Sustained Increase In Intracellular Calcium Research Articles

Heavy metals in cigarette smoke strongly inhibit pancreatic ductal function and promote development of chronic pancreatitis.

Smoking is recognised as an independent risk factor in the development of chronic pancreatitis (CP). Cystic fibrosis transmembrane conductance regulator (CFTR) function and ductal fluid and bicarbonate secretion are also known to be impaired in CP, so it is crucial to understand the relationships between smoking, pancreatic ductal function and the development of CP. We measured sweat chloride (Cl-) concentrations in patients with and without CP, both smokers and non-smokers, to assess CFTR activity. Serum heavy metal levels and tissue cadmium concentrations were determined by mass spectrometry in smoking and non-smoking patients. Guinea pigs were exposed to cigarette smoke, and cigarette smoke extract (CSE) was prepared to characterise its effects on pancreatic HCO3 - and fluid secretion and CFTR function. We administered cerulein to both the smoking and non-smoking groups of mice to induce pancreatitis. Sweat samples from smokers, both with and without CP, exhibited elevated Cl- concentrations compared to those from non-smokers, indicating a decrease in CFTR activity due to smoking. Pancreatic tissues from smokers, regardless of CP status, displayed lower CFTR expression than those from non-smokers. Serum levels of cadmium and mercury, as well as pancreatic tissue cadmium, were increased in smokers. Smoking, CSE, cadmium, mercury and nicotine all hindered fluid and HCO3 - secretion and CFTR activity in pancreatic ductal cells. These effects were mediated by sustained increases in intracellular calcium ([Ca2+]i), depletion of intracellular ATP (ATPi) and mitochondrial membrane depolarisation. Smoking impairs pancreatic ductal function and contributes to the development of CP. Heavy metals, notably cadmium, play a significant role in the harmful effects of smoking. Smoking and cigarette smoke extract diminish pancreatic ductal fluid and HCO3 - secretion as well as the expression and function of CFTR Cd and Hg concentrations are significantly higher in the serum samples of smokers Cd accumulates in the pancreatic tissue of smokers.

Regulation of Renin-Angiotensin System Activity and Blood Pressure by Vascular Smooth Muscle Cullin-3

Mutations in the ubiquitin ligase scaffold protein Cullin 3 (CUL3) in smooth muscle cells (SMCs) cause vascular dysfunction, arterial stiffness, and severe hypertension. We hypothesized that conditional deletion of CUL3 in SMCs causes severe hypertension through a renin-angiotensin system (RAS)-dependent mechanism. Mice carrying a conditional allele of CUL3 were bred to mice expressing a tamoxifen-inducible CRE-recombinase driven by a smooth muscle promoter (ISM-CRE, control). Tamoxifen was administered to generate smooth muscle CUL3 knockout (S-CUL3KO). Primary aortic SMCs were used to elucidate the contribution of CUL3 to intracellular angiotensin II-mediated calcium flux. Calcium response in SMCs was analyzed via ratiometric confocal fluorescent microscopyS-CUL3KO mice demonstrated hypertension following three weeks of tamoxifen, mean arterial pressure (MAP) was 108±1 vs. 140±1 mmHg (ISM-CRE vs. S-CUL3KO). Aorta from S-CUL3KO mice exhibited impaired vasorelaxation to acetylcholine (ACh, max relaxation: 84±4% vs. 16±9%, p<0.05) and sodium nitroprusside (SNP, max relaxation: 95±3% vs. 56±9%, p<0.05). Captopril administration (7 days) markedly reduced MAP to 84±0.3 mmHg and improved vasorelaxation in response to both ACh (72±6%) and SNP (91±4%) in S-CUL3KO mice, suggesting RAS-dependency of the hypertension. Additionally, candesartan treatment (7 days) substantially decreased MAP from 138±1 to 96±3 mmHg suggesting that hypertension in S-CUL3KO group is angiotensin type-1 receptor mediated. Q-PCR and RNAScope analyses demonstrated no difference in renin expression in the kidney between the S-CUL3KO and control mice suggesting a blunting of the baroreceptor mechanism regulating renin mRNA expression. Equilibrium angiotensin II plasma levels in both groups were not different (2517±352 and 2379±549 pmol/L, p>0.05). Preliminary data demonstrated that acute application of 10 μM of angiotensin II to S-CUL3KO SMCs resulted in a sustained increase in intracellular calcium which was abolished by a preincubation with 2 μM of candesartan.Our data suggest that CUL3 may play a vital role in baroreceptor mechanism in the kidney and regulate angiotensin II response in SMCs which are essential for responding to changes in perfusion pressure. Ultimately, understanding CUL3 target proteins may benefit the development of novel treatments for hypertension. R35 HL144807 (to CDS). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Homocysteine-induced membrane currents, calcium responses and changes in mitochondrial potential in rat cortical neurons

Homocysteine, a sulfur-containing amino acid, exhibits neurotoxic effects and is involved in the pathogenesis of several major neurodegenerative disorders. In contrast to well studied excitoxicity of glutamate, the mechanism of homocysteine neurotoxicity is not clearly understood. By using whole-cell patch-clamp, calcium imaging (fluo-3) and measurements of mitochondrial membrane potential (rhodamine 123) we studied transmembrane currents, calcium signals and changes in mitochondrial membrane potential induced by homocysteine versus responses induced by NMDA and glutamate in cultured rat cortical neurons. L-homocysteine (50 µM) induced inward currents that could be completely blocked by the selective antagonist of NMDA receptors - AP-5. In contrast to NMDA-induced currents, homocysteine-induced currents had a smaller steady-state amplitude. Comparison of calcium responses to homocysteine, NMDA or glutamate demonstrated that in all cortical neurons homocysteine elicited short, oscillatory-type calcium responses, whereas NMDA or glutamate induced sustained increase of intracellular calcium. Analysis of mitochondrial changes demonstrated that in contrast to NMDA homocysteine did not cause a drop of mitochondrial membrane potential at the early stages of action. However, after its long-term action, as in the case of NMDA and glutamate, the changes in mitochondrial membrane potential were comparable with the full drop of respiratory chain induced by protonophore FCCP. Our data suggest that in cultured rat cortical neuron homocysteine at the first stages of action induces neurotoxic effects through activation of NMDA-type ionotropic glutamate receptors with strong calcium influx through the channels of these receptors. The long-term action of homocysteine may lead to mitochondrial disfuction and appears as a drop of mitochondrial membrane potential.

Dysbalance of astrocyte calcium under hyperammonemic conditions.

Increased brain ammonium (NH4 +/NH3) plays a central role in the manifestation of hepatic encephalopathy (HE), a complex syndrome associated with neurological and psychiatric alterations, which is primarily a disorder of astrocytes. Here, we analysed the influence of NH4 +/NH3 on the calcium concentration of astrocytes in situ and studied the underlying mechanisms of NH4 +/NH3-evoked calcium changes, employing fluorescence imaging with Fura-2 in acute tissue slices derived from different regions of the mouse brain. In the hippocampal stratum radiatum, perfusion with 5 mM NH4 +/NH3 for 30 minutes caused a transient calcium increase in about 40% of astrocytes lasting about 10 minutes. Furthermore, the vast majority of astrocytes (∼90%) experienced a persistent calcium increase by ∼50 nM. This persistent increase was already evoked at concentrations of 1–2 mM NH4 +/NH3, developed within 10–20 minutes and was maintained as long as the NH4 +/NH3 was present. Qualitatively similar changes were observed in astrocytes of different neocortical regions as well as in cerebellar Bergmann glia. Inhibition of glutamine synthetase resulted in significantly larger calcium increases in response to NH4 +/NH3, indicating that glutamine accumulation was not a primary cause. Calcium increases were not mimicked by changes in intracellular pH. Pharmacological inhibition of voltage-gated sodium channels, sodium-potassium-chloride-cotransporters (NKCC), the reverse mode of sodium/calcium exchange (NCX), AMPA- or mGluR5-receptors did not dampen NH4 +/NH3-induced calcium increases. They were, however, significantly reduced by inhibition of NMDA receptors and depletion of intracellular calcium stores. Taken together, our measurements show that sustained exposure to NH4 +/NH3 causes a sustained increase in intracellular calcium in astrocytes in situ, which is partly dependent on NMDA receptor activation and on release of calcium from intracellular stores. Our study furthermore suggests that dysbalance of astrocyte calcium homeostasis under hyperammonemic conditions is a widespread phenomenon, which might contribute to the disturbance of neurotransmission during HE.

Thromboxane A2 mimetic, U46619, does not induce markers of pathological hypertrophy in cardiomyocytes

We have previously shown that application of the thromboxane (TxA2) receptor agonist, U46619, induces increases in intracellular calcium in isolated primary cardiomyocytes. One of the well‐known effects of sustained increases in intracellular calcium in cardiomyocytes is pathological hypertrophy. Since TxA2 is an inflammatory mediator, we hypothesized that TxA2 release during inflammation would result in cardiac hypertrophy. U46619 (0.1–10 μM) was applied to isolated primary mouse cardiomyocytes, cardiac muscle strips, and cultured HL‐1 cardiomyocytes and markers of hypertrophy were measured. U46619 treatment for 24 hrs did not increase expression of pathological hypertrophy genes (atrial natriuretic peptide, beta myosin heavy chain, skeletal muscle alpha actin) and it did not increase protein synthesis. There was also no increase in HL‐1 cardiomyocyte cell size after 48 hr treatment with U46619 as measured by flow cytometry. Previous reports have suggested that cyclooxygenase products such as TxA2 may play a role in directly inducing hypertrophy and cardiac remodeling. However, our current data would suggest that TxA2 at the doses and times we tested does not induce markers of pathological hypertrophy. (Support was provided by American Heart Association 11SDG5330016 to MJW)

Triptolide-mediated cell death in neuroblastoma occurs by both apoptosis and autophagy pathways and results in inhibition of nuclear factor–kappa B activity

BackgroundNeuroblastoma is an aggressive pediatric malignancy with significant chemotherapeutic resistance. We assessed triptolide as a potential therapy. MethodsSH-SY5Y and IMR-32 neuroblastoma cell lines were treated with triptolide. Viability, intracellular calcium, caspase activation, protein, and mRNA levels were measured. Autophagy was evaluated with confocal microscopy. Nuclear factor–kappa B (NF-κB) activation was measured using a dual luciferase assay. ResultsTriptolide treatment resulted in death in both cell lines within 72 hours, with sustained increases in intracellular calcium. IMR-32 cells underwent cell death by apoptosis. Conversely, light chain 3II (LC3II) protein levels were elevated in SH-SY5Y cells, which is consistent with autophagy. Confocal microscopy confirmed increased LC3 puncta in SH-SY5Y cells compared with control cells. Heat shock pathway protein and mRNA levels decreased with treatment. NF-κB assays demonstrated inhibition of tumor necrosis factor (TNF)-α–induced activity with triptolide. ConclusionsTriptolide treatment induces cell death in neuroblastoma by different mechanisms with multiple pathways targeted. Triptolide may serve a potential chemotherapeutic role in advanced cases of neuroblastoma.

The neuropeptide catestatin promotes vascular smooth muscle cell proliferation through the Ca2+–calcineurin–NFAT signaling pathway

The Chromogranin A-derived neuropeptide catestatin is an endogenous nicotinic cholinergic antagonist that acts as a pleiotropic hormone. Since catestatin shares several functions with other members derived from the chromogranin/secretogranin protein family and other neuropeptides which exert proliferative effects on vascular smooth muscle cells (VSMCs), we therefore hypothesized that catestatin would regulate VSMC proliferation. The present study demonstrates that catestatin caused a dose-dependent induction of proliferation in rat aortic smooth muscle cells and furthermore evoked a sustained increase in intracellular calcium. This subsequently leaded to enhanced activation of the Ca2+/calmodulin-dependent phosphatase, calcineurin and resulted in an activation of the Ca2+-dependent transcription factor, nuclear factor of activated T cells (NFAT), initiating transcription of proliferative genes. In addition, cyclosporin A (CsA), a potent inhibitor of calcineurin, abrogated catestatin-mediated effect on VSMCs, indicating that the calcineurin–NFAT signaling is strongly required for catestatin-induced growth of VSMCs. The present study establishes catestatin as a novel proliferative cytokine on vascular smooth muscle cells and this effect is mediated by the Ca2+–calcineurin–NFAT signaling pathway.

The Pseudomonas aeruginosa Autoinducer 3O-C12 Homoserine Lactone Provokes Hyperinflammatory Responses from Cystic Fibrosis Airway Epithelial Cells

The discovery of novel antiinflammatory targets to treat inflammation in the cystic fibrosis (CF) lung stands to benefit patient populations suffering with this disease. The Pseudomonas aeruginosa quorum sensing autoinducer N-3-oxododecanoyl homoserine lactone (3O-C12) is an important bacterial virulence factor that has been reported to induce proinflammatory cytokine production from a variety of cell types. The goal of this study was to examine the ability of 3O-C12 to induce proinflammatory cytokine production in normal and CF bronchial epithelial cells, and better understand the cellular mechanisms by which this cytokine induction occurs. 3O-C12 was found to induce higher levels of IL-6 production in the CF cell lines IB3-1 and CuFi, compared to their corresponding control cell lines C38 and NuLi. Systems biology and network analysis revealed a high predominance of over-represented innate immune pathways bridged together by calcium-dependant transcription factors governing the transcriptional responses of A549 airway cells to stimulation with 3O-C12. Using calcium-flux assays, 3O-C12 was found to induce larger and more sustained increases in intracellular calcium in IB3-1 cells compared to C38, and blocking this calcium flux with BAPTA-AM reduced the production of IL-6 by IB3-1 to the levels produced by C38. These data suggest that 3O-C12 induces proinflammatory cytokine production in airway epithelial cells in a calcium-dependent manner, and that dysregulated calcium storage or signalling in CF cells results in an increased production of proinflammatory cytokines.

Mechanisms of Microparticle Generation: On the Trail of the Mitochondrion!

Membrane remodeling, phosphatidylserine (PS) exposure, and subsequent microparticle (MP) shedding regulation is a critical step in maintaining vascular homeostasis. Shed MP, more particularly those of platelet origin, could be viewed as a way to increase the catalytic procoagulant surface relying on the essential presence of PS for optimal hemostatic response. Whether "flip-flop" is mandatory for the release of MP is suggested from the phenotype of Scott's syndrome, a rare bleeding disorder in which both PS exposure and MP shedding are deficient. PS exposure results from a specific cytoskeleton degradation pathway involving caspases, tuned by mitochondria permeability changes, and requiring a sustained increase in intracellular calcium. The actual roles of transmembrane ion transport or transient transmembrane pores in PS exposure remain to be more firmly established. Considering that an excess of plasma membrane procoagulant activity is associated with an increased risk of thrombosis, the identification of effectors of PS exposure and MP release appear relevant targets in thrombosis research and focused drug design. In this view, animal models of Scott's syndrome should prove of primary importance for the characterization of the genetic trait(s) accounting for the associated defect that would provide an important hint toward the control of PS exposure and subsequent MP release.

Essential Role of Nuclear Factor of Activated T Cells (NFAT)-mediated Wnt Signaling in Osteoblast Differentiation Induced by Strontium Ranelate

The antiosteoporotic treatment strontium ranelate (SrRan) was shown to increase bone mass and strength by dissociating bone resorption and bone formation. To identify the molecular mechanisms of action of SrRan on osteoblasts, we investigated its effects on calcineurin-NFAT (nuclear factor of activated T cells) signaling, an important calcium sensitive pathway controlling bone formation. Using murine MC3T3-E1 and primary murine osteoblasts, we demonstrate that SrRan induces NFATc1 nuclear translocation, as shown by immunocytochemical and Western blot analyses. Molecular analysis showed that SrRan increased NFATc1 transactivation in osteoblasts, an effect that was fully abrogated by the calcineurin inhibitors cyclosporin A or FK506, confirming that SrRan activates NFATc1 signaling in osteoblasts. This has functional implications because calcineurin inhibitors blunted the enhanced osteoblast replication and expression of the osteoblast phenotypic markers Runx2, alkaline phosphatase, and type I collagen induced by SrRan. We further found that SrRan increased the expression of Wnt3a and Wnt5a as well as beta-catenin transcriptional activity in osteoblasts, and these effects were abolished by calcineurin inhibitors. The Wnt inhibitors sFRP1 and DKK1 abolished SrRan-induced osteoblast gene expression. Furthermore, blunting the Wnt5a receptor Ryk or RhoA that acts downstream of Ryk abrogated cell proliferation and osteoblast gene expression induced by SrRan. These results indicate that activation of NFATc1 and downstream canonical and non-canonical Wnt signaling pathways mediate SrRan-induced osteoblastic cell replication and differentiation, which provides novel insights into the mechanisms of action of this antiosteoporotic agent in osteoblastogenesis.

Structurally diverse peroxisome proliferator‐activated receptor agonists induce apoptosis in human uro‐epithelial cells by a receptor‐independent mechanism involving store‐operated calcium channels

Peroxisome proliferator-activated receptors (PPARs) are implicated in epithelial cell proliferation and differentiation, but investigation has been confounded by potential off-target effects of some synthetic PPAR ligands. Our aim was to determine mechanisms underlying the pro-apoptotic effect of synthetic PPAR agonists in normal human bladder uro-epithelial (urothelial) cells and to reconcile this with the role of PPARs in urothelial cytodifferentiation. Normal human urothelial (NHU) cells were grown as non-immortal lines in vitro and exposed to structurally diverse agonists ciglitazone, troglitazone, rosiglitazone (PPARgamma), ragaglitazar (PPARalpha/gamma), fenofibrate (PPARalpha) and L165041 (PPARbeta/delta). NHU cells underwent apoptosis following acute exposure to ciglitazone, troglitazone or ragaglitazar, but not fenofibrate, L165041 or rosiglitazone, and this was independent of ERK or p38 MAP-kinase activation. Pro-apoptotic agonists induced sustained increases in intracellular calcium, whereas removal of extracellular calcium altered the kinetics of ciglitazone-mediated calcium release from sustained to transient. Cell death was accompanied by plasma-membrane disruption, loss of mitochondrial membrane-potential and caspase-9/caspase-3 activation. PPARgamma-mediated apoptosis was unaffected following pre-treatment with PPARgamma antagonist T0070907 and was strongly attenuated by store-operated calcium channel (SOC) inhibitors 2-APB and SKF-96365. Our results provide a mechanistic basis for the ability of some PPAR agonists to induce death in NHU cells and demonstrate that apoptosis is mediated via PPAR-independent mechanisms, involving intracellular calcium changes, activation of SOCs and induction of the mitochondrial apoptotic pathway.

Physiology and pathophysiology of canonical transient receptor potential channels.

The existence of a mammalian family of TRPC ion channels, direct homologues of TRP, the visual transduction channel of flies, was discovered during 1995-1996 as a consequence of research into the mechanism by which the stimulation of the receptor-Gq-phospholipase Cbeta signaling pathway leads to sustained increases in intracellular calcium. Mammalian TRPs, TRPCs, turned out to be nonselective, calcium-permeable cation channels, which cause both a collapse of the cell's membrane potential and entry of calcium. The family comprises 7 members and is widely expressed. Many cells and tissues express between 3 and 4 of the 7 TRPCs. Despite their recent discovery, a wealth of information has accumulated, showing that TRPCs have widespread roles in almost all cells studied, including cells from excitable and nonexcitable tissues, such as the nervous and cardiovascular systems, the kidney and the liver, and cells from endothelia, epithelia, and the bone marrow compartment. Disruption of TRPC function is at the root of some familial diseases. More often, TRPCs are contributing risk factors in complex diseases. The present article reviews what has been uncovered about physiological roles of mammalian TRPC channels since the time of their discovery. This analysis reveals TRPCs as major and unsuspected gates of Ca(2+) entry that contribute, depending on context, to activation of transcription factors, apoptosis, vascular contractility, platelet activation, and cardiac hypertrophy, as well as to normal and abnormal cell proliferation. TRPCs emerge as targets for a thus far nonexistent field of pharmacological intervention that may ameliorate complex diseases.

Discrimination of cell types in mixed cortical culture using calcium imaging: A comparison to immunocytochemical labeling

Neuronal–glial interactions in the central nervous system are important for both normal function and response to pathological states. Differences in calcium processing between these cell types may be exploited to allow dynamic differentiation using calcium-imaging protocols without the need to fix and immunostain the study population. Mixed rat primary cortical cultures were grown on coverslips, incubated for 30min in 2μM fluo-3 AM and mounted in a devised, low volume imaging chamber. Calcium influx was measured over the duration of a 50s exposure to 30μM glutamate in all cells. Cells were then fixed in situ, and immunostained for NeuN and GFAP. Direct comparison between live calcium dynamics and cell type markers were made. Over the duration of the glutamate exposure, those cells that subsequently stained for NeuN exhibited a sustained increase in intracellular calcium, whereas GFAP positive and non-staining cells exhibited a decline over the duration of the glutamate exposure. We found that examining the average calcium fluorescence over the last 10s of glutamate exposure allowed the identification of cells as neuronal if the average was >85% of the maximal calcium change, or non-neuronal if the average was <85% of the maximal calcium change. This technique compares very favourably to the established technique of immunocytochemical labeling for the identification of cell types; both techniques agreed in their classification of cells as neuronal or non-neuronal 96.83% of the time. However, this technique cannot reliably distinguish between non-neuronal cell types.

Activation of Store‐Operated Calcium Entry Channels Stably Increases Membrane‐Localized Calcium

Inflammatory agonists deplete calcium from the endoplasmic reticulum and activate calcium entry in a process referred to as store‐operated calcium (SOC) entry. Calcium that permeates through SOC channels is sufficient to increase permeability although it is not clear why calcium entry, and not calcium release, is necessary to increase permeability. In our present studies, we tested the hypothesis that calcium entry is necessary to produce a sustained increase in intracellular calcium that is localized at the cell membrane. To test this hypothesis, pulmonary artery endothelial cells were loaded with either a cytosol‐localized calcium fluorophore (Fura‐2AM) or a membrane‐localized fluorophore (FFP‐18). Membrane and cytosolic calcium concentrations were calculated using the Grynkiewicz equation. Baseline membrane calcium concentrations were higher than cytosolic calcium concentrations, indicating a calcium gradient from the plasma membrane to the bulk cytosol. Upon thapsigargin‐induced SOC entry, both cytosolic and membrane calcium increased. However, cytosolic calcium decayed over time whereas membrane calcium was sustained. Collectively, these observations indicate that calcium entry through SOC channels is necessary to stably increase membrane‐associated calcium, likely necessary to induce the inter‐endothelial cell gaps that precede edema formation. Supported by HL60024.

CALCIUM: A Role for Neuroproduction and Sustained Adaptation

Sustained increases in intracellular calcium following prolonged seizures or other neurological insults have been thought to be responsible for neuronal cell death for well over two decades. For example, a seizure or a stroke can lead to excessive release of glutamate, an endogenous excitotoxin. Overactivation of receptors that interact with glutamate will raise calcium levels to stimulate a variety of signaling pathways that can impair neuronal respiration and eventually kill neurons. On the contrary, recent evidence shows that under numerous conditions calcium can prevent neurons from dying. Experimental epilepsy and ischemia models show that protection of neurons appears to depend upon the age of the animal, the amount and route of calcium elevation, timing of initial insults, and brain regions involved. This review will discuss novel findings on the protective signaling role of calcium under a wide range of pathological conditions.

Fetuin-A uptake in bovine vascular smooth muscle cells is calcium dependent and mediated by annexins

Fetuin-A is a known inhibitor of vascular calcification in vitro. In arteries with calcification, there is increased immunostaining for fetuin-A. However, vascular smooth muscle cells (VSMC) do not synthesize fetuin-A, suggesting fetuin-A may be endocytosed to exert its inhibitory effects. To examine the mechanism by which fetuin-A is taken up in bovine VSMC (BVSMC), we examined living cells by confocal microscopy and determined the uptake of Cy5-labeled fetuin-A. The results demonstrated that fetuin-A was taken up in BVSMC only in the presence of extracellular calcium, whereas phosphorus had no effect. Additional studies demonstrated the calcium-dependent uptake was specific for fetuin-A and only observed in BVSMC and osteoblasts, but not epithelial, endothelial, or adipose cells. The uptake was dose dependent, but could not be inhibited by excess unlabeled fetuin-A, suggesting a fluid phase rather than a receptor-mediated process. Fetuin-A also induced a sustained increase in intracellular calcium in BVSMC in the presence of extracellular calcium, whereas there was no increase in the absence of extracellular calcium. To further characterize the uptake, we utilized an inhibitor of annexin calcium channel activity, demonstrating inhibition of both fetuin-A uptake and intracellular calcium increase. Finally, we demonstrate that fetuin-A binds to annexin II at the cell membrane of BVSMC. In summary, our study demonstrates calcium- and annexin-dependent uptake of fetuin-A that leads to a sustained rise in intracellular calcium. This regulated uptake may be a mechanism by which fetuin-A inhibits VSMC calcification in the presence of excess calcium.

Intoxicating Fatty Acids

Ethanol excess can cause severe pancreatic injury, such as acute pancreatitis. Organ necrosis, eventual failure, and even death may occur. Criddle et al. report that ethanol itself may not be the major culprit of pancreatic necrosis, but rather a fatty acid ethyl ester (FAEE) metabolite of ethanol. Exposure of cultured pancreatic acinar cells to the nonoxidative FAEE metabolite palmitoleic acid ethyl ester (POAEE) caused a sustained, global increase in intracellular calcium. The rise in calcium activates intracellular digestive enzymes and vacuolization that mark the early stages of pancreatic cell injury. Even at 850 mM, ethanol generally caused only a transient increase in intracellular calcium. In contrast, treatment of cells with POAEE (from 10 μM to 100 μM) resulted in a global, sustained increase in intracellular calcium and cell necrosis. A pharmacological inhibitor indicated that calcium was released from intracellular stores. However, the absence of extracellular calcium blocked a rise in intracellular calcium in response to low concentrations of POA (a deesterification product), which implicated a store-operated calcium entry channel in generating the calcium signal. When people are intoxicated, FAEE concentration in their blood may reach as high as 50 μM, and its accumulation is highest in the pancreas. D. N. Criddle, M. G. T. Raraty, J. P. Neoptolemos, A. V. Tepikin, O. H. Petersen, R. Sutton, Ethanol toxicity in pancreatic acinar cells: Mediation by nonoxidative fatty acid metabolites. Proc. Natl. Acad. Sci. U.S.A. 101 , 10738-10743 (2004). [Abstract] [Full Text]

Involvement of Calcineurin in Transforming Growth Factor-β-mediated Regulation of Extracellular Matrix Accumulation

Calcineurin is a calcium-dependent, serine/threonine phosphatase that functions as a signaling intermediate. In this study, we investigated the role of calcineurin in transforming growth factor-beta (TGF-beta)-mediated cellular effects and examined the signaling pathway involved in activation of calcineurin. Calcineurin is activated by TGF-beta in a time- and dose-dependent manner. Consistent with increased phosphatase activity, the calcineurin substrate, NFATc1, is dephosphorylated and transported to the nucleus. Inhibition of calcineurin prior to the addition of TGF-beta revealed that calcineurin is required for TGF-beta-mediated accumulation of extracellular matrix (ECM) proteins but not cell hypertrophy. Conversely, overexpression of constitutively active calcineurin was sufficient to induce ECM protein expression. The mechanism of calcineurin activation by TGF-beta was found to be induction of a low, sustained increase of intracellular calcium. Chelation of extracellular calcium blocked both TGF-beta-mediated calcium influx and calcineurin activity. Finally, calcium entry was found to be dependent upon generation of reactive oxygen species (ROS) including superoxide anion and hydrogen peroxide. Accordingly, inhibition of ROS generation also blocked TGF-beta-mediated calcineurin phosphatase activity and decreased ECM accumulation. In conclusion, this study describes a new pathway for TGF-beta-mediated regulation of ECM via generation of ROS, calcium influx, and activation of calcineurin.

Saying NO to Pressure

Increases in arterial blood pressure stimulate vasoconstriction. In small cerebral arteries, this involves a graded depolarization in the membrane potential of arterial smooth muscle cells and the consequent influx of calcium through voltage-dependent calcium channels (VDCCs). Nuclear factor of activated T cells (NFAT), a transcription factor that undergoes translocation to the nucleus in response to sustained increases in intracellular calcium ([Ca 2+ ] i ), has been implicated in long-term cardiovascular processes such as vasculogenesis and cardiac hypertrophy. Gonzalez Bosc et al. used immunofluorescence microscopy to investigate possible effects of pressure on transcription and found that increased intraluminal pressure led to nuclear accumulation of NFATc3 in smooth muscle cells of isolated mouse cerebral arteries, which was inhibited by blockers of VDCCs. [This group previously found that Ca 2+ influx after depolarization with high concentrations of extracellular K + (HK) does not lead to nuclear accumulation of NFATc3 in cerebral vascular smooth muscle.] NFATc3 accumulation in response to pressure depended on endothelial cells [which produce nitric oxide (NO)]; pharmacological analysis indicated that accumulation depended on NO production and the ensuing activation of PKG [cyclic guanosine 3´,5´-monophosphate (cGMP)-dependent protein kinase] and was independent of vasoconstriction. (Indeed, NO and PKG promote smooth muscle relaxation.) Moreover, NO plus HK elicited nuclear accumulation of NFATc3. Increased pressure was associated with diminished phosphorylation of JNK2 (c-Jun N-terminal kinase 2, which promotes NFATc3 export from the nucleus), an effect that depended on PKG. In myocytes from mice that lacked JNK2, the effects of pressure were independent of PKG. Thus, in cerebral vascular smooth muscle, NO and Ca 2+ appear to enhance nuclear accumulation of NFAT cooperatively in response to pressure through respective effects on export and import. L. V. Gonzalez Bosc, M. K. Wilkerson, K. N. Bradley, D. M. Eckman, D. C. Hill-Eubanks, M. T. Nelson, Intraluminal pressure is a stimulus for NFATc3 nuclear accumulation. J. Biol. Chem. 279 , 10702-10709 (2004). [Abstract] [Full Text]

Intraluminal pressure is a stimulus for NFATc3 nuclear accumulation: role of calcium, endothelium-derived nitric oxide, and cGMP-dependent protein kinase.

The transcription factor NFAT (nuclear factor of activated T-cells) is implicated in cardiac hypertrophy and vasculogenesis. NFAT activation, reflecting dephosphorylation by the calcium-dependent phosphatase, calcineurin, and subsequent nuclear localization, is generally thought to require a sustained increase in intracellular calcium. However, in smooth muscle we have found that elevation of calcium by membrane depolarization fails to induce an increase in nuclear localization of the NFATc3 isoform. Here, we demonstrate that physiological intravascular pressure (100 mm Hg) induces an increase in NFATc3 nuclear localization in mouse cerebral arteries. Pressure-induced NFATc3 nuclear accumulation is abrogated by endothelial denudation and by nitric-oxide synthase, cGMP-dependent kinase (PKG), and voltage-dependent calcium channels inhibition. We further show that exogenous nitric oxide, in combination with an elevation in calcium, is an effective stimulus for NFATc3 nuclear accumulation. c-Jun terminal kinase 2 (JNK) activity, which has been shown to regulate NFATc3 nuclear export, is also reduced by pressure, an effect that is prevented by pretreatment with a PKG inhibitor. Consistent with this, pressure-induced NFATc3 nuclear accumulation is independent of PKG in arteries from JNK2(-/-) mice. Collectively, our results indicate that both activation of the NO/PKG pathway and elevation of smooth muscle calcium are required for NFATc3 nuclear accumulation and that PKG inhibits JNK2 to decrease NFAT nuclear export. Our findings suggest that at physiological intravascular pressures NFATc3 is localized to the nucleus in smooth muscle cells of intact arteries and indicate a novel and unexpected role for nitric oxide/PKG in NFAT activation.