphate and its receptors in inducing calcium increases during hematopoiesis is discussed. Lastly, the participation of this ion in myeloid proliferation and differentiation by cytokines and P2 receptors is also discussed.

Up-Regulation of the Gap Junction Intercellular Communication by Tea Polyphenol in the Human Metastatie Lung Carcinoma Cell Line

Our previous study has proven that tea polyphenol has a role in lung neoplasms. The present communication was to investage the anti-proliferation effect of tea polyphenol on the PG cells, which was a high metastatic human lung carcinoma cell line, by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide (MTT) cell viability assay, and to study the change of intracellular calcium concentration, connexin43 (Cx43) expression, gap junctional intercellular communication (GJIC) and cell cycle distribution after the tea polyphenol treatment by laser scanning confocal microscopy and flow cytometry. The results showed that 1) tea polyphenol could kill the PG cells in a dose-depent manner via inhibiting the PG cell proliferation and blocking the PG cell cycle progression staying in G0/G1 phase and not transfering in S and G2/M phases to reduce the PG cell proliferation index; 2) the increases of intracellular calcium concentration, GJIC and Cx43 expression were related with the tea polyphenol doses. The data suggested that tea polyphenol could inhibit the growth of PG cells, which mechanism was associated with the up-regulation of GJIC.

Identification of the First Mutations in the Human Triadin Gene, Associated to Catecholaminergic Tachycardia, a Pathology of the Cardiac Calcium Release Complex

Cardiac contraction is triggered when a membrane depolarisation induces a massive increase in intracellular calcium concentration. This process called “excitation-contraction (E-C) coupling” relies on a multimolecular protein complex, the calcium release complex (CRC) organized around the sarcoplasmic reticulum calcium channel, the ryanodine receptor (RyR2). Among the proteins involved in the efficient function of the CRC, calsequestrin, triadin and junctin are sarcoplasmic reticulum proteins able to interact with RyR2 and regulate calcium release.Mutations in RyR2 and calsequestrin are associated to a rare but fatal cardiac arrhythmia: catecholaminergic polymorphic ventricular tachycardia (CPVT). Nevertheless, variations in these two genes (RYR2 and CASQ2) account so far for only 50 to 70% of the cases, suggesting that other genes are most probably involved. To reveal new genes involved in CPVT, we have based a candidate gene approach on the hypothesis that the pathology could be considered as a disease of the calcium release complex. We therefore searched for variations in the genes encoding proteins of the CRC in a large French cohort of CPVT patients with no detected mutations in RYR2 or CASQ2. We have identified for the first time mutations in the human triadin gene TRDN, and studied the functional consequences of a missense mutation both in a cell model and in vivo after expression in triadin KO mice. Our results confirmed the hypothesis that CPVT can be more generally considered as a defect in the CRC.

Endothelin receptor antagonist activity of (R)-(-)-2-(benzo[1,3]dioxol-5-yl)-N-(4-isopropylphenylsulfonyl)-2-(6-methyl- 2-propylpyridin-3-yloxy)acetamide hydrochloride (PABSA) in rat aortic smooth muscle cells and isolated rat thoracic aorta.

Antagonist activities of (R)-(-)-2-(benzo[1,3]dioxol-5-yl)-N-(4-isopropylphenylsulfonyl)-2-(6-methyl- 2-propylpyridin-3-yloxy) acetamide hydrochloride (CAS 188710-94-3, PABSA), a novel endothelin (ET) receptor antagonist, for ETA and ETB receptors were evaluated using rat aortic smooth muscle A7r5 cells and isolated rat thoracic aorta. PABSA concentration-dependently inhibited the ET-1-induced increase in intracellular calcium concentration ([Ca2+]i) mediated via ETA receptors in A7r5 cells with an IC50 of 0.17 nmol/l. PABSA antagonized the ETA receptor-mediated contraction induced by ET-1 in endothelium-denuded rat aorta with a Kb of 0.74 nmol/l. The potency of PABSA in inhibiting ETA receptor-mediated vasocontraction was approximately 40- and 100-fold greater than those of BQ-123, a selective ETA antagonist, and bosentan, a mixed ETA/ETB receptor antagonist, respectively. ETB receptor-mediated endothelium-dependent vasorelaxation induced by ET-3 in the aorta was also antagonized by PABSA, with a Kb of 9.8 nmol/l. In contrast, PABSA affected neither the vasocontraction induced by KCl or norepinephrine nor the vasorelaxation induced by acetylcholine or prostaglandin I2 in the aorta. These results suggest that PABSA is a highly potent and selective ETA receptor antagonist.

Calcium Signaling in Live Cells on Elastic Gels under Mechanical Vibration at Subcellular Levels

A new device was designed to generate a localized mechanical vibration of flexible gels where human umbilical vein endothelial cells (HUVECs) were cultured to mechanically stimulate these cells at subcellular locations. A Fluorescence Resonance Energy Transfer (FRET)-based calcium biosensor (an improved Cameleon) was used to monitor the spatiotemporal distribution of intracellular calcium concentrations in the cells upon this mechanical stimulation. A clear increase in intracellular calcium concentrations over the whole cell body (global) can be observed in the majority of cells under mechanical stimulation. The chelation of extracellular calcium with EGTA or the blockage of stretch-activated calcium channels on the plasma membrane with streptomycin or gadolinium chloride significantly inhibited the calcium responses upon mechanical stimulation. Thapsigargin, an endoplasmic reticulum (ER) calcium pump inhibitor, or U73122, a phospholipase C (PLC) inhibitor, resulted in mainly local calcium responses occurring at regions close to the stimulation site. The disruption of actin filaments with cytochalasin D or inhibition of actomyosin contractility with ML-7 also inhibited the global calcium responses. Therefore, the global calcium response in HUVEC depends on the influx of calcium through membrane stretch-activated channels, followed by the release of inositol trisphosphate (IP3) via PLC activation to trigger the ER calcium release. Our newly developed mechanical stimulation device can also provide a powerful tool for the study of molecular mechanism by which cells perceive the mechanical cues at subcellular levels.

AMPA receptor potentially participates in the mediation of the increased brain-derived neurotrophic factor following chronic ketamine use

We read with great interest the recently published article “Chronic ketamine use increases serum levels of brain-derived neurotrophic factor” by Ricci et al. (2011). In this study, the authors considered that the mechanism underlying the increased serum levels of BDNF in chronic ketamine use are attributed to the antagonistic action of N-methyl-D-aspartate (NMDA) receptor. However, the intracellular glutamate release increased by ketamine administration preferentially favors α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor over NMDA receptor because the latter is occupied by ketamine (Machado-Vieira et al. 2009). Moreover, several studies have demonstrated that AMPA receptor mediates the increase of BDNF via two distinct signaling pathways. The first pathway involves the increases in intracellular calcium concentration, which subsequently leads to the upregulation of BDNF gene expression through calcium response elements (Tao et al. 1998). The second pathway depends on the activation of Lyn, a member of the src family of protein tyrosine kinases, which upregulates the mitogen-activated protein kinase pathway and then leads to the increases of BDNF expression (Hayashi et al. 1999).

Protective role of antidiabetic drug metformin against gentamicin induced apoptosis in auditory cell line

Besides their prominent function in cellular energy metabolism, the central role of mitochondria has been focused on control of cellular death in last decades. The mitochondrial permeability transition pore (PTP) is involved in the intrinsic pathway of apoptosis via the release of cytochrome c into cytosol. Metformin, a drug widely used in the treatment of type II diabetes, has recently received attention owing to new findings regarding its effect on apoptosis through mitochondrial permeability transition and cytochrome c release. The modulation of PTP is still unknown, but calcium is certainly the most important known inducer. In the present study, the preventive effects of metformin on gentamicin ototoxicity were investigated through the changes of intracellular calcium concentrations using calcium imaging in HEI-OC1 cells. Calcium imaging traced the changes of intracellular calcium concentration after the application of 50 mM of gentamicin in both 100 uM of metformin pretreated group and non-pretreated group. These calcium reactions were compared and analyzed with the results of cell viability test, Hoechst staining, intracellular reactive oxygen species level and expression of caspase-3, and poly-ADP-ribose polymerase (PARP). Continuous increase of intracellular calcium concentration (increase of 380/340 ratio) occurred after application of 50 mM of gentamicin. However, there was no change of intracellular calcium concentration in 100 uM metformin pretreated group. Cell viability was significantly higher in 100 uM metformin pretreated group and also, metformin pretreated HEI-OC1 cells produced less ROS that gentamicin alone treated group. Gentamicin increased cleaved PARP and caspase-3, but metformin inhibited the expression of caspase-3 and cleavage of PARP. This study demonstrated that metformin prevented gentamicin induced apoptosis through the calcium modulating and ROS reducing anti-apoptotic effects.

Cyclic nucleotide-dependent relaxation pathways in vascular smooth muscle.

Vascular smooth muscle tone is controlled by a balance between the cellular signaling pathways that mediate the generation of force (vasoconstriction) and release of force (vasodilation). The initiation of force is associated with increases in intracellular calcium concentrations, activation of myosin light-chain kinase, increases in the phosphorylation of the regulatory myosin light chains, and actin-myosin crossbridge cycling. There are, however, several signaling pathways modulating Ca(2+) mobilization and Ca(2+) sensitivity of the contractile machinery that secondarily regulate the contractile response of vascular smooth muscle to receptor agonists. Among these regulatory mechanisms involved in the physiological regulation of vascular tone are the cyclic nucleotides (cAMP and cGMP), which are considered the main messengers that mediate vasodilation under physiological conditions. At least four distinct mechanisms are currently thought to be involved in the vasodilator effect of cyclic nucleotides and their dependent protein kinases: (1) the decrease in cytosolic calcium concentration ([Ca(2+)]c), (2) the hyperpolarization of the smooth muscle cell membrane potential, (3) the reduction in the sensitivity of the contractile machinery by decreasing the [Ca(2+)]c sensitivity of myosin light-chain phosphorylation, and (4) the reduction in the sensitivity of the contractile machinery by uncoupling contraction from myosin light-chain phosphorylation. This review focuses on each of these mechanisms involved in cyclic nucleotide-dependent relaxation of vascular smooth muscle under physiological conditions.

Purinergic receptor antagonists inhibit odorant-mediated CREB phosphorylation in sustentacular cells of mouse olfactory epithelium

BackgroundExtracellular nucleotides have long been known to play neuromodulatory roles and to be involved in intercellular signalling. In the olfactory system, ATP is released by olfactory neurons, and exogenous ATP can evoke an increase in intracellular calcium concentration in sustentacular cells, the nonneuronal supporting cells of the olfactory epithelium. Here we investigate the hypothesis that olfactory neurons communicate with sustentacular cells via extracellular ATP and purinergic receptor activation.ResultsHere we show that exposure of mice to a mixture of odorants induced a significant increase in the levels of the transcription factor CREB phosphorylated at Ser-133 in the nuclei of both olfactory sensory neurons and sustentacular cells. This activation was dependent on adenylyl cyclase III-mediated olfactory signaling and on activation of P2Y purinergic receptors on sustentacular cells. Purinergic receptor antagonists inhibited odorant-dependent CREB phosphorylation specifically in the nuclei of the sustentacular cells.ConclusionOur results point to a possible role for extracellular nucleotides in mediating intercellular communication between the neurons and sustentacular cells of the olfactory epithelium in response to odorant exposure. Maintenance of extracellular ionic gradients and metabolism of noxious chemicals by sustentacular cells may therefore be regulated in an odorant-dependent manner by olfactory sensory neurons.

No-scanning type of confocal microscope for a fast functional imaging of neuronal circuit

The properties of dorsal root ganglion (DRG) neurons have been mostly investigated in culture of dissociated cells, and it is uncertain whether these cells maintain the electrophysiological properties of the intact DRG neurons. Few attempts have been made to record from DRG neurons in the intact ganglion using the patch clamp technique. In this study, rat DRGs were dissected and incubated for at least 1 h at 37 °C in collagenase (10 mg/ml). We used oblique epi-illumination to visualize DRG neurons and perform patch clamp recordings. All DRG neurons exhibited strong delayed rectifier potassium current and a high threshold for spike generation (−15 mV) that rendered the cells very weakly excitable, generating only one action potential upon strong current injection (>300 pA). It is therefore possible that cultured DRG neurons, commonly used in studies of pain processing, may be hyperexcitable because they acquired “neuropathic” properties due to the injury induced by their dissociation. Electrical stimulation of the attached root produced an antidromic spike in the soma that could be blocked by intracellular hyperpolarization or high frequency stimulation. Imaging intracellular calcium concentration with Oregon Green BAPTA-1 indicates that antidromic stimulation caused a long-lasting increase in intracellular calcium concentration mostly near the cell membrane. This study describes a simple approach to examine the electrophysiological and pharmacological properties and intracellular calcium signaling in DRG neurons in the intact ganglion where the effects of somatic spike invasion can be studied as well.