Implication of Transient Receptor Potential Vanilloid Type 1 in 14,15-Epoxyeicosatrienoic Acid-induced Angiogenesis

14,15-epoxyeicosatrienoic acid (14,15-EET) is implicated in regulating physiological functions of endothelial cells (ECs), yet the potential molecular mechanisms underlying the beneficial effects in ECs are not fully understood. In this study, we investigated whether transient receptor potential vanilloid receptor type 1 (TRPV1) is involved in 14,15-EET-mediated Ca²⁺ influx, nitric oxide (NO) production and angiogenesis. In human microvascular endothelial cells (HMECs), 14,15-EET time-dependently increased the intracellular level of Ca²⁺. Removal of extracellular Ca²⁺, pharmacological inhibition or genetic disruption of TRPV1 abrogated 14,15-EET-mediated increase of intracellular Ca²⁺ level in HMECs or TRPV1-transfected HEK293 cells. Furthermore, removal of extracellular Ca²⁺ or pharmacological inhibition of TRPV1 decreased 14,15-EET-induced NO production. 14,15-EET-mediated tube formation was abolished by TRPV1 pharmacological inhibition. In an animal experiment, 14,15-EET-induced angiogenesis was diminished by inhibition of TRPV1 and in TRPV1-deficient mice. TRPV1 may play a crucial role in 14,15-EET-induced Ca²⁺ influx, NO production and angiogenesis.     

Plasma membrane stretch activates transient receptor potential vanilloid and ankyrin channels in Merkel cells from hamster buccal mucosa

Merkel cells (MCs) have been proposed to form a part of the MC-neurite complex with sensory neurons. Many transient receptor potential (TRP) channels have been identified in mammals; however, the activation properties of these channels in oral mucosal MCs remain to be clarified. We investigated the biophysical and pharmacological properties of TRP vanilloid (TRPV)-1, TRPV2, TRPV4, TRP ankyrin (TRPA)-1, and TRP melastatin (TRPM)-8 channels, which are sensitive to osmotic and mechanical stimuli by measurement of intracellular free Ca²⁺ concentration ([Ca²⁺]_i) using fura-2. We also analyzed their localization patterns through immunofluorescence. MCs showed immunoreaction for TRPV1, TRPV2, TRPV4, TRPA1, and TRPM8 channels. In the presence of extracellular Ca²⁺, the hypotonic test solution evoked Ca²⁺ influx. The [Ca²⁺]_i increases were inhibited by TRPV1, TRPV2, TRPV4, or TRPA1 channel antagonists, but not by the TRPM8 channel antagonist. Application of TRPV1, TRPV2, TRPV4, TRPA1, or TRPM8 channel selective agonists elicited transient increases in [Ca²⁺]_i only in the presence of extracellular Ca²⁺. The results indicate that membrane stretching in MCs activates TRPV1, TRPV2, TRPV4, and TRPA1 channels, that it may be involved in synaptic transmission to sensory neurons, and that MCs could contribute to the mechanosensory transduction sequence.     

Modulation of TRPV4 gating by intra- and extracellular Ca

We have studied the modulation of gating properties of the Ca²⁺-permeable, cation channel TRPV4 transiently expressed in HEK293 cells. The phorbol ester 4αPDD transiently activated a current through TRPV4 in the presence of extracellular Ca²⁺. Increasing the concentration of extracellular Ca²⁺ ([Ca²⁺]_e) reduced the current amplitude and accelerated its decay. This decay was dramatically delayed in the absence of [Ca²⁺]_e. It was also much slower in the presence of [Ca²⁺]_e in a mutant channel, obtained by a point mutation in the 6th transmembrane domain, F707A. Mutant channels, containing a single mutation in the C-terminus of TRPV4 (E797), were constitutively open. In conclusion, gating of the 4αPDD-activated TRPV4 channel depends on both extra- and intracellular Ca²⁺, and is modulated by mutations of single amino acid residues in the 6th transmembrane domain and the C-terminus of the TRPV4 protein.     

Persistent Na+ influx drives L-type channel resting Ca2+ entry in rat melanotrophs

Rat melanotrophs express several types of voltage-gated and ligand-gated calcium channels, although mechanisms involved in the maintenance of the resting intracellular Ca²⁺ concentration ([Ca²⁺]_i) remain unknown. We analyzed mechanisms regulating resting [Ca²⁺]_i in dissociated rat melanotrophs by Ca²⁺-imaging and patch-clamp techniques. Treatment with antagonists of L-type, but not N- or P/Q-type voltage-gated Ca²⁺ channels (VGCCs) as well as removal of extracellular Ca²⁺ resulted in a rapid and reversible decrease in [Ca²⁺]_i, indicating constitutive Ca²⁺ influx through L-type VGCCs. Reduction of extracellular Na⁺ concentration (replacement with NMDG⁺) similarly decreased resting [Ca²⁺]_i. When cells were champed at –80 mV, decrease in the extracellular Na⁺ resulted in a positive shift of the holding current. In cell-attached voltage-clamp and whole-cell current-clamp configurations, the reduction of extracellular Na⁺ caused hyperpolarisation. The holding current shifted in negative direction when extracellular K⁺ concentration was increased from 5 mM to 50 mM in the presence of K⁺ channel blockers, Ba²⁺ and TEA, indicating cation nature of persistent conductance. RT-PCR analyses of pars intermedia tissues detected mRNAs of TRPV1, TRPV4, TRPC6, and TRPM3-5. The TRPV channel blocker, ruthenium red, shifted the holding current in positive direction, and significantly decreased the resting [Ca²⁺]_i. These results indicate operation of a constitutive cation conductance sensitive to ruthenium red, which regulates resting membrane potential and [Ca²⁺]_i in rat melanotrophs.     

P2Y13 Receptor-Mediated Rapid Increase in Intracellular Calcium Induced by ADP in Cultured Dorsal Spinal Cord Microglia

P2Y receptors have been implicated in the calcium mobilization by the response to neuroexcitatory substances in neurons and astrocytes, but little is known about P2Y receptors in microglia cells. In the present study, the effects of ADP on the intracellular calcium concentration ([Ca²⁺]i) in cultured dorsal spinal cord microglia were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescence indicator that could monitor real-time alterations of [Ca²⁺]i. Here we show that ADP (0.01–100 μM) causes a rapid increase in [Ca²⁺]i with a dose-dependent manner in cultured microglia. The action of ADP on [Ca²⁺]i was significantly blocked by MRS2211 (a selective P2Y₁₃ receptor antagonist), but was unaffected by MRS2179 (a selective P2Y₁ receptor antagonist) or MRS2395 (a selective P2Y₁₂ receptor antagonist), which suggest that P2Y₁₃ receptor may be responsible for ADP-evoked Ca²⁺ mobilization in cultured microglia. P2Y₁₃-evoked Ca²⁺ response can be obviously inhibited by BAPTA-AM and U-73122, respectively. Moreover, removal of extracellular Ca²⁺ (by EGTA) also can obvious suppress the Ca²⁺ mobilization. These results means both intracellular calcium and extracellular calcium are potentially important mechanisms in P2Y₁₃ receptor-evoked Ca²⁺ mobilization. However, P2Y₁₃ receptor-evoked Ca²⁺ response was not impaired after CdCl₂ and verapamil administration, which suggest that voltage-operated Ca²⁺ channels may be not related with P2Y₁₃-evoked Ca²⁺ response. In addition, Ca²⁺ mobilization induced by ADP was abolished by different store-operated Ca²⁺ channels (SOCs) blocker, 2-APB (50 μM) and SKF-96365 (1 mM), respectively. These observations suggest that the activation of P2Y₁₃ receptor might be involved in the effect of ADP on [Ca²⁺]i in cultured dorsal spinal cord microglia. Furthermore, our results raise a possibility that P2Y₁₃ receptor activation causes Ca²⁺ release from Ca²⁺ store, which leads to the opening of SOCs.     

Activation of TRPV4 channel in pancreatic INS-1E beta cells enhances glucose-stimulated insulin secretion via calcium-dependent mechanisms

Transient receptor potential channel vanilloid type 4 (TRPV4) is a Ca²⁺- and Mg²⁺-permeable cation channel that influences oxidative metabolism and insulin sensitivity. The role of TRPV4 in pancreatic beta cells is largely unknown. Here, we characterize the role of TRPV4 in controlling intracellular Ca²⁺ and insulin secretion in INS-1E beta cells. Osmotic, thermal or pharmacological activation of TRPV4 caused a rapid rise of intracellular Ca²⁺ and enhanced glucose-stimulated insulin secretion. In the presence of the TRPV channel blocker ruthenium red (RuR) or after suppression of TRPV4 protein production, TRPV4 activators failed to increase [Ca²⁺]_i and insulin secretion in INS-1E cells.     

Store-operated calcium entry (SOCE) contributes to phosphorylation of p38 MAPK and suppression of TNF-α signalling in the intestinal epithelial cells

Calcium influx via store-operated calcium entry (SOCE) has an important role for regulation of vast majority of cellular physiological events. MAPK signalling is also another pivotal modulator of many cellular functions. However, the relationship between SOCE and MAPK is not well understood. In this study, we elucidated the involvement of SOCE in Gα_q/11 protein-mediated activation of p38 MAPK in an intestinal epithelial cell line HT-29/B6. In this cell line, we previously showed that the stimulation of M3 muscarinic acetylcholine receptor (M3-mAChR) but not histamine H1 receptor (H1R) led to phosphorylation of p38 MAPK which suppressed tumor necrosis factor-α (TNF-α)-induced NF-κB signalling through ADAM17 protease-mediated shedding of TNF receptor-1 (TNFR1). First, we found that stimulation of M3-mAChR and protease-activated receptor-2 (PAR-2) but not H1R induced persistent upregulation of cytosolic Ca²⁺ concentration through SOCE. Activation of M3-mAChR or PAR-2 also suppressed TNF-α-induced NF-κB phosphorylation, which was dependent on the p38 MAPK activity. Time course experiments revealed that M3-mAChR stimulation evoked intracellular Ca²⁺-dependent early phase p38 MAPK phosphorylation and extracellular Ca²⁺-dependent later phase p38 MAPK phosphorylation. This later phase p38 MAPK phosphorylation, evoked by M3-mAChRs or PAR-2, was abolished by inhibition of SOCE. Thapsigargin or ionomycin also phosphorylate p38 MAPK by Ca²⁺ influx through SOCE, leading to suppression of TNF-α-induced NF-κB phosphorylation. Finally, we showed that p38 MAPK was essential for thapsigargin-induced cleavage of TNFR1 and suppression of TNF-α-induced NF-κB phosphorylation. In conclusion, SOCE is important for p38 MAPK phosphorylation and is involved in TNF-α signalling suppression.     

6-Gingerol inhibits ROS and iNOS through the suppression of PKC-α and NF-κB pathways in lipopolysaccharide-stimulated mouse macrophages

Inflammation is involved in numerous diseases, including chronic inflammatory diseases and the development of cancer. Many plants possess a variety of biological activities, including antifungal, antibacterial and anti-inflammatory activities. However, our understanding of the anti-inflammatory effects of 6-gingerol is very limited. We used lipopolysaccharide (LPS)-stimulated macrophages as a model of inflammation to investigate the anti-inflammatory effects of 6-gingerol, which contains phenolic structure. We found that 6-gingerol exhibited an anti-inflammatory effect. 6-Gingerol could decrease inducible nitric oxide synthase and TNF-α expression through suppression of I-κBα phosphorylation, NF-κB nuclear activation and PKC-α translocation, which in turn inhibits Ca²⁺ mobilization and disruption of mitochondrial membrane potential in LPS-stimulated macrophages. Here, we demonstrate that 6-gingerol acts as an anti-inflammatory agent by blocking NF-κB and PKC signaling, and may be developed as a useful agent for the chemoprevention of cancer or inflammatory diseases.     

Activation of Glutamate Receptors Stimulates the Formation of Nitrite in Synaptosomes from Rat Cerebellum

Abstract: Synaptosomes from rat cerebellum were used to investigate the involvement of different glutamate receptor subtypes in the control of the synthesis of nitric oxide (NO), measured as its breakdown product nitrite (NO₂^-). Synaptosomes incubated in the presence of NAD|PH and l -arginine produced measurable levels of NO₂^-, which were reduced by addition of Nω-nitro-l -arginine methyl ester, an inhibitor of nitric oxide synthase. The selective ionotropic glutamate receptor agonist N-methyl-d -aspartate (NMDA) induced a pronounced increase in NO₂^-formation, which was prevented by Nω-nitro-l -arginine methyl ester and by the specific NMDA receptor antagonist Dl -2-amino-5-phosphonovaleric acid (AP-5). The NMDA-induced increase in NO₂^-formation was blocked by chelation of extracellular Ca²⁺ with EGTA. Both l -glutamate and the selective agonist for the metabotropic glutamate receptors (β)-1-aminocyclopentane-trans-1,3-dicarboxylic acid raised NO₂^-production, which retumed to control levels after addition of Nω-nitro-l -arginine methyl ester. The selective glutamate ionotropic receptor agonist (R,S)-α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid did not cause any change in NO₂ formation. The stimulatory effect of l -glutamate was blocked by the metabotropic glutamate receptor antagonist Dl -2-amino-4-phosphonobutyric acid but was unaffected by the selective NMDA receptor blocker AP-5. Removal of extracellular Ca²⁺ by EGTA did not affect the action of l -glutamate; whereas W-7, an inhibitor of calmodulin, and dantrolene, a compound that blocks the mobilization of Ca²⁺ from intracellular stores, abolished the effect of l -glutamate on NO₂^-formation. It is suggested that stimulation of ionotropic NMDA receptors activates NO metabolism by causing an influx of Ca²⁺ from the extracellular space, whereas activation of metabotropic receptors by l -glutamate provokes a mobilization of Ca²⁺ from intracellular stores, which stimulates nitric oxide synthase activity by forning Ca²⁺/calmodulin complexes.     

Different mechanisms are involved in intracellular calcium increase by insulin-like growth factors 1 and 2 in articular chondrocytes: Voltage-gated calcium channels,and/or phospholipase C coupled to a pertussis-sensitive G-protein

This study describes the mechanisms involved in the IGF-1 and IGF-2-induced increases in intracellular calcium concentration [Ca²⁺]i in cultured chondrocytes and the involvement of type 1 IGF receptors. It shows that IGF-1, IGF-2, and insulin increased the cytosolic free calcium concentration [Ca²⁺]i in a dose-dependent manner, with a plateau from 25 to 100 ng/ml for both IGF-1 and IGF-2 and from 1 to 2 μg/ml for insulin. The effect of IGF-1 was twice as great as the one of IGF-2, and the effect of insulin was 40% lower than IGF-1 effect. Two different mechanisms are involved in the intracellular [Ca²⁺]i increase. 1) IGF-1 and insulin but not IGF-2 involved a Ca²⁺ influx through voltage-gated calcium channels: pretreatment of the cells by EGTA and verapamil diminished the IGF-1 or insulin-induced[Ca²⁺]i but did not block the effect of IGF-2.2)IGF-1, IGF-2, and insulin also induced a Ca²⁺ mobilization from the endoplasmic reticulum: phospholipase C (PLC) inhihitors, neomycin, or U-73122 partially blocked the intracellular [Ca²⁺]i increase induced by IGF-1 and insulin and totally inhibited the effect of IGF-2. This Ca²⁺ mobilization was pertussis toxin (PTX) dependent, suggesting an activation of a PLC coupled to a PTX-sensitive G-protein. Lastly, preincubation of the cells with IGF₁ receptor antibodies diminished the IGF-1-induced Ca²⁺ spike and totally abolished the Ca²⁺ influx, but did not modify the effect of IGF-2. These results suggest that IGF-1 action on Ca²⁺ influx involves the IGF₁ receptor, while part of IGF-1 and all of IGF-2 Ca²⁺ mobilization do not implicate this receptor. J. Cell. Biochem. 64:414–422. © 1997 Wiley-Liss, Inc.     

Inhibitors of the Metabolism of Arachidonic Acid Suppress Ca<Superscript>2+</Superscript> Responses Induced by Trifluoperazine in Macrophages

The influence of the neuroleptic trifluoperazine on the intracellular concentration of Ca²⁺ in macrophages of rats was studied using a Fura-2AM fluorescent Ca²⁺ probe. It was found that trifluoperazine causes a dose-dependent increase in the intracellular Ca²⁺ concentration associated with Ca²⁺ mobilization from intracellular Ca²⁺ stores and subsequent entry of Ca²⁺ into peritoneal macrophages of rats. It was also shown that inhibitors of phospholipase A₂ (4-bromophenacyl bromide, prednisolone, and dexamethasone), cyclooxygenases (aspirin and indomethacin), and lipoxygenases (caffeic acid, zileuton, and baicalein) suppress Ca²⁺ responses induced by trifluoperazine in macrophages. The data obtained indicate the participation of enzymes and/or products of the cascade of arachidonic acid metabolism in the influence of trifluoperazine on the intracellular concentration of Ca²⁺ in peritoneal macrophages.     

Ajmalicine production in methyl jasmonate-induced Catharanthus roseus cell cultures depends on Ca2+ level

Cytosolic Ca²⁺ and jasmonate mediate signals that induce defense responses in plants. In this study, the interaction between Ca²⁺ and methyl jasmonate (MJ) in modulating defense responses was investigated by monitoring ajmalicine production in Catharanthus roseus suspension cultures. C. roseus suspensions were treated with nine combinations of CaCl₂ (3, 23, and 43 mM) and MJ (0, 10, and 100 μM) on day 6 of growth. Increased Ca²⁺ influx through the addition of extracellular CaCl₂ suppressed ajmalicine production in MJ-induced cultures. The highest ajmalicine production (4.75 mg/l) was observed when cells were treated with a low level of calcium (3 mM) combined with a high level of MJ (100 μM). In the presence of 3 mM CaCl₂ in the medium, the addition of Ca²⁺ chelator EGTA (1, 2.5, and 5 mM) or Ca²⁺ channel blocker verapamil (1, 10, and 50 μM) to MJ-induced (100 μM) cultures on day 6 also inhibited ajmalicine production at higher levels of the Ca²⁺ inhibitors. Hence, ajmalicine production in MJ-induced C. roseus cultures depended on the intracellular Ca²⁺ concentration and a low extracellular Ca²⁺ concentration (3 mM) enhanced MJ-induced ajmalicine production.     

Thapsigargin and EGTA inhibit endothelin-1-induced glucose transport

We have previously demonstrated that ET-1 may enhance glucose transport in 3T3-L1 adipocytes, secondarily to its stimulatory effect on GLUT1 gene expression by a mitogen-activated protein kinase (MAPK)-dependent pathway. In the present study, we further tested the involvement of Ca²⁺ in glucose uptake in response to ET-1. Among a variety of Ca²⁺-related agents tested, EGTA and thapsigargin were found to suppress both the glucose uptake and intracellular Ca²⁺ mobilization induced by ET-1, as determined by Fura-2 analysis. However, a phospholipase C inhibitor, U73122, also eliminated the intracellular calcium mobilization induced by ET-1, but had no effect on ET-1-stimulated glucose uptake. The finding that neither EGTA nor thapsigargin had any influence on ET-1-induced MAPK activation implies that some mechanism downstream of MAPK activation is involved. Further investigation showed that both agents exerted global inhibitory effects on protein and RNA syntheses. Since both thapsigargin and EGTA may deplete endoplasmic reticulum (ER) Ca²⁺ stores, our results suggest that (1) ET-1-induced glucose transport is independent of ET-1's effect on Ca²⁺ mobilization and (2) depletion of ER Ca²⁺ stores per se may interfere with ET-1's effect on GLUT1 expression.     

The effect of intestinal alkaline phosphatase on intestinal epithelial cells,macrophages and chronic colitis in mice

Aims

Intestinal alkaline phosphatase (IAP) is an intestinal brush border enzyme that is shown to function as a gut mucosal defense factor, but its defensive mechanism remains unclear. The aims of this study were to evaluate the effect of IAP on intestinal epithelial cells and macrophages, and on chronic colitis in interleukin-10-deficient (IL-10^−/−) mice.

Main methods

Human intestinal epithelial cells COLO 205 and peritoneal macrophages from IL-10^−/− mice were pretreated with IAP and then stimulated with lipopolysaccharide (LPS). IL-8 secretion from COLO205 cells and TNF-α, IL-6, IL-12 from peritoneal macrophages were measured by ELISA. Electrophoretic mobility shift assay was used to assess the DNA binding activity of NF-κB and IκBα phosphorylation/degradation was evaluated by immunoblot assay in COLO 205. For the in vivo study, colitis was induced in IL-10^−/− mice with piroxicam, the mice were then treated with 100 or 300 units of IAP by oral gavage for 2 weeks. Colitis was quantified by histopathologic scoring, and the phosphorylation of IκBα in the colonic mucosa was assessed using immunohistochemistry.

Key findings

IAP significantly inhibited LPS-induced inflammatory cytokine production in both IECs and peritoneal macrophages. IAP also attenuated LPS-induced NF-κB binding activity and IκBα phosphorylation/degradation in IECs. Oral administration of IAP significantly reduced the severity of colitis and down-regulated colitis-induced IκBα phosphorylation in IL-10^−/− mice.

Significance

IAP may inhibit the activation of intestinal epithelial cells and peritoneal macrophages, and may attenuate chronic murine colitis. This finding suggests that IAP supplementation is a potential therapeutic option for inflammatory bowel disease.     

Oleic acid-induced Ca2+ mobilization in human platelets: is oleic acid an intracellular messenger?

The purpose of this study was to explore the effect of oleic acid (OA) on intracellular Ca²⁺ mobilization in human platelets. When applied extracellularly, OA produced a concentration dependent rise in cytosolic [Ca²⁺] [Ca²⁺]_cyt) when extracellular [Ca²⁺] ([Ca²⁺]_ext was zero (presence of EGTA), suggesting that OA caused an intracellular release of Ca²⁺. Intracellular Ca²⁺ release was directly proportional to entry of OA into platelets and OA entry was indirectly proportional to [Ca²⁺]_ext. In permeabilized platelets, OA caused the release of ⁴⁵Ca²⁺ from ATP dependent intracellular stores. Finally, our results show that thrombin stimulated the release of [³H]OA from platelet phospholipids. The saturated fatty acids stearic and palmitic acid did not stimulate an increase in [Ca²⁺]_cyt under these conditions, but the unsaturated fatty acid, linolenic acid produced effects similar to those of OA, suggesting specificity among fatty acids for effects on [Ca²⁺]_cyt. Taken together, our experiments suggest that OA which has been incorporated into platelet phospholipids was released intothe cytosol by thrombinstimulation. Our experiments also show that OA stimulates Ca²⁺ release from intracellular stores. These results support the hypothesis that OA may serve as an intracellular messenger in human platelets.     

Hyperglycemia and Diabetes Downregulate the Functional Expression of TRPV4 Channels in Retinal Microvascular Endothelium

Retinal endothelial cell dysfunction is believed to play a key role in the etiology and pathogenesis of diabetic retinopathy. Numerous studies have shown that TRPV4 channels are critically involved in maintaining normal endothelial cell function. In the current paper, we demonstrate that TRPV4 is functionally expressed in the endothelium of the retinal microcirculation and that both channel expression and activity is downregulated by hyperglycaemia. Quantitative PCR and immunostaining demonstrated molecular expression of TRPV4 in cultured bovine retinal microvascular endothelial cells (RMECs). Functional TRPV4 activity was assessed in cultured RMECs from endothelial Ca2+-responses recorded using fura-2 microfluorimetry and electrophysiological recordings of membrane currents. The TRPV4 agonist 4α-phorbol 12,13-didecanoate (4-αPDD) increased [Ca²⁺]_i in RMECs and this response was largely abolished using siRNA targeted against TRPV4. These Ca²⁺-signals were completely inhibited by removal of extracellular Ca²⁺, confirming their dependence on influx of extracellular Ca²⁺. The 4-αPDD Ca²⁺-response recorded in the presence of cyclopiazonic acid (CPA), which depletes the intracellular stores preventing any signal amplification through store release, was used as a measure of Ca²⁺-influx across the cell membrane. This response was blocked by HC067047, a TRPV4 antagonist. Under voltage clamp conditions, the TRPV4 agonist GSK1016790A stimulated a membrane current, which was again inhibited by HC067047. Following incubation with 25mM D-glucose TRPV4 expression was reduced in comparison with RMECs cultured under control conditions, as were 4αPDD-induced Ca²⁺-responses in the presence of CPA and ion currents evoked by GSK1016790A. Molecular expression of TRPV4 in the retinal vascular endothelium of 3 months’ streptozotocin-induced diabetic rats was also reduced in comparison with that in age-matched controls. We conclude that hyperglycaemia and diabetes reduce the molecular and functional expression of TRPV4 channels in retinal microvascular endothelial cells. These changes may contribute to diabetes induced endothelial dysfunction and retinopathy.     

Differential kinetics for induction of interleukin-6 mRNA expression in murine peritoneal macrophages: Evidence for calcium-dependent and independent-signalling pathways

It is presently unclear what role elevations in intracellular calcium concentration ([Ca²⁺]_i) play in the control of monokine secretion, or whether such alterations underlie the ability of physiologic stimuli to induce production of these important signalling molecules. To address these issues, we have performed experiments in murine peritoneal macrophages to determine whether lipopolysaccharide (LPS) or interferon gamma (IFN-γ) initiate production of the proinflammatory monokine interleukin 6 (IL-6) concomitant with elevations in [Ca²⁺]_i and with kinetics similar to that seen with known Ca²⁺ mobilizing agents. Alterations in [Ca²⁺]_i after treatment with LPS, IFN-γ, platelet activating factor (PAF), or thapsigargin were measured by fluorimetric methods. These effects were compared with the ability of each to induce IL-6 mRNA expression as measured by semiquantitative reverse-transcribed polymerase chain reactions. We report that neither LPS nor IFN-γ elicited detectable elevations in [Ca²⁺]_i but that both up-regulated expression of IL-6 mRNA expression within 60 min. In contrast, experiments using either thapsigargin or PAF showed rapid and dramatic elevations in [Ca²⁺]_i with marked increases in IL-6 mRNA expression, as quickly as 15 min after initial exposure. Elevations in mRNA encoding IL-6 by thapsigargin and PAF were found to occur in a dose-dependent manner, mirroring their ability to elicit elevations in [Ca²⁺]_i. These data demonstrate that LPS and IFN-γ induce IL-6 message expression by means of Ca²⁺-independent signalling pathways. Furthermore, Ca²⁺-mobilizing agents that evoke monokine message expression do so far more rapidly than do LPS or IFN-γ. Taken in concert, these data are consistent with the hypothesis that multiple signalling pathways exist by which production of proinflammatory monokines are initiated. J. Cell. Physiol. 177:232–240, 1998. © 1998 Wiley-Liss, Inc.