Type 2 Diabetes Alters Intracellular Ca2+ Handling in Native Endothelium of Excised Rat Aorta.

An increase in intracellular Ca2+ concentration ([Ca2+]i) plays a key role in controlling endothelial functions; however, it is still unclear whether endothelial Ca2+ handling is altered by type 2 diabetes mellitus, which results in severe endothelial dysfunction. Herein, we analyzed for the first time the Ca2+ response to the physiological autacoid ATP in native aortic endothelium of obese Zucker diabetic fatty (OZDF) rats and their lean controls, which are termed LZDF rats. By loading the endothelial monolayer with the Ca2+-sensitive fluorophore, Fura-2/AM, we found that the endothelial Ca2+ response to 20 µM and 300 µM ATP exhibited a higher plateau, a larger area under the curve and prolonged duration in OZDF rats. The "Ca2+ add-back" protocol revealed no difference in the inositol-1,4,5-trisphosphate-releasable endoplasmic reticulum (ER) Ca2+ pool, while store-operated Ca2+ entry was surprisingly down-regulated in OZDF aortae. Pharmacological manipulation disclosed that sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) activity was down-regulated by reactive oxygen species in native aortic endothelium of OZDF rats, thereby exaggerating the Ca2+ response to high agonist concentrations. These findings shed new light on the mechanisms by which type 2 diabetes mellitus may cause endothelial dysfunction by remodeling the intracellular Ca2+ toolkit.

Resolvin D1, but not resolvin E1, transactivates the epidermal growth factor receptor to increase intracellular calcium and glycoconjugate secretion in rat and human conjunctival goblet cells.

PURPOSE: To identify interactions of the epidermal growth factor receptor (EGFR) with the pro-resolving mediator receptors for RvD1 and RvE1 to stimulate an increase in intracellular [Ca2+] ([Ca2+]i) and mucin secretion from cultured human and rat conjunctival goblet cells. METHODS: Goblet cells from human and rat conjunctiva were grown in culture using RPMI media. Cultured goblet cells were pre-incubated with inhibitors, and then stimulated with RvD1, RvE1, EGF or the cholinergic agonist carbachol (Cch). Increase in [Ca2+]i was measured using fura-2/AM. Goblet cell secretion was measured using an enzyme-linked lectin assay with UEA-1. Western blot analysis was performed with antibodies against AKT and ERK 1/2. RESULTS: In cultured human conjunctival goblet cells RvE1 -stimulated an increase in [Ca2+]i. RvD1-, but not the RvE1-, stimulated increase in [Ca2+]i and mucin secretion was blocked by the EGFR inhibitor AG1478 and siRNA for the EGFR. RvD1-, but not RvE1-stimulated an increase in [Ca2+]i that was also inhibited by TAPI-1, an inhibitor of the matrix metalloprotease ADAM 17. Inhibition of the EGFR also blocked RvD1-stimulated increase in AKT activity and both RvD1-and RvE1-stimulated increase in ERK 1/2 activity. Pretreatment with either RvD1 or RvE1 did not block the EGFR-stimulated increase in [Ca2+]i. CONCLUSIONS: We conclude that in cultured rat and human conjunctival goblet cells, RvD1 activates the EGFR, increases [Ca2+]i, activates AKT and ERK1/2 to stimulate mucin secretion. RvE1 does not transactivate the EGFR to increase [Ca2+]I and stimulate mucin secretion, but does interact with the receptor to increase ERK 1/2 activity.

Rapid screening of drug candidates against EGFR/HER2 signaling pathway using fluorescence assay.

Over the recent decade, the calcium-based assays have gained much popularity in order to discover new drugs. Since breast cancer is the second cause of death in the female population, rapid and effective methods are needed to screen drug compounds with fewer side effects. Human epidermal growth factor receptor 2 (HER2) increases intracellular free Ca2+ on its signaling pathways. In the present study, BT474 cell line, which overexpresses HER2 receptor, was selected and using fura-2-AM, intracellular Ca2+ release was investigated. The changes in the concentration of intracellular Ca2+ were evaluated by variation in the amount of fluorescence intensity. In the presence of epidermal growth factor (EGF), an increase in fluorescence intensity was observed so that after 20 min it raised to the maximum level. After treatment of BT474 cells by lapatinib, as a tyrosine kinase inhibitor (TKI), the signaling pathway of EGFR/HER2 heterodimer was significantly inhibited, which resulted in a decrease in Ca2+ entry into the cytoplasm and fluorescence emission decreased. The IC50 value for the effect of lapatinib on BT474 cells was 113.2 nmol/L. Our results suggest this method is a simple, efficient and specific approach and can potentially be useful for screening new drug candidates against EGFR/HER2 heterodimer signaling pathways. Graphical abstract ᅟ.

Activation of the EGF Receptor by Histamine Receptor Subtypes Stimulates Mucin Secretion in Conjunctival Goblet Cells.

Purpose: The purpose of this study was to determine if histamine receptors interact with the epidermal growth factor receptor (EGFR) in cultured rat conjunctival goblet cells. Methods: Goblet cells from rat conjunctiva were grown in organ culture. First-passage goblet cells were used in all experiments. Phosphorylated (active) and total EGFR, AKT, and extracellular signal-regulated kinase (ERK)1/2 were measured by Western blot analysis. Cells were preincubated with the EGFR antagonist AG1478 for 30 minutes or small interfering RNA specific to the EGFR for 3 days prior to stimulation with histamine or agonists specific for histamine receptor subtypes for 2 hours. Goblet cell secretion was measured using an enzyme-linked lectin assay. Goblet cells were incubated for 1 hour with the calcium indicator molecule fura-2/AM, and intracellular [Ca2+] ([Ca2+]i) was determined. Data were collected in real time and presented as the actual [Ca2+]i with time and as the change in peak [Ca2+]i. Results: Histamine increased the phosphorylation of the EGFR. Mucin secretion and increase in [Ca2+]i stimulated by histamine, and agonists specific for each histamine receptor subtype were blocked by inhibition of the EGFR. Increase in [Ca2+]i stimulated by histamine and specific agonists for each histamine receptor was also inhibited by TAPI-1, a matrix metalloproteinase (MMP) inhibitor. The histamine-stimulated increase in activation of AKT, but not ERK1/2, was blocked by AG1478. Conclusions: In conjunctival goblet cells, histamine, using all four receptor subtypes, transactivates the EGFR via an MMP. This in turn phosphorylates AKT to increase [Ca2+]i and stimulate mucin secretion.

Poly-arginine R18 and R18D (D-enantiomer) peptides reduce infarct volume and improves behavioural outcomes following perinatal hypoxic-ischaemic encephalopathy in the P7 rat.

We examined the neuroprotective efficacy of the poly-arginine peptide R18 and its D-enantiomer R18D in a perinatal hypoxic-ischaemic (HI) model in P7 Sprague-Dawley rats. R18 and R18D peptides were administered intraperitoneally at doses of 30, 100, 300 or 1000 nmol/kg immediately after HI (8% O2/92%N2 for 2.5 h). The previously characterised neuroprotective JNKI-1-TATD peptide at a dose of 1000 nmol/kg was used as a control. Infarct volume and behavioural outcomes were measured 48 h after HI. For the R18 and R18D doses examined, total infarct volume was reduced by 25.93% to 43.80% (P = 0.038 to < 0.001). By comparison, the JNKI-1-TATD reduced lesion volume by 25.27% (P = 0.073). Moreover, R18 and R18D treatment resulted in significant improvements in behavioural outcomes, while with JNKI-1-TATD there was a trend towards improvement. As an insight into the likely mechanism underlying the effects of R18, R18D and JNKI-1-TATD, the peptides were added to cortical neuronal cultures exposed to glutamic acid excitotoxicity, resulting in up to 89, 100 and 71% neuroprotection, respectively, and a dose dependent inhibition of neuronal calcium influx. The study further confirms the neuroprotective properties of poly-arginine peptides, and suggests a potential therapeutic role for R18 and R18D in the treatment of HIE.

5-Lipoxygenase inhibitor zileuton inhibits Ca(2+)-responses induced by glutoxim and molixan in macrophages.

Using Fura-2AM microfluorimetry, we have shown for the first time that 5-lipoxygenase specific inhibitor antiasthmatic agent zileuton significantly inhibits Ca(2+)-responses induced by glutoxim and molixan in macrophages. The results support 5-lipoxygenase involvement in the effect of glutoxim and molixan on intracellular Ca(2+) concentration in macrophages and indicate the inadvisability of a combined use of drugs glutoxim and molixan and antiasthmatic agent zileuton.

Methyl-ß-cyclodextrin inhibits Ca2+-responses induced by glutoxim and molixan in macrophages.

Using Fura-2AM microfluorimetry, we have shown for the first time that methyl-ß-cyclodextrin, inducing cholesterol extraction from membranes and raft disruption, significantly inhibits glutoxim- and molixan-induced Ca2+-responses in rat peritoneal macrophages. The results suggest that intact rafts are necessary for signaling cascade induced by glutoxim or molixan and leading to intracellular Ca2+ concentration increase in macrophages.

Involvement of the Arp2/3 complex and WASP proteins in the effect of glutoxim and molixan on intracellular Ca(2+) concentration in macrophages.

The Fura-2AM fluorescent Ca(2+) probe was used to study the possibility that the Arp2/3 complex and WASP proteins are involved in the effects of glutoxim and molixan on the intracellular Ca(2+) concentration in macrophages. It has been demonstrated that preincubation of macrophages with inhibitors of the Arp2/3 complex or WASP proteins (CK-0944666 or wiskostatin, respectively) results in a significant suppression of Ca(2+)-responses induced by glutoxim or molixan. This suggests that polymerization of actin filaments is a process involved in the effect of glutoxim or molixan on intracellular Ca(2+) concentration in macrophages.

Nitric oxide stress and activation of AMP-activated protein kinase impair ß-cell sarcoendoplasmic reticulum calcium ATPase 2b activity and protein stability.

The sarcoendoplasmic reticulum Ca(2+) ATPase 2b (SERCA2b) pump maintains a steep Ca(2+) concentration gradient between the cytosol and ER lumen in the pancreatic ß-cell, and the integrity of this gradient has a central role in regulated insulin production and secretion, maintenance of ER function and ß-cell survival. We have previously demonstrated loss of ß-cell SERCA2b expression under diabetic conditions. To define the mechanisms underlying this, INS-1 cells and rat islets were treated with the proinflammatory cytokine interleukin-1ß (IL-1ß) combined with or without cycloheximide or actinomycin D. IL-1ß treatment led to increased inducible nitric oxide synthase (iNOS) gene and protein expression, which occurred concurrently with the activation of AMP-activated protein kinase (AMPK). IL-1ß led to decreased SERCA2b mRNA and protein expression, whereas time-course experiments revealed a reduction in protein half-life with no change in mRNA stability. Moreover, SERCA2b protein but not mRNA levels were rescued by treatment with the NOS inhibitor l-NMMA (NG-monomethyl L-arginine), whereas the NO donor SNAP (S-nitroso-N-acetyl-D,L-penicillamine) and the AMPK activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) recapitulated the effects of IL-1ß on SERCA2b protein stability. Similarly, IL-1ß-induced reductions in SERCA2b expression were rescued by pharmacological inhibition of AMPK with compound C or by transduction of a dominant-negative form of AMPK, whereas ß-cell death was prevented in parallel. Finally, to determine a functional relationship between NO and AMPK signaling and SERCA2b activity, fura-2/AM (fura-2-acetoxymethylester) Ca(2+) imaging experiments were performed in INS-1 cells. Consistent with observed changes in SERCA2b expression, IL-1ß, SNAP and AICAR increased cytosolic Ca(2+) and decreased ER Ca(2+) levels, suggesting congruent modulation of SERCA activity under these conditions. In aggregate, these results show that SERCA2b protein stability is decreased under inflammatory conditions through NO- and AMPK-dependent pathways and provide novel insight into pathways leading to altered ß-cell calcium homeostasis and reduced ß-cell survival in diabetes.

Vibrio vulnificus RtxA1 modulated calcium flux contributes reduced internalization in phagocytes.

AIMS: Vibrio vulnificusis an opportunistic pathogen that causes primary septicemia and wound infection with high mortality rate. This pathogen produces an RTX toxin (RtxA1) which can cause host cell rounding, cell death and interference with internalization by host phagocytes. However, the mechanism of RtxA1-induced phagocyte paralysis is not clear. MAIN METHODS: Using the murine macrophage cell line RAW264.7, we measured cytotoxicity and phagocytosis of V. vulnificusin normal and calcium-depleted media. To deplete extracellular and cytosolic Ca(2+), cells were exposed to the calcium chelators ethylene glycol tetraacetic acid (EGTA) and 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl esteris (BAPTA-AM), respectively. The cytotoxicity was examined by measuring the activity of lactate dehydrogenase (LDH) released from the damaged cells. The gentamicin protection assay was conducted to determine the number of internalized bacteria, while acridine orange staining was applied to visualize the intracellular bacteria. The fluorescent indicator fura-2-acetoxymethyl ester (fura 2-AM) was used to measure the Ca(2+)signal post-infection. KEY FINDINGS: We revealed that extracellular Ca(2+)was essential for phagocytes to internalize V. vulnificus. Meanwhile, cytosolic Ca(2+)flux in RAW264.7 cells induced by an RtxA1 isogenic mutant was repressed by the parent strain. Furthermore, depletion of extracellular Ca(2+)level by EGTA significantly reduced the cytotoxicity but did not affect the antiphagocytic activity of RtxA1 toxin. SIGNIFICANCE: Our results indicated that RtxA1 may interfere with cytosolic Ca(2+)flux of phagocyte to promote bacteria colonization.

Effect of deoxycholic acid on Ca2+ movement, cell viability and apoptosis in human gastric cancer cells.

Deoxycholic acid (DOA) is one of the secondary bile acids used as a mild detergent for the isolation of membrane associated proteins. This study examined whether the secondary bile acid, DOA, altered Ca(2+) movement, cell viability and apoptosis in SCM1 human gastric cancer cells. The Ca(2+)-sensitive fluorescent dye fura-2 was used to measure [Ca(2+)]i. DOA-evoked [Ca(2+)]i rises concentration dependently. The response was reduced by removing extracellular Ca(2+). DOA-evoked Ca(2+) entry was inhibited by store-operated Ca(2+) channel inhibitors (nifedipine, econazole and SKF96365), the protein kinase C (PKC) activator phorbol 12-myristate 13 acetate (PMA) and the PKC inhibitor GF109203X. In Ca(2+)-free medium, treatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (TG) abolished DOA-evoked [Ca(2+)]i rises. Conversely, treatment with DOA abolished TG-evoked [Ca(2+)]i rises. Inhibition of phospholipase C with U73122 abolished DOA-evoked [Ca(2+)]i rises. At 100-500 µM, DOA decreased cell viability, which was not changed by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). DOA between 100 and 300 µM also induced apoptosis. Collectively, in SCM1 cells, DOA-induced [Ca(2+)]i rises by evoking phospholipase C-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via store-operated Ca(2+) channels. DOA also caused Ca(2+)-independent apoptosis.

NAD derived second messengers: Role in spontaneous diastolic Ca(2+) transients in murine cardiac myocytes.

Strong ß-adrenergic stimulation induced spontaneous diastolic Ca(2+) transients (SCTs) in electrically paced murine cardiac myocytes [28]. To obtain further insights into the underlying mechanism, we developed a method for a simultaneous analysis, in which the free luminal Ca(2+) concentration in the sarcoplasmic reticulum (SR) ([Ca(2+)]SR) and the free cytosolic Ca(2+) concentration ([Ca(2+)]i) were measured in parallel in the same cell. Each spontaneous diastolic Ca(2+) transient was exactly mirrored by a decrease of [Ca(2+)]SR. Since antagonism of the Ca(2+) mobilizing second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) was shown to block SCTs in single cardiac myocytes [28], we analyzed the effect of the novel ADP-ribosyl cyclase inhibitor SAN4825 on both cytosolic and intra-luminal Ca(2+) transients upon strong ß-adrenergic stimulation. A strong antagonist effect of SAN4825 on SCTs at low micromolar concentrations was observed. Our results suggest that the underlying mechanism of spontaneous diastolic Ca(2+) transients observed upon strong ß-adrenergic stimulation is sensitization of type 2 ryanodine receptor by the Ca(2+) releasing activity of the products of ADP-ribosyl cyclase activity.

Glibenclamide decreases ATP-induced intracellular calcium transient elevation via inhibiting reactive oxygen species and mitochondrial activity in macrophages.

Increasing evidence has revealed that glibenclamide has a wide range of anti-inflammatory effects. However, it is unclear whether glibenclamide can affect the resting and adenosine triphosphate (ATP)-induced intracellular calcium ([Ca(2+)]i) handling in Raw 264.7 macrophages. In the present study, [Ca(2+)]i transient, reactive oxygen species (ROS) and mitochondrial activity were measured by the high-speed TILLvisION digital imaging system using the indicators of Fura 2-am, DCFDA and rhodamine-123, respectively. We found that glibenclamide, pinacidil and other unselective K(+) channel blockers had no effect on the resting [Ca(2+)]i of Raw 264.7 cells. Extracellular ATP (100 µM) induced [Ca(2+)]i transient elevation independent of extracellular Ca(2+). The transient elevation was inhibited by an ROS scavenger (tiron) and mitochondria inhibitor (rotenone). Glibenclamide and 5-hydroxydecanoate (5-HD) also decreased ATP-induced [Ca(2+)]i transient elevation, but pinacidil and other unselective K(+) channel blockers had no effect. Glibenclamide also decreased the peak of [Ca(2+)]i transient induced by extracellular thapsigargin (Tg, 1 µM). Furthermore, glibenclamide decreased intracellular ROS and mitochondrial activity. When pretreated with tiron and rotenone, glibenclamide could not decrease ATP, and Tg induced maximal [Ca(2+)]i transient further. We conclude that glibenclamide may inhibit ATP-induced [Ca(2+)]i transient elevation by blocking mitochondria KATP channels, resulting in decreased ROS generation and mitochondrial activity in Raw 264.7 macrophages.

Acute disturbance of calcium homeostasis in PC12 cells as a novel mechanism of action for (sub)micromolar concentrations of organophosphate insecticides.

Organophosphates (OPs) and carbamates are widely used insecticides that exert their neurotoxicity via inhibition of acetylcholine esterase (AChE) and subsequent overexcitation. OPs can induce additional neurotoxic effects at concentrations below those for inhibition of AChE, indicating other mechanisms of action are also involved. Since tight regulation of the intracellular calcium concentration ([Ca(2+)]i) is essential for proper neuronal development and function, effects of one carbamate (carbaryl) and two OPs (chlorpyrifos, parathion-ethyl) as well as their -oxon metabolites on [Ca(2+)]i were investigated. Effects of acute (20min) exposure to (mixtures of) insecticides on basal and depolarization-evoked [Ca(2+)]i were measured in fura-2-loaded PC12 cells using single-cell fluorescence microscopy. Acute exposure to chlorpyrifos and its metabolite chlorpyrifos-oxon (10µM) induced a modest increase in basal [Ca(2+)]i. More importantly, the tested OPs concentration-dependently inhibited depolarization-evoked [Ca(2+)]i. Chlorpyrifos already induced a ∼30% inhibition at 0.1µM and a 100% inhibition at 10µM (IC50=0.43µM), whereas parathion-ethyl inhibited the depolarization-evoked [Ca(2+)]i increase with ∼70% at 10µM. Interestingly, -oxon metabolites were more potent inhibitors of AChE, but were less potent inhibitors of depolarization-evoked [Ca(2+)]i compared to their parent compound (chlorpyrifos-oxon) or were even without effect (paraoxon-ethyl and -methyl). Similarly, acute exposure to carbaryl had no effect on [Ca(2+)]i. Exposure to mixtures of chlorpyrifos with its oxon-analog or with parathion-ethyl did not increase the degree of inhibition, indicating additivity does not apply. These data demonstrate that concentration-dependent inhibition of depolarization-evoked [Ca(2+)]i is a novel mechanism of action of (sub)micromolar concentrations of OPs that could partly underlie OP-induced neurotoxicity.

Reporter dyes demonstrate functional expression of multidrug resistance proteins in the marine flatworm Macrostomum lignano: the sponge-derived dye Ageladine A is not a substrate of these transporters.

The marine plathyhelminth Macrostomum lignano was recently isolated from Adriatic shore sediments where it experiences a wide variety of environmental challenges, ranging from hypoxia and reoxygenation, feeding on toxic algae, to exposure to anthropogenic contaminants. As multidrug resistance transporters constitute the first line of defense against toxins and toxicants we have studied the presence of such transporters in M. lignano in living animals by applying optical methods and pharmacological inhibitors that had been developed for mammalian cells. Application of the MDR1 inhibitor Verapamil or of the MRP1 inhibitors MK571 or Probenecid increased the intracellular fluorescence of the reporter dyes Fura-2 am, Calcein am, Fluo-3 am in the worms, but did not affect their staining with the dyes Rhodamine B, CMFDA or Ageladine A. The marine sponge alkaloid Ageladine A remained intracellularly trapped for several days in the worms, suggesting that it does not serve as substrate of multidrug resistance exporters. In addition, Ageladine A did not affect multidrug resistance-associated protein (MRP)-mediated dye export from M. lignano or the MRP1-mediated glutathione (GSH) export from cultured rat brain astrocytes. The data obtained demonstrate that life-imaging is a useful tool to address physiological drug export from intact marine transparent flatworms by using multiphoton scanning microscopy.

The loss of sustained Ca(2+) signaling underlies suppressed endothelial nitric oxide production in preeclamptic pregnancies: implications for new therapy.

Approximately 8% of pregnancies are complicated by preeclampsia (PE), a hypertensive condition characterized by widespread endothelial dysfunction. Reduced nitric oxide (NO) output in PE subjects has been inferred but not directly measured, and there is little understanding of why this occurs. To address this we have used direct imaging of changes in intracellular Ca(2+) concentration ([Ca(2+)]i) and NO in umbilical vein endothelium of normal and PE subjects that is still intact and on the vessel luminal surface. This was achieved by dissection and preloading with fura 2 and DAF-2 imaging dyes, respectively, before subsequent challenge with ATP (100 µM, 30 min). As a control to reveal the content of active endothelial nitric oxide synthase (eNOS) per vessel segment, results were compared with a maximal stimulus with ionomycin (5 µM, 30 min). We show for the first time that normal umbilical vein endothelial cells respond to ATP with sustained bursting that parallels sustained NO output. Furthermore, in subjects with PE, a failure of sustained [Ca(2+)]i bursting occurs in response to ATP and is associated with blunted NO output. In contrast, NO responses to maximal [Ca(2+)]i elevation using ionomycin and the levels of eNOS protein are more similar between groups than the responses to ATP. When the endothelial cells from PE subjects are isolated and allowed to recover in culture, they regain the ability under fura 2 imaging to show multiple [Ca(2+)]i bursts otherwise seen in the cells from normal subjects. Thus novel clinical therapy aimed at restoring function in vivo may be possible.

Signaling pathways used by EGF to stimulate conjunctival goblet cell secretion.

The purpose of this study was to identify the signaling pathways that epidermal growth factor (EGF) uses to stimulate mucin secretion from cultured rat conjunctival goblet cells and to compare the pathways used by EGF with those used by the known secretagogue muscarinic, cholinergic agonists. To this end, goblet cells from rat conjunctiva were grown in culture using RPMI media. For immunofluorescence experiments, antibodies against EGF receptor (EGFR) and ERK 2 as well as muscarinic receptors (M(1)AchR, M(2)AchR, and M(3)AchR) were used, and the cells viewed by fluorescence microscopy. Intracellular [Ca(2+)] ([Ca(2+)](i)) was measured using fura 2/AM. Glycoconjugate secretion was determined after cultured goblet cells were preincubated with inhibitors, and then stimulated with EGF or the cholinergic agonist carbachol (Cch). Goblet cell secretion was measured using an enzyme-linked lectin assay with UEA-I or ELISA for MUC5AC. In cultured goblet cells EGF stimulated an increase in [Ca(2+)](i) in a concentration-dependent manner. EGF-stimulated increase in [Ca(2+)](i) was blocked by inhibitors of the EGF receptor and removal of extracellular Ca(2+). Inhibitors against the EGFR and ERK 1/2 blocked EGF-stimulated mucin secretion. In addition, cultured goblet cells expressed M(1)AchR, M(2)AchR, and M(3)AchRs. Cch-stimulated increase in [Ca(2+)](i) was blocked by inhibitors for the M(1)AchRs, matrix metalloproteinases, and EGF receptors. Inhibitors against the EGF receptor and ERK 1/2 also blocked Cch-stimulated mucin secretion. We conclude that in conjunctival goblet cells, EGF itself increases [Ca(2+)](i) and activates ERK 1/2 to stimulate mucin secretion. EGF-stimulated secretion is dependent on extracellular Ca(2+). This mechanism of action is similar to cholinergic agonists that use muscarinic receptors to transactivate the EGF receptor, increase [Ca(2+)](i), and activate ERK 1/2 leading to an increase in mucin secretion.

CRH activation of different signaling pathways results in differential calcium signaling in human pregnant myometrium before and during labor.

CONTEXT: Our previous study has demonstrated that CRH has differential effects on human uterine contractility before and after onset of labor. Intracellular Ca2+ concentration ([Ca2+]i) mobilization plays an important role in the control of uterine contraction. OBJECTIVE: Our objective was to investigate the effects of CRH on [Ca2+]i homeostasis in laboring and nonlaboring myometrial cells and determine subsequent signaling involved in [Ca2+]i regulation by CRH. DESIGN: The myometrial tissues were obtained from pregnant women who were undergoing or not undergoing labor at term. [Ca2+]i was determined by Ca2+ imaging system using the fluorescent dye fura-2-acetoxymethyl ester. Western blot analysis, ELISA, and RIA were used to determine the signaling pathways induced by CRH. RESULTS: CRH induced Ca2+ transient in laboring cells, which was blocked by CRH receptor type 1 (CRHR1) antagonist antalarmin. CRHR1 knockdown impaired this effect of CRH. CRH activated Gi protein, decreased cAMP production, and induced phosphorylated phospholipase C-ß3 and inositol-1,4,5-triphosphate production. Phospholipase C and inositol-1,4,5-triphosphate receptor inhibitors blocked the CRH-induced Ca2+ transient in laboring cells. CRH did not induce whereas antalarmin induced the Ca2+ transient in nonlaboring cells. Knockdown of CRHR1 impaired the effect of antalarmin. CRH acted on CRHR1 to activate Gs in nonlaboring cells. Forskolin blocked antalarmin-induced Ca2+ transient. CONCLUSIONS: CRH acts on CRHR1 to activate different signaling pathways before and after onset of labor, thereby resulting in differential calcium signaling in response to CRH. The signaling pathways of CRHR1 might serve as a target for the development of new therapeutic strategies for preterm birth.

High-throughput assays to measure intracellular Ca²âº mobilization in cells that express recombinant S1P receptor subtypes.

Intracellular Ca(2+) mobilization is a useful readout to screen for agonists or antagonists of G-protein -coupled receptors (GPCRs). Here, we describe methods to conduct high-throughput screening of stably or transiently transfected HTC4 cells expressing the individual S1P1-5 receptor subtypes. The cells are grown in 96-well plates and loaded with the cell permeable fluorescent Ca(2+) indicator dye Fura-2-AM. Changes in intracellular Ca(2+) levels in response to S1P or test compounds are detected using a FlexStation II scanning fluorometer with integrated fluidics transfer capabilities.

GPER regulates endothelin-dependent vascular tone and intracellular calcium.

AIMS: An increase in intracellular vascular smooth muscle cell calcium concentration (VSMC [Ca(2+)](i)) is essential for endothelin-1 (ET-1)-induced vasoconstriction. Based on previous findings that activation of the G protein-coupled estrogen receptor (GPER) inhibits vasoconstriction in response to ET-1 and regulates [Ca(2+)](i) in cultured VSMC, we investigated whether endogenous GPER regulates ET-1-induced changes in VSMC [Ca(2+)](i) and constriction of intact arteries. MAIN METHODS: Pressurized carotid arteries of GPER-deficient (GPER(0)) and wildtype (WT) mice were loaded with the calcium indicator fura 2-AM. Arteries were stimulated with the GPER-selective agonist G-1 or solvent followed by exposure to ET-1. Changes in arterial diameter and VSMC [Ca(2+)](i) were recorded simultaneously. Vascular gene expression levels of ET(A) and ET(B) receptors were determined by qPCR. KEY FINDINGS: ET-1-dependent vasoconstriction was increased in arteries from GPER(0) compared to arteries from WT mice. Despite the more potent vasoconstriction to ET-1, GPER deficiency was associated with a marked reduction in the ET-1-stimulated VSMC [Ca(2+)](i) increase, suggesting an increase in myofilament force sensitivity to [Ca(2+)](i). Activation of GPER by G-1 had no effect on vasoconstriction or VSMC [Ca(2+)](i) responses to ET-1, and expression levels of ET(A) or ET(B) receptor were unaffected by GPER deficiency. SIGNIFICANCE: These results demonstrate that endogenous GPER inhibits ET-1-induced vasoconstriction, an effect that may be associated with reduced VSMC Ca(2+) sensitivity. This represents a potential mechanism through which GPER could contribute to protective effects of endogenous estrogen in the cardiovascular system.