Total Inositol Phosphates Research Articles

Phytase dose-dependent response of kidney inositol phosphate levels in poultry.

Phytases, enzymes that degrade phytate present in feedstuffs, are widely added to the diets of monogastric animals. Many studies have correlated phytase addition with improved animal productivity and a subset of these have sought to correlate animal performance with phytase-mediated generation of inositol phosphates in different parts of the gastro-intestinal tract or with release of inositol or of phosphate, the absorbable products of phytate degradation. Remarkably, the effect of dietary phytase on tissue inositol phosphates has not been studied. The objective of this study was to determine effect of phytase supplementation on liver and kidney myo-inositol and myo-inositol phosphates in broiler chickens. For this, methods were developed to measure inositol phosphates in chicken tissues. The study comprised wheat/soy-based diets containing one of three levels of phytase (0, 500 and 6,000 FTU/kg of modified E. coli 6-phytase). Diets were provided to broilers for 21 D and on day 21 digesta were collected from the gizzard and ileum. Liver and kidney tissue were harvested. Myo-inositol and inositol phosphates were measured in diet, digesta, liver and kidney. Gizzard and ileal content inositol was increased progressively, and total inositol phosphates reduced progressively, by phytase supplementation. The predominant higher inositol phosphates detected in tissues, D-and/or L-Ins(3,4,5,6)P4 and Ins(1,3,4,5,6)P5, differed from those (D-and/or L-Ins(1,2,3,4)P4, D-and/or L-Ins(1,2,5,6)P4, Ins(1,2,3,4,6)P5, D-and/or L-Ins(1,2,3,4,5)P5 and D-and/or L-Ins(1,2,4,5,6)P5) generated from phytate (InsP6) degradation by E. coli 6-phytase or endogenous feed phytase, suggesting tissue inositol phosphates are not the result of direct absorption. Kidney inositol phosphates were reduced progressively by phytase supplementation. These data suggest that tissue inositol phosphate concentrations can be influenced by dietary phytase inclusion rate and that such effects are tissue specific, though the consequences for physiology of such changes have yet to be elucidated.

The impact of dietary calcium content on phosphorus absorption and retention in growing pigs is enhanced by dietary microbial phytase supplementation.

Sixty growing male pigs were used to test the hypothesis that high dietary Ca content reduces P absorption to a greater extent in microbial phytase-supplemented diets via reducing inositol phosphate (IP) degradation and enhancing P precipitation. Pigs were equally allotted over diets with three Ca contents 2·0, 5·8 and 9·6 g/kg with or without microbial phytase (0 v. 500 FTU/kg) in a 2 × 3 factorial arrangement. Faeces and urine were collected at the end of the 21-d experimental period. Subsequently, pigs were euthanised and digesta quantitatively collected from different gastrointestinal tract (GIT) segments. Increasing dietary Ca content reduced apparent P digestibility in all GIT segments posterior to the stomach (P < 0·001), with greater effect in phytase-supplemented diets in the distal small intestine (Pinteraction = 0·007) and total tract (Pinteraction = 0·023). Nonetheless, increasing dietary Ca to 5·8 g/kg enhanced P retention, but only in phytase-supplemented diets. Ileal IP6 degradation increased with phytase (P < 0·001) but decreased with increasing dietary Ca content (P = 0·014). Proportion of IP esters in total IP (∑IP) indicated that IP6/∑IP was increased while IP4/∑IP and IP3/∑IP were reduced with increasing dietary Ca content and also with a greater impact in phytase-supplemented diets (Pinteraction = 0·025, 0·018 and 0·009, respectively). In all GIT segments, P solubility was increased with phytase (P < 0·001) and tended to be reduced with dietary Ca content (P < 0·096). Measurements in GIT segments showed that increasing dietary Ca content reduced apparent P digestibility via reducing IP degradation and enhancing P precipitation, with a greater impact in phytase-supplemented diets due to reduced IP degradation.

Effect of Different Technological Factors on the Gelation of a Low-Lectin Bean Protein Isolate

Gelling ability of a bean protein isolate (BPI) obtained from a naturally low-lectin variety (Phaseolus vulgaris var. Almonga) was analysed. For that purpose differences on gels processing: concentration (14% and 17%), salt addition (0 and 2%), and pH (6.5 –lot A- and 7 –lot B), were studied to obtain suitable colour, mechanical and viscoelastic properties for making appropriate meat and seafood analogues. Gelation at pH 7 at both 14 and 17% BPI concentrations, produced less rigid, more flexible, time-stable and cohesive gel networks. Colour of the resulting gels was white enough to be considered as an adequate base for making plant-based analogues. The content of total galactoside, inositol phosphates and trypsin inhibitors (bioactive compounds) present in one serving (100 g) of these BPI gels were up to 0.80 mg/g, 8.06 mg/g and 239 TIUs, respectively.

Identification of lower-order inositol phosphates (IP&amp;lt;sub&amp;gt;5&amp;lt;/sub&amp;gt; and IP&amp;lt;sub&amp;gt;4&amp;lt;/sub&amp;gt;) in soil extracts as determined by hypobromite oxidation and solution &amp;lt;sup&amp;gt;31&amp;lt;/sup&amp;gt;P NMR spectroscopy

Abstract. Inositol phosphates (IPs) are a major pool of identifiable organic phosphorus (P) in soil. However, insight into their distribution and cycling in soil remains limited, particularly of lower-order IP (IP5 and IP4). This is because the quantification of lower-order IP typically requires a series of chemical extractions, including hypobromite oxidation to isolate IP, followed by chromatographic separation. Here, for the first time, we identify the chemical nature of organic P in four soil extracts following hypobromite oxidation using solution 31P NMR spectroscopy and transverse relaxation (T2) experiments. Soil samples analysed include A horizons from a Ferralsol (Colombia), a Cambisol and a Gleysol from Switzerland, and a Cambisol from Germany. Solution 31P nuclear magnetic resonance (NMR) spectra of the phosphomonoester region in soil extracts following hypobromite oxidation revealed an increase in the number of sharp signals (up to 70) and an on average 2-fold decrease in the concentration of the broad signal compared to the untreated soil extracts. We identified the presence of four stereoisomers of IP6, four stereoisomers of IP5, and scyllo-IP4. We also identified for the first time two isomers of myo-IP5 in soil extracts: myo-(1,2,4,5,6)-IP5 and myo-(1,3,4,5,6)-IP5. Concentrations of total IP ranged from 1.4 to 159.3 mg P per kg soil across all soils, of which between 9 % and 50 % were comprised of lower-order IP. Furthermore, we found that the T2 times, which are considered to be inversely related to the tumbling of a molecule in solution and hence its molecular size, were significantly shorter for the underlying broad signal compared to for the sharp signals (IP6) in soil extracts following hypobromite oxidation. In summary, we demonstrate the presence of a plethora of organic P compounds in soil extracts, largely attributed to IPs of various orders, and provide new insight into the chemical stability of complex forms of organic P associated with soil organic matter.

Comparison of four methods for phytate analysis in plant-based foods

Plant-based staple diets common in low-income countries (LICs) often contain high levels of phytic acid (myo-inositol hexaphosphate, IP6), an inhibitor of zinc absorption. Depending on food source, handling and preparation, varying amounts of IP6 may be broken down to less phosphorylated inositol phosphates such as inositol tetra- and pentaphosphates (IP4, IP5) which differ from IP6 in their impact on zinc absorption. IP4 has little impact, whereas IP5’s impact is moderately reduced as compared with IP6. Therefore to accurately estimate the risk of zinc deficiency, values both for phytic acid and IP5 may be required. High-Performance Liquid chromatography (HPLC) can separate and quantify IP6 and its breakdown products but requires specialized equipment and expertise and is relatively time-consuming and expensive. We compared HPLC and three low-tech and widely accessible alternative methods via an analysis of a sample of typical LIC staple foods. The “ferric precipitation” method and the much simpler “HL” colorimetric assay provide accurate data but they only assay total inositol phosphates. A Polyacrylamide Gel Electrophoresis (PAGE) method can separate IP5 and IP6 from IP4 and other breakdown products, and can provide valid quantitative data for these compounds. Therefore PAGE, alone or in combination with a simple colorimetric assay like the HL method, is an accessible, low-tech alternative to HPLC for estimating dietary total phytate, IP6 and IP5, and represents an appropriate technology for LIC laboratories.

Cooking Effect on the Bioactive Compounds, Texture, and Color Properties of Cold-Extruded Rice/Bean-Based Pasta Supplemented with Whole Carob Fruit.

Pasta is considered as the ideal vehicle for fortification; thus, different formulations of gluten-free pasta have been developed (rice 0–100%, bean 0–100%, and carob fruit 0% or 10%). In this article, the content of individual inositol phosphates, soluble sugars and α-galactosides, protease inhibitors, lectin, phenolic composition, color, and texture were determined in uncooked and cooked pasta. The highest total inositol phosphates and protease inhibitors contents were found in the samples with a higher bean percentage. After cooking, the content of total inositol phosphates ranged from 2.12 to 7.97 mg/g (phytic acid or inositol hexaphosphate (IP6) was the major isoform found); the protease inhibitor activities showed values up to 12.12 trypsin inhibitor (TIU)/mg and 16.62 chymotrypsin inhibitor (CIU)/mg, whereas the competitive enzyme-linked immunosorbent assay (ELISA) showed the elimination of lectins. Considering the different α-galactosides analyzed, their content was reduced up to 70% (p < 0.05) by the cooking process. The total phenols content was reduced around 17–48% after cooking. The cooked samples fortified with 10% carob fruit resulted in darker fettuccine with good firmness and hardness and higher antioxidant activity, sucrose, and total phenols content than the corresponding counterparts without this flour. All of the experimental fettuccine can be considered as functional and healthy pasta mainly due to their bioactive compound content, compared to the commercial rice pasta.

Phosphatidylinositol 4-phosphate is a major source of GPCR-stimulated phosphoinositide production.

Phospholipase C (PLC) enzymes hydrolyze the plasma membrane (PM) lipid phosphatidylinositol 4,5-bisphosphate (PI4,5P2) to generate the second messengers inositol trisphosphate (IP3) and diacylglycerol (DAG) in response to receptor activation in almost all mammalian cells. We previously found that stimulation of G protein-coupled receptors (GPCRs) in cardiac cells leads to the PLC-dependent hydrolysis of phosphatidylinositol 4-phosphate (PI4P) at the Golgi, a process required for the activation of nuclear protein kinase D (PKD) during cardiac hypertrophy. We hypothesized that GPCR-stimulated PLC activation leading to direct PI4P hydrolysis may be a general mechanism for DAG production. We measured GPCR activation-dependent changes in PM and Golgi PI4P pools in various cells using GFP-based detection of PI4P. Stimulation with various agonists caused a time-dependent reduction in PI4P-associated, but not PI4,5P2-associated, fluorescence at the Golgi and PM. Targeted depletion of PI4,5P2 from the PM before GPCR stimulation had no effect on the depletion of PM or Golgi PI4P, total inositol phosphate (IP) production, or PKD activation. In contrast, acute depletion of PI4P specifically at the PM completely blocked the GPCR-dependent production of IPs and activation of PKD but did not change the abundance of PI4,5P2 Acute depletion of Golgi PI4P had no effect on these processes. These data suggest that most of the PM PI4,5P2 pool is not involved in GPCR-stimulated phosphoinositide hydrolysis and that PI4P at the PM is responsible for the bulk of receptor-stimulated phosphoinositide hydrolysis and DAG production.

Long-term Changes in Grassland Soil Phosphorus with Fertilizer Application and Withdrawal.

Long-term phosphorus (P) applications can increase soil P concentrations in excess of agronomic optima, posing a risk to water quality. Once fertilization stops, however, it may take time for soil P concentrations to decline. Whereas P fertilization adds orthophosphate, little is known about changes in other soil P forms during P buildup and drawdown. This study examined changes in P pools (total P, Olsen P, Mehlich P, and water-extractable P) and P forms determined by P-nuclear magnetic resonance spectroscopy (P-NMR) in grazed grassland plots from Northern Ireland. Between 1994 and 1999, all plots received 8.3 kg P ha yr with variable rates of nitrogen (100-500 kg N ha yr). From 2000 to 2005, plots received 0, 20, 40, or 80 kg P ha yr and 250 kg N ha yr; from 2005 to 2010, no P fertilizer was applied to any plots. In 2005, soil P pool concentrations at the highest P fertilization rates were significantly elevated compared with those in 2000 but had decreased to 2000 concentrations by 2010. In soils receiving no P, soil P pool concentrations were significantly lower than those in 1994 only in 2010. There were few changes in P forms determined by P-NMR. Orthophosphate followed the same trend observed for the soil P pools; total organic P, total inositol phosphates, and total orthophosphate monoesters and diesters were highest in 2010 in the soil receiving no P fertilizer for 10 yr. For these soils, fertilizer application and cessation influenced inorganic P more than organic P.

PLC‐Dependent Hydrolysis of Phosphatidylinositol 4‐Phosphate is Responsible for the Majority of Total Inositol Phosphate Production and PKC/D Activation Downstream of Multiple GPCRs

Receptor activation of phospholipase C (PLC) is important for many physiological processes, including neurotransmission and cell proliferation. PLC enzymes are 6 families of proteins that hydrolyze the plasma membrane lipid, phosphatidylinositol 4,5‐bisphosphate (PIP2) into inositol trisphosphate and diacylglycerol (DAG). Our group identified a novel pathway in cardiac cells, where G protein coupled receptor (GPCR) stimulation leads to PLC‐dependent hydrolysis of the PIP2‐precursor, phosphatidylinositol 4‐phosphate (PI4P) on intracellular Golgi membranes. We proposed that this localized DAG production was essential for nuclear processes involved in cardiac hypertrophy.Our hypothesis is that receptor‐stimulated PI4P hydrolysis expands beyond cardiac cells as a mechanism for DAG production without calcium signaling. We measured PI4P in various cell types using PI4P specific binding domains fused to GFP and found that PI4P was depleted both at the Golgi and plasma membrane (PM) in response to GPCR‐stimulation. Since PI4P is the precursor of PIP2, one could argue that depletion of PI4P happens to replenish PIP2 used by PLC and not as an actual substrate. To test this, we depleted PIP2 prior to GPCR stimulation. We used the PM‐targeted FRB/FKBP‐5‐phosphatase rapamycin‐induced dimerization system. After rapamycin addition, the PI‐5‐phosphatase is recruited to the PM where it removes the 5 phosphate from the head group of PIP2. As predicted, depletion of PIP2 did not inhibit GPCR‐stimulated PM or Golgi PI4P depletion, total IP production or PKD activation. In contrast, pharmacological inhibition of PI4‐kinases to deplete PI4P specifically at the PM, without affecting PIP2 levels, completely blocked GPCR‐dependent total IP production and PKD activation. These data strongly suggest that PI4P hydrolysis is the major reaction catalyzed by PLCs in terms of phosphoinositide mass and is a key source of DAG for regulation of protein kinases.While we have identified PI4P hydrolysis in various cell types, it is not clear which PLC isoforms catalyze this reaction. PLC epsilon performs this function at the Golgi‐Nuclear Envelope interface, but all PLC isoforms hydrolyze PI4P in vitro and may participate in PI4P hydrolysis in different cell types. To determine the identity of PLC isoforms responsible for PI4P hydrolysis, we used CRISPR technology to knockout individual PLC isoforms and will assess their roles in total IP production and PKD activation. Ultimately, finding which PLC isoforms are involved may provide new and more specific therapeutic targets for diseases.Support or Funding InformationSupport from NIH, RO1 grants GM053536 and GM111735 (A.V.S.) and NRSA predoctoral grant F31 GM116557 (R.G.).

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.

A novel mutation in the calcium-sensing receptor in a French family with familial hypocalciuric hypercalcaemia

The calcium-sensing receptor (CASR) has an important role in calcium homoeostasis by controlling PTH secretion and renal calcium handling. Inactivating mutations in the CASR gene (HGNC ID: 1514) cause familial hypocalciuric hypercalcaemia (FHH). We present a case of FHH patient to describe a novel mutation in the CASR. A 34-year-old patient was referred because of recurrent hypercalcaemia after resection of two hyperplastic parathyroids. Extensive evaluation found elevated PTH and low calcium/creatinine clearance ratio. One of her three children had high serum calcium concentrations. Genetic studies were performed by PCR amplification of CASR coding exons and direct sequencing of PCR products. Transient transfection of the wild-type (WT) CASR and the mutant CASR into COS-7 was performed to assess functional impact of the mutation and the capacity of either protein to mediate increases in cellular levels of inositol phosphates (IPs). CASR sequencing found a previously undescribed heterozygous base substitution, determining a change of threonine to isoleucine at codon 550 (p.T550I) in the sixth exon. In contrast to those transfected with WT CASR, which showed a five- to eightfold increase in total IPs at high levels of calcium, COS-7 cells transfected with the (p.T550I) mutant showed no increase confirming to the inactivating nature of the mutation. COS-7 cells co-transfected with the WT and the (p.T550I) mutant showed an intermediate response suggesting a possible dominant negative effect. This case report presents a not-yet-described mutation in the cysteine-rich region of the CASR extracellular domain, a mutation with a possible dominant negative effect.

Temporal Profiling of Orexin Receptor-Arrestin-Ubiquitin Complexes Reveals Differences between Receptor Subtypes

Orexin G protein-coupled receptors (OxRs) and their cognate agonists have been implicated in a number of disorders since their recent discovery, ranging from narcolepsy to formation of addictive behavior. Bioluminescence resonance energy transfer assays of agonist-occupied OxRs provided evidence for a strong dose-dependent interaction with both trafficking proteins β-arrestin 1 and 2 that required unusually high agonist concentrations compared with inositol phosphate signaling. This appears to be reflected in functional differences in potency with respect to orexin A (OxA) and OxR2-dependent ERK1/2 phosphorylation after 90 min compared with 2 min, potentially consistent with β-arrestin-mediated versus G protein-mediated signaling, respectively. Furthermore, extended bioluminescence resonance energy transfer kinetic data monitoring OxA-dependent receptor-β-arrestin and β-arrestin-ubiquitin proximity suggested subtype-specific differences in receptor trafficking, with OxR2 activation resulting in more sustained receptor-β-arrestin-ubiquitin complex formation than elicited by OxR1 activation. Enzyme-linked immunosorbent assay (ELISA) data also revealed that OxR1 underwent significantly more rapid recycling compared with OxR2. Finally, we have observed sustained OxA-dependent ERK1/2 phosphorylation in the presence of OxR2 compared with OxR1. Although both OxR subtypes could be classified as class B receptors for β-arrestin usage based on the initial strength of interaction with both β-arrestins, our temporal profiling revealed tangible differences between OxR subtypes. Consequently, OxR1 appears to fit uneasily into the commonly used β-arrestin classification scheme. More importantly, it is hoped that this improved profiling capability, enabling the subtleties of protein complex formation, stability, and duration to be assessed in live cells, will help unlock the therapeutic potential of targeting these receptors.

The interaction between noradrenaline and ATP upon polyphosphoinositide metabolism and contraction in tail arteries from normo- and hypertensive rats.

The effects of, and interaction between, noradrenaline and alpha,beta-methylene ATP upon polyphosphoinositide (PPI) breakdown, investigated by measuring the accumulation of inositol phosphates, and contraction, were studied in tail arteries from normo- (WKY) and spontaneously-hypertensive (SHR) rats. Noradrenaline (10(-7)-10(-3) M) evoked a prazosin (10(-6) M)-sensitive, concentration-dependent increase in total inositol phosphate accumulation in both WKY and SHR rats. No significant differences were observed in either the maximal response or in the concentration range over which noradrenaline evoked this response, between these two populations. Noradrenaline (5 x 10(-7)-5 x 10(-5) M) evoked a concentration-dependent contraction of arteries from both SHR and WKY rats. The responses to noradrenaline were about 2-fold greater at all effective concentrations of noradrenaline in SHR compared with WKY rats. alpha,beta-Methylene ATP (10(-6) M) did not alter noradrenaline-stimulated total inositol phosphate accumulation, in arteries from either SHR or WKY rats, measured either as the maximal response or as the EC50. alpha,beta-Methylene ATP (5 x 10(-6) M), by itself, evoked a contractile response, which was quantitatively similar in SHR and WKY rats, and was additive with the contractile responses to noradrenaline (5 x 10(-7)-5 x 10(-5) M). The maximum response produced by a combination of noradrenaline and alpha,beta-Methylene ATP was quantitatively similar to that produced by noradrenaline alone. No evidence of synergism between alpha,beta-Methylene ATP and noradrenaline upon contraction was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential Regulation of Phospholipase C-β2 Activity and Membrane Interaction by Gαq, Gβ1γ2, and Rac2

We combined fluorescence recovery after photobleaching (FRAP) beam-size analysis with biochemical assays to investigate the mechanisms of membrane recruitment and activation of phospholipase C-beta(2) (PLCbeta(2)) by G protein alpha(q) and betagamma dimers. We show that activation by alpha(q) and betagamma differ from activation by Rac2 and from each other. Stimulation by alpha(q) enhanced the plasma membrane association of PLCbeta(2), but not of PLCbeta(2)Delta, which lacks the alpha(q)-interacting region. Although alpha(q) resembled Rac2 in increasing the contribution of exchange to the FRAP of PLCbeta(2) and in enhancing its membrane association, the latter effect was weaker than with Rac2. Moreover, the membrane recruitment of PLCbeta(2) by alpha(q) occurred by enhancing PLCbeta(2) association with fast-diffusing (lipid-like) membrane components, whereas stimulation by Rac2 led to interactions with slow diffusing membrane sites. On the other hand, activation by betagamma shifted the FRAP of PLCbeta(2) and PLCbeta(2)Delta to pure lateral diffusion 3- to 5-fold faster than lipids, suggesting surfing-like diffusion along the membrane. We propose that these different modes of PLCbeta(2) membrane recruitment may accommodate contrasting functional needs to hydrolyze phosphatidylinositol 4,5-bisphosphate (PtdInsP(2)) in localized versus dispersed populations. PLCbeta(2) activation by Rac2, which leads to slow lateral diffusion and much faster exchange, recruits PLCbeta(2) to act locally on PtdInsP(2) at specific domains. Activation by alpha(q) leads to lipid-like diffusion of PLCbeta(2) accompanied by exchange, enabling the sampling of larger, yet limited, areas prior to dissociation. Finally, activation by betagamma recruits PLCbeta(2) to the membrane by transient interactions, leading to fast "surfing" diffusion along the membrane, sampling large regions for dispersed PtdInsP(2) populations.

A Phosphoinositide 3-Kinase/Phospholipase Cgamma1 Pathway Regulates Fibroblast Growth Factor-Induced Capillary Tube Formation

BackgroundThe fibroblast growth factors (FGFs) are key regulators of embryonic development, tissue homeostasis and tumour angiogenesis. Binding of FGFs to their receptor(s) results in activation of several intracellular signalling cascades including phosphoinositide 3-kinase (PI3K) and phospholipase C (PLC)γ1. Here we investigated the basic FGF (FGF-2)-mediated activation of these enzymes in human umbilical vein endothelial cells (HUVECs) and defined their role in FGF-2-dependent cellular functions.Methodology/Principal FindingsWe show that FGF-2 activates PLCγ1 in HUVECs measured by analysis of total inositol phosphates production upon metabolic labelling of cells and intracellular calcium increase. We further demonstrate that FGF-2 activates PI3K, assessed by analysing accumulation of its lipid product phosphatidylinositol-3,4,5-P3 using TLC and confocal microscopy analysis. PI3K activity is required for FGF-2-induced PLCγ1 activation and the PI3K/PLCγ1 pathway is involved in FGF-2-dependent cell migration, determined using Transwell assay, and in FGF-2-induced capillary tube formation (tubulogenesis assays in vitro). Finally we show that PI3K-dependent PLCγ1 activation regulates FGF-2-mediated phosphorylation of Akt at its residue Ser473, determined by Western blotting analysis. This occurs through protein kinase C (PKC)α activation since dowregulation of PKCα expression using specific siRNA or blockade of its activity using chemical inhibition affects the FGF-2-dependent Ser473 Akt phosphorylation. Furthermore inhibition of PKCα blocks FGF-2-dependent cell migration.Conclusion/SignificanceThese data elucidate the role of PLCγ1 in FGF-2 signalling in HUVECs demonstrating its key role in FGF-2-dependent tubulogenesis. Furthermore these data unveil a novel role for PLCγ1 as a mediator of PI3K-dependent Akt activation and as a novel key regulator of different Akt-dependent processes.

Muscarinic cholinoceptor activation by pilocarpine triggers apoptosis in human skin fibroblast cells

The aim of the present work was to examine the role of muscarinic acetylcholine receptors (mAChRs) on apoptosis in human skin fibroblast cells. Neonatal human skin fibroblast cultures were stimulated with pilocarpine in the presence or absence of specific antagonists. Pilocarpine stimulates apoptosis, total inositol phosphates (InsP) accumulation and nitric oxide synthase (NOS) activity. All these effects were inhibited by atropine, mustard hydrochloride (4-DAMP) and pirenzepine, indicating that M(1) and M(3) mAChRs are implicated in pilocarpine action. Pilocarpine apoptotic action is accompanied by caspase-3 and JNK activation. The intracellular pathway leading to pilocarpine-induced biological effects involved phospholipase C, calcium/calmodulin and extracellular calcium as U-73122, W-7, verapamil, BAPTA and BAPTA-AM blocked pilocarpine effects. L-NMMA, a NOS inhibitor, had no effect, indicating that the enzyme does not participate in the apoptosis phenomenon. These results may contribute to a better understanding of the modulatory role of the parasympathetic muscarinic system on the apoptotic human skin fibroblast process.

Characterization of Phospholipase Cγ Enzymes with Gain-of-Function Mutations

Phospholipase C gamma isozymes (PLC gamma 1 and PLC gamma 2) have a crucial role in the regulation of a variety of cellular functions. Both enzymes have also been implicated in signaling events underlying aberrant cellular responses. Using N-ethyl-N-nitrosourea (ENU) mutagenesis, we have recently identified single point mutations in murine PLC gamma 2 that lead to spontaneous inflammation and autoimmunity. Here we describe further, mechanistic characterization of two gain-of-function mutations, D993G and Y495C, designated as ALI5 and ALI14. The residue Asp-993, mutated in ALI5, is a conserved residue in the catalytic domain of PLC enzymes. Analysis of PLC gamma 1 and PLC gamma 2 with point mutations of this residue showed that removal of the negative charge enhanced PLC activity in response to EGF stimulation or activation by Rac. Measurements of PLC activity in vitro and analysis of membrane binding have suggested that ALI5-type mutations facilitate membrane interactions without compromising substrate binding and hydrolysis. The residue mutated in ALI14 (Tyr-495) is within the spPH domain. Replacement of this residue had no effect on folding of the domain and enhanced Rac activation of PLC gamma 2 without increasing Rac binding. Importantly, the activation of the ALI14-PLC gamma 2 and corresponding PLC gamma 1 variants was enhanced in response to EGF stimulation and bypassed the requirement for phosphorylation of critical tyrosine residues. ALI5- and ALI14-type mutations affected basal activity only slightly; however, their combination resulted in a constitutively active PLC. Based on these data, we suggest that each mutation could compromise auto-inhibition in the inactive PLC, facilitating the activation process; in addition, ALI5-type mutations could enhance membrane interaction in the activated state.

In situ ruminal degradation of phytic acid in formaldehyde-treated rice bran

Rice bran has a very high content of phytic acid (IP6), which is a nutritional antagonist of Ca. Microbial phytase degrades IP6, but ruminal degradation of nutrients can be reduced by formaldehyde treatment. Milk fever in dairy cows can be prevented by reducing available dietary Ca to stimulate Ca homeostasis. In the present study, effects of formaldehyde treatment on ruminal degradation of IP6 in rice bran were investigated. Two samples of full-fat rice bran were treated with four levels of formaldehyde ( i.e., 0, 1000, 2500 and 5000 ppm fresh weight) and ruminally incubated in situ for 0, 4, 8, 16, 32, 64 and 336 h in three rumen fistulated lactating dairy cows. Dry matter (DM) disappearance was determined, residues were analysed for P and, for one of the products, also for inositol phosphate (IP) forms. Degradation parameters were calculated for DM, P, total IP, and IP6. The in situ washable fraction ( W), undegradable fraction ( U) and degradable fraction ( D) were measured and the rate of degradation (kd) was calculated by exponential regression to the equation: Y( t) = U + D × exp(−kd × t). HPLC analyses confirmed that most P in the original sample and residues was phytate, mostly IP6. DM and P degraded differently in the two rice brans and formaldehyde treatment reduced degradability, lowering W and increasing D and kd. The calculated rumen escape (kp = 0.05/h) for P increased from 0.082 at 0 ppm to 0.136, 0.284 and 0.398 at 1000, 2500 and 5000 ppm of formaldehyde treatment, respectively. Degradation of total IP forms and IP6 corresponded with P disappearance. Formaldehyde treatment reduced W in total IP and IP6, proportionally increasing D, while U was 0 in both. kd decreased with formaldehyde treatment in total IP and IP6, which decreased from 0.309/h at 0 ppm to 0.217, 0.116 and 0.071%/h as formaldehyde treatment level increased. The calculated rumen escape (kp = 0.05/h) of IP6 were 0.079, 0.126, 0.229 and 0.318 for the increasing formaldehyde treatment levels. Formaldehyde treatment reduced rumen degradability of IP6 in rice bran. One kg of formaldehyde-treated rice bran could bind 7 g of dietary Ca post-ruminally, making it a potentially feasible tool to decrease intestinal Ca availability to aid in the prevention of milk fever.

Constitutively Active Mutant N111G of Angiotensin II Type 1 (AT1) Receptor Induces Homologous Internalization Through Mediation of AT1-Receptor Antagonist

The present study investigated the internalization behavior of the constitutively active mutant (CAM) N111G of angiotensin II type 1 (AT1) receptor and correlated the result with the mechanism of the constitutive activity of the mutant. The inverse agonist activity of valsartan, losartan, candesartan, and telmisartan was also examined by inositol phosphate (IP) accumulation study as well as receptor-internalization assay. Both wild-type (WT) and N111G mutant receptors were transiently expressed in COS-7 cells and the binding affinities towards the agonist and these four AT1 antagonists were determined. Production of total IP was measured in the presence and absence of the compounds. The agonist-induced receptor internalization of both WT and N111G mutant receptors was also investigated. Although the mutant showed similar binding characteristics with agonist and the antagonists used as WT, the internalization of the mutant was much lower (19.56 ± 2.87%) than that of the WT receptor (74.63 ± 1.00%). Internalization of the mutant significantly increased (63.22 ± 0.03%) in the presence of valsartan, which also showed significant inverse agonist activity in the N111G mutant. The results indicate that internalization of CAM N111G of the AT1 receptor is induced by the use of valsartan, which may be an important characteristic of inverse agonist activities of AT1 antagonists in N111G.

Switching of G-protein Usage by the Calcium-sensing Receptor Reverses Its Effect on Parathyroid Hormone-related Protein Secretion in Normal Versus Malignant Breast Cells

The calcium-sensing receptor (CaR) is a G-protein-coupled receptor that signals in response to extracellular calcium and regulates parathyroid hormone secretion. The CaR is also expressed on normal mammary epithelial cells (MMECs), where it has been shown to inhibit secretion of parathyroid hormone-related protein (PTHrP) and participate in the regulation of calcium and bone metabolism during lactation. In contrast to normal breast cells, the CaR has been reported to stimulate PTHrP production by breast cancer cells. In this study, we confirmed that the CaR inhibits PTHrP production by MMECs but stimulates PTHrP production by Comma-D cells (immortalized murine mammary cells) and MCF-7 human breast cancer cells. We found that changes in intracellular cAMP, but not phospholipase C or MAPK signaling, correlated with the opposing effects of the CaR on PTHrP production. Pharmacologic stimulation of cAMP accumulation increased PTHrP production by normal and transformed breast cells. Inhibition of protein kinase A activity mimicked the effects of CaR activation on inhibiting PTHrP secretion by MMECs and blocked the effects of the CaR on stimulating PTHrP production in Comma-D and MCF-7 cells. We found that the CaR coupled to Galphai in MMECs but coupled to Galphas in Comma-D and MCF-7 cells. Thus, the opposing effects of the CaR on PTHrP production are because of alternate G-protein coupling of the receptor in normal versus transformed breast cells. Because PTHrP contributes to hypercalcemia and bone metastases, switching of G-protein usage by the CaR may contribute to the pathogenesis of breast cancer.