Inositol Phosphate Formation Research Articles

Tracing G-Protein-Mediated Contraction and Relaxation in Vascular Smooth Muscle Cell Spheroids.

Analyses of G-protein-mediated contraction and relaxation of vascular smooth muscle cells (VSMCs) are usually hampered by a rigid growth surface and culture conditions promoting cell proliferation and a less contractile phenotype. Our studies indicated that mouse aortic VSMCs cultured in three-dimensional spheroids acquire a quiescent contractile status while decreasing the baseline G-protein-dependent inositolphosphate formation and increasing the expression of endothelin receptor type A (Ednra). Endothelin-1 (ET-1) promoted inositolphosphate formation in VSMC spheroids, but not in VSMCs cultured under standard conditions. To trace ET-1-mediated contraction of VSMC spheroids, we developed an assay by adhering them to collagen hydrogels and recording structural changes by time-lapse microscopy. Under these conditions, mouse and human VSMC spheroids contracted upon treatment with ET-1 and potassium chloride or relaxed in response to caffeine and the prostacyclin analogue Iloprost. ET-1 activated AKT-, MKK1-, and MKK3/6-dependent signaling cascades, which were inhibited by an overexpressing regulator of G-protein signaling 5 (Rgs5) to terminate the activity of Gα subunits. In summary, culture of VSMCs in three-dimensional spheroids lowers baseline G-protein activity and enables analyses of both contraction and relaxation of mouse and human VSMCs. This model serves as a simple and versatile tool for drug testing and investigating G-protein-depending signaling.

Kinetics of phytate hydrolysis during storage of red kidney beans and the implication in hard-to-cook development

In this study, the kinetics of phytate (inositol hexaphosphate, InsP6) hydrolysis by endogenous phytase in red kidney beans stored at varying conditions of temperature (25–42 °C) and moisture content (6.9–14.5%) was determined and the potential role in hard-to-cook (HTC) development was evaluated. In addition, the concept of glass transition temperature (Tg) was assessed and correlated against the rate of phytate hydrolysis. Under the conditions studied, phytate hydrolysis during storage was mainly influenced by storage temperature and time with limited influence of storage moisture content whereby the highest and lowest storage temperatures (42 °C and 25 °C) resulted in the highest and lowest hydrolysis rates (0.058 ± 0.003 and 0.003 ± 0.001 week−1). Hydrolysis of phytate resulted in formation of lower inositol phosphates, inositol pentaphosphate (InsP5) representing an intermediate whose concentration increased with storage time and temperature. The relationship between the rate of InsP6 hydrolysis and storage above the overall Tg (T − Tg) was moisture content dependent implying that this difference did not fully explain InsP6 hydrolysis. Nevertheless, for each moisture content, the rates of InsP6 hydrolysis during storage were strongly correlated (r > 0.98, p < 0.05) with rates of HTC development signifying that InsP6 hydrolysis facilitates HTC development in beans.

PH-Sensing G Protein-Coupled Receptor OGR1 (GPR68) Expression and Activation Increases in Intestinal Inflammation and Fibrosis.

Local extracellular acidification occurs at sites of inflammation. Proton-sensing ovarian cancer G-protein-coupled receptor 1 (OGR1, also known as GPR68) responds to decreases in extracellular pH. Our previous studies show a role for OGR1 in the pathogenesis of mucosal inflammation, suggesting a link between tissue pH and immune responses. Additionally, pH-dependent signalling is associated with the progression of intestinal fibrosis. In this study, we aimed to investigate OGR1 expression and OGR1-mediated signalling in patients with inflammatory bowel disease (IBD). Our results show that OGR1 expression significantly increased in patients with IBD compared to non-IBD patients, as demonstrated by qPCR and immunohistochemistry (IHC). Paired samples from non-inflamed and inflamed intestinal areas of IBD patients showed stronger OGR1 IHC staining in inflamed mucosal segments compared to non-inflamed mucosa. IHC of human surgical samples revealed OGR1 expression in macrophages, granulocytes, endothelial cells, and fibroblasts. OGR1-dependent inositol phosphate (IP) production was significantly increased in CD14+ monocytes from IBD patients compared to healthy subjects. Primary human and murine fibroblasts exhibited OGR1-dependent IP formation, RhoA activation, F-actin, and stress fibre formation upon an acidic pH shift. OGR1 expression and signalling increases with IBD disease activity, suggesting an active role of OGR1 in the pathogenesis of IBD.

Phytate degradation and phosphorus utilisation by broiler chickens fed diets containing wheat with increased phytase activity

ABSTRACT 1. The objective of this study was to investigate wheat genotypes bred for increased intrinsic phytase activity for InsP6 disappearance and the formation of lower inositol phosphates in such wheat-fed broiler chickens. The influence of monocalcium phosphate (MCP) supplementation on these characteristics and the utilisation of P and Ca were also determined. A three-step in vitro assay and a broiler trial were performed. 2. In the 63 wheat genotypes tested in vitro, phytase activity varied from 1900 FTU/kg to 5200 FTU/kg, and InsP6 disappearance increased with higher phytase activity of wheat in a linear manner. The addition of MCP significantly reduced in vitro InsP6 disappearance by one-third, independent of the inclusion level of wheat in the feed. When exogenous phytase was added to wheat, in vitro InsP6 disappearance increased independently of the phytase activity of the wheat used. 3. In the broiler trial, four wheat genotypes with phytase activities between 2400 and 3700 FTU/kg were included at 400 g/kg in diets with and without MCP. The diets were not pelleted. Separately, wheat 1, without MCP, was tested with the addition of exogenous phytase. Unsexed Ross 308 broiler chickens were allocated to 72 metabolic units of 10 birds each and assigned one of the nine diets. Mineral utilisation was measured based on excreta collection from 20 to 23 d of age. Digesta from the crop and terminal ileum were collected on d 24. 4. In the crop and ileum, InsP6 disappearance was not affected by the wheat genotypes, but the addition of MCP significantly decreased InsP6 disappearance. Precaecal P disappearance was significantly reduced by the addition of MCP, with wheat genotypes also exerting an effect. Wheat genotypes and the addition of exogenous phytase significantly affected P utilisation. Exogenous phytase had no effect on InsP6 disappearance in the crop but did up to the terminal ileum, the precaecal InsP6 and P disappearance increased with the addition of exogenous phytase. 5. Although the intrinsic wheat phytase activity exerted distinct effects on in vitro InsP6 disappearance, no such effect was found in the broiler trial. The addition of MCP significantly inhibited InsP6 degradation in vitro and in vivo.

Noncatalytic Bruton's tyrosine kinase activates PLCγ2 variants mediating ibrutinib resistance in human chronic lymphocytic leukemia cells

Treatment of patients with chronic lymphocytic leukemia (CLL) with inhibitors of Bruton's tyrosine kinase (BTK), such as ibrutinib, is limited by primary or secondary resistance to this drug. Examinations of CLL patients with late relapses while on ibrutinib, which inhibits BTK's catalytic activity, revealed several mutations in BTK, most frequently resulting in the C481S substitution, and disclosed many mutations in PLCG2, encoding phospholipase C-γ2 (PLCγ2). The PLCγ2 variants typically do not exhibit constitutive activity in cell-free systems, leading to the suggestion that in intact cells they are hypersensitive to Rac family small GTPases or to the upstream kinases spleen-associated tyrosine kinase (SYK) and Lck/Yes-related novel tyrosine kinase (LYN). The sensitivity of the PLCγ2 variants to BTK itself has remained unknown. Here, using genetically-modified DT40 B lymphocytes, along with various biochemical assays, including analysis of PLCγ2-mediated inositol phosphate formation, inositol phospholipid assessments, fluorescence recovery after photobleaching (FRAP) static laser microscopy, and determination of intracellular calcium ([Ca2+] i ), we show that various CLL-specific PLCγ2 variants such as PLCγ2S707Y are hyper-responsive to activated BTK, even in the absence of BTK's catalytic activity and independently of enhanced PLCγ2 phospholipid substrate supply. At high levels of B-cell receptor (BCR) activation, which may occur in individual CLL patients, catalytically-inactive BTK restored the ability of the BCR to mediate increases in [Ca2+] i Because catalytically-inactive BTK is insensitive to active-site BTK inhibitors, the mechanism involving the noncatalytic BTK uncovered here may contribute to preexisting reduced sensitivity or even primary resistance of CLL to these drugs.

OGR1 mediates the inhibitory effects of acidic environment on proliferation and angiogenesis of endothelial progenitor cells.

Ovarian cancer G-protein-coupled receptor 1 (OGR1), an acid-sensitive receptor, plays a key proton-sensing role through stimulation of inositol phosphate formation. Avascular necrosis of the femoral head is characterized by apoptosis of bone cells mainly resulting from deficient local blood perfusion, eventually leading to acidification with disruption of endothelial progenitor cells' (EPCs) function. However, whether EPCs express OGR1 has not been demonstrated. This study attempted to whether OGR1 mediates the effects of acid on proliferation, migration, and angiogenesis in EPCs. FITC-UEA-I and Dil-Ac-LDL double-staining methods were used to identify EPCs. Expression of OGR1 was analyzed by RT-PCR (reverse transcription PCR) and western blot after incubation in media ranging in pH, cell counting kit-8 and cell cycle analysis were used to analyze proliferation and cell cycle distribution. Scratch test, transwell migration assay, and tube formation experiments were performed to analyze migration and vascularization of EPCs after silencing OGR1 with small interfering RNA (siRNA). The result show EPCs were positive for FITC-UEA-I and Dil-Ac-LDL double-staining and expressed OGR1. The expression of OGR1 increased gradually with decreased pH and was highest in pH 6.4 medium. Incubation in pH 6.4 medium inhibited proliferation of EPCs and caused cell cycle arrest. Silencing of OGR1 using siRNA partially reversed the effect of acidic environment on EPCs. Migration and angiogenesis of EPCs were inhibited in pH 6.4 medium, and silencing of OGR1 partially reversed this effect. The results demonstrated expression of OGR1 in EPCs, and the OGR1 mediated the effects of acidic environment on proliferation, migration, and angiogenesis of EPCs.

β‐arrestins mediate rapid 5‐HT2A receptor endocytosis to regulate intensity and duration of signaling

Serotonin 5‐HT2A receptors (5‐HT2AR) regulate important physiological processes including platelet aggregation, smooth muscle contraction, mood and perception. 5‐HT2AR are the site of action for most hallucinogens and are principle molecular targets for atypical antipsychotic drugs, as well as targets of antidepressants and anxiolytics. The 5‐HT2AR canonically activates heterotrimeric Gaq G‐proteins which activate phospholipase C (PLC) and formation of inositol phosphates to stimulate intracellular release of Ca2+. The 5‐HT2AR also interact with β‐arrestin proteins; however, the importance of β‐arrestins for controlling the efficacy and duration of 5‐HT2AR signaling remains largely undefined. Here we delineate contributions of β‐arrestin isoforms in 5‐HT2AR signaling and trafficking using CRISPR/Cas9 genome editing to stably knockout (KO) β‐arrestins. We first examined if agonist activation of 5‐HT2AR receptors caused plasma membrane recruitment of β‐arrestin using confocal imaging. Agonist activation of HA‐5‐HT2AR with 10 uM 5‐HT, or the selective agonist DOI, induced robust and rapid (within 30 sec) translocation of β‐arrestin2‐GFP from cytoplasm to the plasma membrane, where it strongly colocalized with HA‐5‐HT2AR. Live cell confocal imaging of HA‐5‐HT2AR determined rapid endocytosis within 3 mins of agonist stimulation. To determine if β‐arrestins control this rapid receptor endocytosis to impact signaling, we studied HEK293 cells lacking β‐arrestins. Western blots confirmed a complete loss of the β‐arrestin 1 and 2 in KO cells vs parent cells. Using a receptor cell surface ELISA assay, we confirmed 5 min agonist treatments resulted in rapid (~35%) loss of receptors from the cell surface; however, endocytosis (3 min to 45 min) was significantly reduced in β‐arrestin 1/2 KO cells. By measuring kinetic live‐cell Ca2+ release using the FLIPR assay and dose responses of selective 5‐HT2AR agonists, we determined prolonged duration of Ca2+ release in β‐arrestin 1/2 KO cells. The maximal 5‐HT2AR calcium signaling was significantly elevated by 45% (5‐HT) and 46 % (DOI) in KO cells vs. WT cells; however, agonist potency was unchanged. Re‐expression of β‐arrestin 1 or 2 in KO cells, reduced the elevated 5‐HT2AR Ca2+ responses to that of parent cells, indicating a specific effect of the genetic knockout. In addition, knockout of β‐arrestin1/2 increased and prolonged the duration of 5‐HT2AR‐mediated ERK phosphorylation. Taken together, this study indicates β‐arrestins rapidly interact with 5‐HT2AR receptors and profoundly limit both intensity and duration of Gaq‐mediated signal transduction. Moreover, these results indicate rapid 5‐HT2AR endocytosis during agonist activation is dependent on β‐arrestins, which likely limits receptor and agonist signaling. Using the robust CRISPR/Cas9 KO model, future studies will examine downstream signaling events to further define the role of β‐arrestins in regulating 5‐HT2AR agonist and antagonist signaling and pharmacology.Support or Funding InformationRising Star Award, UT System (JAA); UTMB institutional funding (JAA); T32 DA07287 (DEF)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

In Vitro Functional Characterization of GET73 as Possible Negative Allosteric Modulator of Metabotropic Glutamate Receptor 5.

The present study was aimed to further characterize the pharmacological profile of N-[4-(trifluoromethyl) benzyl]-4-methoxybutyramide (GET73), a putative negative allosteric modulator (NAM) of metabotropic glutamate subtype 5 receptor (mGluR5) under development as a novel medication for the treatment of alcohol dependence. This aim has been accomplished by means of a series of in vitro functional assays. These assays include the measure of several down-stream signaling [intracellular Ca++ levels, inositol phosphate (IP) formation and CREB phosphorylation (pCREB)] which are generally affected by mGluR5 ligands. In particular, GET73 (0.1 nM–10 μM) was explored for its ability to displace the concentration-response curve of some mGluR5 agonists/probes (glutamate, L-quisqualate, CHPG) in different native preparations. GET73 produced a rightward shift of concentration-response curves of glutamate- and CHPG-induced intracellular Ca++ levels in primary cultures of rat cortical astrocytes. The compound also induced a rightward shift of concentration response curve of glutamate- and L-quisqualate-induced increase in IP turnover in rat hippocampus slices, along with a reduction of CHPG (10 mM)-induced increase in IP formation. Moreover, GET73 produced a rightward shift of concentration-response curve of glutamate-, CHPG- and L-quisqualate-induced pCREB levels in rat cerebral cortex neurons. Although the engagement of other targets cannot be definitively ruled out, these data support the view that GET73 acts as an mGluR5 NAM and support the significance of further investigating the possible mechanism of action of the compound.

FUNCTIONAL CHARACTERIZATION OF A RARE GENETIC VARIANT IN PHOSPHOLIPASE Cγ2 WHICH IS ASSOCIATED WITH A BENEFICIAL EFFECT ON THE PROGRESSION OF ALZHEIMER’S DISEASE

PLCG2 is an important modulator of T- and B –lymphocyte mediated immune response and plays a crucial role in signal transmission by cleavage of 1-phosphatidylinositol-4,5-bisphosphate to diacylglycerol and inositol-3 phosphate, which further activates various PKC mediated signaling cascades. Receptor mediated activation of PLCG2 is controlled by receptor tyrosine kinases like BTK, LYN, GAB2, and TREM2, which makes of PLCG2 a good downstream modulator of signal cascades related to Alzheimer's disease (AD). Here, we functionally characterize a mutPLCG2 variant by cell biological and biochemical approaches. We also explore the effect of this variant on AD-relevant cerebrospinal fluid (CSF) biomarkers. Effect of mutPLCG2 construct was explored in transient transfected COS cell. Activation of PLCG2 variants were analyzed by both transfections with Btk or Rac2-G12V, or treatment with recombinant epidermal growth factor and a subsequent measurement of inositol phosphate level. In order to reduce clonal artefacts and ensure equal expression levels, stably PLCG2 WT and Mut expressing Hek FlpIn cells were generated for cell biological and biochemical experiments. Variant on PLCG2 was genotyped in a cohort of 1000 individuals with values for amyloid-beta 42, Tau and pTau levels on CSF. COS cells expressing transiently either PLCG2 WT or mutPLCG2 showed significant differences in inositol phosphate formation when transfected with bruton's tyrosine kinase or the constitutively active Rho GTPase, Rac2-G12V. Similar results were observed when PLCG2 activation was induced by EGF. Following experiments in stable HEK FlpIn cells showed no difference in protein expression level or migration between WT and mutPLCG2under control conditions. Further analysis of the PKCθ/NFκB pathway showed also no effects under control conditions. Next, we analyses the effect of this genetic variant on CSF levels of different biomarkers relevant for AD. We observed that carriers of the rare variant showed lower levels of pTau compared to non-carriers. Interestingly, this difference in pTau was stronger in mild cognitive impartments than in AD patients. Here we functionally characterized a rare variant in PLCG2 providing a potential link to the protective effect seen in AD. Additional experiments are currently underway and will be presented at the AAIC in London.

Modification and application of an in vitro assay to examine inositol phosphate degradation in the digestive tract of poultry.

An in vitro assay was modified to study the disappearance of inositol hexakisphosphate (InsP6 ) and the formation of lower inositol phosphate (InsP) isomers in the poultry digestive tract, and three experiments investigated the influence of diets with different ingredients and additives. Using the poultry diet as a matrix, the assay simulated the conditions (e.g. pH, temperature, proteolytic enzymes, water content, and retention time) of the crop, stomach, and small intestine, and extraction and analysis of InsP isomers were immediately conducted. The assay produced highly reproducible results with coefficients of variation ≤10% for an InsP isomer concentration ≥0.4 µmol g-1 DM (n = 3), and it was sensitive to the factors that varied in the three experiments. The described assay is a suitable tool that can be used to screen feed enzymes and to investigate the effects of supplements in the absence of endogenous phytases. The ease of handling and high reproducibility of the assay indicated that the assay is a rapid and feasible method that can be used to examine the degradation pathway of phytate in feed under gastrointestinal conditions. © 2017 Society of Chemical Industry.

NaCl-CaCl2 treatment enhancing nutritional and functional quality of mung bean sprouts

In this study, the effects of NaCl, CaCl2 and NaCl-CaCl2 treatments on the growth profiles, nutritional quality, phytic acid degradation-lower inositol phosphate formation system of germinated mung bean were investigated. Mung bean seeds were germinated at 30 oC in darkness for 4 days and sprayed solutions containing distilled water, 1.6 mM NaCl, 6 mM CaCl2, and 1.6 mM NaCl plus 6 mM CaCl2 every 1 h . Samples were collected every 2 days in liquid nitrogen for experiments and each experiment was repeated three times. The results showed that the NaCl-CaCl2 treatment improved nutritional quality significantly for the higher content of ascorbic acid, soluble sugar and free amino acid content in mung bean sprouts. Meanwhile, NaCl-CaCl2 treatment was also the most effective way to improve the degradation of phytic acid by enhancing phytase activity and the related gene expressions. This led to a higher level of the lower inositol phosphate content in mung bean sprouts. These results suggest that NaCl-CaCl2 treatment had a better influence on improving nutritional and functional quality of mung bean sprouts than NaCl and CaCl2.

The impact of phosphorus on the immune system and the intestinal microbiota with special focus on the pig.

There is increasing interest in dietary ingredients that are appropriate to support digestive and immune functions, but also maintain a stable microbial ecosystem in the gastrointestinal tract (GIT), particularly in weaned pigs. P is an essential nutrient for both microbes and their host, as it is involved, for example, in bone formation, energy metabolism, cellular signalling and stabilisation of cell membranes. Non-ruminant animals have limited access to phytate, the main storage form of P in plant seeds. The release of P bound to phytate requires phytase activity of plant or microbial origin, resulting in the formation of variable phosphorylated inositol phosphates (InsPs). The present review focuses on interactions between variations in dietary P supply, the immune system of the host, and the intestinal microbial ecosystem. Although results on the interaction between P and the immune system are inconsistent, several studies in different species have shown a positive impact of dietary P and phytase addition on the adaptive immune response. Recent studies with pigs suggest that P supply may influence intestinal microbial composition and activity. Individual InsPs or phosphate may also affect properties of pathogenic micro-organisms, such as metabolism or virulence. In conclusion, P may be considered as part of an integrated approach to support immune functions and maintain a stable microbial ecosystem in the GIT, thereby providing a barrier against potential pathogens. Within this regard, differences in phytate-P content and intrinsic phytase activity of plant feedstuffs, as well as the formation of individual InsPs, have to be taken into account.

PDZK1/NHERF3 Differentially Regulates Corticotropin-releasing Factor Receptor 1 and Serotonin 2A Receptor Signaling and Endocytosis

The corticotropin-releasing factor receptor 1 (CRFR1) and serotonin 2A receptor (5-HT2AR) are linked to cellular mechanisms underlying stress anxiety and depression. Both receptors are members of the G protein-coupled receptor (GPCR) superfamily and encode class I PSD-95/DiscsLarge/Zona Occludens 1 (PDZ) binding motifs (-S/T-x-V/I/L) at the end of their carboxyl-terminal tails. We have identified PDZK1, also referred to as Na(+)/H(+) exchange regulatory cofactor 3 (NHERF3) as both a CRFR1- and 5-HT2AR-interacting protein. We have examined whether PDZK1 plays a role in regulating both CRFR1 and 5-HT2AR activity. We find that while PDZK1 interactions with CRFR1 are PDZ binding motif-dependent, PDZK1 associates with 5-HT2AR in a PDZ binding motif-independent manner and CRFR1 expression, but not 5-HT2AR expression, redistributes PDZK1 to the plasma membrane in PDZ binding motif-dependent manner. PDZK1, negatively regulates 5-HT2AR endocytosis and has no effect upon 5-HT2AR-mediated ERK1/2 phosphorylation. In contrast, PDZK1 overexpression does not affect CRFR1 endocytosis, but selectively increases CRFR1-stimulated ERK1/2 phosphorylation. Similar to what has been previously reported for PSD-95 and SAP97, PDZK1 positively influences 5-HT2AR-stimulated inositol phosphate formation, but does not contribute to the regulation of CRFR1-mediated cAMP signaling. Taken together, these results indicate that PDZK1 differentially regulates the signaling and trafficking of CRFR1 and 5-HT2AR via PDZ-dependent and -independent mechanisms, respectively.

Hydrolysis of phytate and formation of inositol phosphate isomers without or with supplemented phytases in different segments of the digestive tract of broilers.

The objective was to characterise degradation of myo-inositol 1,2,3,4,5,6-hexakis (dihydrogen phosphate) (InsP6) and formation of inositol phosphate (InsP) isomers in different segments of the broiler digestive tract. Influence of an Aspergillus niger (PhyA) and two Escherichia coli-derived (PhyE1 and PhyE2) phytases was also investigated. A total of 600 16-d-old broilers were allocated to forty floor pens (ten pens per treatment). Low-P (5·2g/kg DM) maize-soyabean meal-based diets were fed without (basal diet; BD) or with a phytase added. On day 25, digesta from different digestive tract segments were pooled per segment on a pen-basis, freeze-dried and analysed for P, InsP isomers and the marker TiO2. InsP6 degradation until the lower ileum (74 %) in BD-fed birds showed a high potential of broilers and their gut microbiota to hydrolyse InsP6 in low-P diets. Different InsP patterns in different gut segments suggested the involvement of phosphatases of different origin. Supplemented phytases increased InsP6 hydrolysis in the crop (P<0·01) but not in the lower ileum. Measurements in the crop and proventriculus/gizzard confirmed published in vitro degradation pathways of 3- and 6-phytases for the first time. In the intestinal segments, specifically formed InsP4-5 isomers of supplemented phytases were still present, indicating further activity of these enzymes. Myo-inositol tetrakisphosphate (InsP4) accumulation differed between PhyE1 and PhyE2 compared with PhyA in the anterior segments of the gut (P<0·01). Thus, the hydrolytic cleavage of the first phosphate group is not the only limiting step in phytate degradation in broilers.

Abstract 16606: Cardiac Fibroblast-Restricted Enhancement of G q Signaling Prolongs Calcium Transients and Increases Spontaneous Activity in Biomimetic Cardiac Microtissues

Introduction: Enhanced G q signaling in response to hemodynamic stress and injury is well known to induce remodeling in both cardiac myocytes (CMs) and fibroblasts (CFs). However, the effect of CF activation and fibrosis on CMs and integrated myocardial function cannot be easily discerned from concomitant effects on CMs. Hypothesis: Enhanced G q signaling in CFs influences CM function in a 3D microtissue model previously developed in our lab, in which neonatal rat ventricular CMs and CFs are highly interspersed as seen in the myocardium. Methods: CFs infected in suspension (2 hrs, 10 MOI) with empty adenovirus (Ad-Ctr) or Ad encoding wild-type (WT) or constitutively active (Q209L or QL) Gα q were co-seeded with uninfected CMs (CM:CF 1:1) into non-adhesive hydrogels at ~1200 cells per recess (400/800 μm wide/deep). After 2-4 days, we assessed tissue size by microscopy, G q and extracellular matrix proteins proteins by Western Blot analysis, and Ca 2+ transients ([Ca 2+ ] i ) with Rhod 2/AM. Results: Overexpression of Gα q in CFs was associated with a 3-fold (WT) and 7-fold (QL) increase in total inositolphosphate formation, indicating enhanced basal phospholipase β activity. After 4 days, CM:CF QL tissues were larger in size by 30-40% and expressed more laminin and fibronectin. After 3 days, CM:CF CTR paced at 1 Hz had [Ca 2+ ] i duration of 218±3 ms, decay of 141±2 ms, and rise time of 35±4 ms (n=55-69). In CM:CF QL , duration and decay were prolonged by 160 ms and 21 ms, resp., and rise time was decreased by 5 ms (n=10-27). In CM:CF WT with less G q signaling, [Ca 2+ ] i duration and decay were also prolonged but only by 30 ms and 7 ms, resp. (n=46-51). These effects were similar but less pronounced after 2 days and/or at 2 Hz. Spontaneous [Ca 2+ ] i activity was seen in 50% of CM:CF QL , 11% of CM:CF WT , but only 2% of CM:CF CTR microtissues. The gap junction inhibitor carbenoxolone (100 μM) decreased this activity in CM:CF QL by 80% and in CM:CF WT by 15%. Conclusion: We show that enhanced CF-restricted G q signaling in biomimetic cardiac microtissues increases tissue size, prolongs [Ca 2+ ] i duration and decay, and increases spontaneous Ca 2+ activity. Our findings suggest that depending on the CF activation state, CFs can greatly modulate CM calcium handling and potentially influence arrhythmogenesis.

Role of SAP97 in the regulation of 5-HT2AR endocytosis and signaling.

Serotonin (5-HT) interacts with a wide variety of 5-HT receptors (5-HTR) of which 5-HT2AR plays an important target for antidepressant and atypical antipsychotic drugs. The carboxyl-terminal tail of 5-HT2AR encodes a motif that mediates interactions with PSD-95/disc large/zona occludens (PDZ) domain-containing proteins. In the present study, we found that 5-HT2AR interacts with synapse-associated protein 97 (SAP97; also known as DLG1) by coimmunoprecipitation in human embryonic 293 (HEK 293) cells and cortical brain lysates. We found that 5-HT2AR expression results in the recruitment of SAP97 from the cytosol to the plasma membrane and that this recruitment is dependent on an intact 5-HT2AR PDZ binding motif. We also show that 5-HT2AR interacts with SAP97 using bioluminescence energy transfer and that overexpression of SAP97 retards 5-HT2AR endocytosis, while single hairpin RNA knockdown facilitates 5-HT2AR internalization. The knockdown of SAP97 in HEK 293 cells results in a reduction in the maximum efficacy for 5-HT2AR-stimulated inositol phosphate formation and that the deletion of the 5-HT2AR PDZ motif also impairs 5-HT2AR signaling. Similarly to what has been observed for the corticotropin-releasing factor receptor 1 (CRFR1), SAP97 expression is essential for 5-HT2AR-stimulated extracellular-regulated protein kinase 1/2 (ERK1/2) phosphorylation by a PDZ interaction-independent mechanism. Moreover, we find that SAP97 is not responsible for CRFR1-mediated sensitization of 5-HT2AR signaling. Taken together, our studies show that SAP97 plays a conserved role in regulating 5-HT2AR endocytosis and ERK1/2 signaling, but plays a novel role in regulating 5-HT2AR G protein coupling.

The effect of acteoside on intracellular Ca2+ mobilization and phospholipase C activity in RBL-2H3 cells stimulated by melittin

This study was performed to investigate the effects of acteoside on various cellular functions such as, intracellular Ca(2+) mobilization, phospholipase C activity, and exocytosis induced by melittin. Melittin (0.1-1μM) dose-dependently increased intracellular Ca(2+) mobilization in the presence of extracellular Ca(2+), but was not affected by 1μM U73122, a specific PLC inhibitor. In the absence of extracellular Ca(2+), melittin (1μM) did not induce a change in intracellular Ca(2+) mobilization, which suggests that melittin-induced intracellular Ca(2+) mobilization may be dependent on the influx of extracellular Ca(2+) rather than on the release of intracellular Ca(2+) storage. Acteoside (10μM) significantly inhibited 1μM melittin-induced Ca(2+) mobilization by 33%. In [(3)H]inositol-labeled cells, 1μM melittin did not increase inositol phosphate formation, but more than 5μM melittin significantly increased inositol phosphate formation, which was significantly inhibited by acteoside. Melittin (1μM) significantly increased histamine release from RBL 2H3 cells in the presence or absence of extracellular Ca(2+). Acteoside significantly inhibited 1-μM-melittin-induced histamine release by 74% in the presence of extracellular Ca(2+) and by 71% in the absence of extracellular Ca(2+). These data suggest that the inhibitory effect of acteoside on 1μM-melittin-induced histamine release may be related to blockage of the calcium-independent pathway. Taken together, these data suggest that melittin has an influence on cellular functions such as intracellular Ca(2+) mobilization, the PLC pathway, and exocytosis via various independent signalling pathways in RBL-2H3 cells, and was significantly inhibited by acteoside.

Succinate receptor GPR91, a Gαi coupled receptor that increases intracellular calcium concentrations through PLCβ

Succinate has been reported as the endogenous ligand for GPR91. In this study, succinate was confirmed to activate GPR91 resulting in both 3′–5′-cyclic adenosine monophosphate (cAMP) inhibition and inositol phosphate formation in a pertussis toxin (PTX)-sensitive manner. GPR91 agonist-mediated effects detected using dynamic mass redistribution (DMR) were inhibited with PTX, edelfosine and U73122 demonstrating the importance of not only the Gαi pathway but also PLCβ. These results show that GPR91 when expressed in HEK293s cells couples exclusively through the Gαi pathway and acts through Gαi not only to inhibit cAMP production but also to increase intracellular Ca2+ in an inositol phosphate dependent mechanism via PLCβ activation.

Role of metabotropic glutamate receptor 5 signaling and homer in oxygen glucose deprivation-mediated astrocyte apoptosis

BackgroundGroup I metabotropic glutamate receptors (mGluR) are coupled via Gαq/11 to the activation of phospholipase Cβ, which hydrolyzes membrane phospholipids to form inositol 1,4,5 trisphosphate and diacylglycerol. In addition to functioning as neurotransmitter receptors to modulate synaptic activity, pathological mGluR5 signaling has been implicated in a number of disease processes including Fragile X, amyotrophic lateral sclerosis, multiple sclerosis, Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, epilepsy, and drug addiction. The expression of mGluR5 in astrocytes has been shown to be increased in several acute and chronic neurodegenerative conditions, but little is known about the functional relevance of mGluR5 up-regulation in astrocytes following injury.ResultsIn the current study, we investigated primary mouse cortical astrocyte cell death in response to oxygen glucose deprivation (OGD) and found that OGD induced both necrotic and apoptotic cell death of astrocytes. OGD resulted in an increase in astrocytic mGluR5 protein expression, inositol phosphate formation and extracellular regulated kinase (ERK1/2) phosphorylation, but only inositol phosphate formation was blocked with the mGluR5 selective antagonist MPEP. Cortical astrocytes derived from mGluR5 knockout mice exhibited resistance to OGD-stimulated apoptosis, but a lack of mGluR5 expression did not confer protection against necrotic cell death. The antagonism of the inositol 1,4,5 trisphosphate receptor also reduced apoptotic cell death in wild-type astrocytes, but did not provide any additional protection to astrocytes derived from mGluR5 null mice. Moreover, the disruption of Homer protein interactions with mGluR5 also reduced astrocyte apoptosis.ConclusionTaken together these observations indicated that mGluR5 up-regulation contributed selectively to the apoptosis of astrocytes via the activation of phospholipase C and the release of calcium from intracellular stores as well as via the association with Homer proteins.

Differential Inhibitory Effects of 2-Azafluorenones on PI-PLC Activation but not on PC-PLC- or PC-PLD-Activation Induced by Histamine, PAF, PMA or A23187 in C6 Glioma Cells

In this study, C6 glioma cells were used to test the effects of 2-azafluorenone and its related compounds on membrane phosphatidylinositol (PI) and phosphatidylcholine (PC) turnover. An increase of [³H]-labeled inositol phosphate (IP1) formation by histamine (100 μM) or A23187 (100 nM) via the activation of phosphatidylinositol-specific phospholipase C (PI-PLC) to breakdown labeled substrate was observed, and this effect could be partially blocked by about half at 100 μM of 2-azafluorenones. Histamine induced the increase of IP1 formation, but failed to cause an increase in extracellularly releasing of [3H]choline metabolites, or intracellular accumulation of [³H]phosphscholine. However, platelet activation factor (PAF) from 0.2 to 1 μM, and phorbol 12-myristate-13-acetate (PMA) at 1 μM caused an increase in extracellularly releasing of [³H]choline metabolites, and intracellular accumulation of [³H]phosphocholine via the activation on phosphatidylcholine (PC)-PLC. These responses of PAF and PMA were not affected by 2-azafluorenone or 4-methyl-2-azafluorenone even at high concentration (10⁻⁴ M). A23187 induced an increase of intracellular [³H]choline release via the activation of PCphospholipase D (PLD). This increasing effect of 100 nM A23187 was not affected by 2-azafluorenone or 4-methyl-2-azafluorenone even at a high concentration of 10⁻⁴ M. In summary, the inhibitory effect of 2-azafluorenone and its related compound 4-methyl-2-azafluorenone was observed selectively on PIPLC, but not on PC-PLC or PC-PLD based on changes of products after the activation of these enzymes.