Detection of Inositol Phosphates by Split PH Domains.

The pleckstrin homology (PH) domain is a family of structurally conserved proteins which can bind inositol phosphate derivatives. Some proteins involved in cellular signaling and cytoskeletal organization possess split PH domains that assemble into a structure which can bind specific inositol phosphates. Here we describe the design of split PH domain from a structurally well-characterized PH domain of phospholipase C (PLC) δ1 and Bruton's tyrosine kinase (Btk), which selectively bind Ins(1,4,5)P3 and Ins(1,3,4,5)P4, respectively. The PH domains fold into a functional structure when the split halves are brought to close proximity, and can be utilized to detect specific inositol phosphate of interest.

Competitive Fluorescent Ligand Assay for Inositol 1,4,5-Trisphosphate.

We present a novel method, termed competitive fluorescent ligand assay for inositol 1,4,5-trisphosphate (CFLA-IP3), to measure inositol 1,4,5-trisphosphate (IP3). This method is based on fluorescence resonance energy transfer (FRET) between two fluorescent molecules, a fluorescent IP3-binding protein and its fluorescent ligand. Binding of these fluorescent molecules generates a FRET signal, and the IP3-dependent decrease in the FRET signal due to displacement of the fluorescent ligand is detected by fluorescence microscopy.

The association between the expression of PAR2 and TMEM16A and neuropathic pain.

Chronic constriction injury (CCI) of the sciatic nerve may induce dorsal root ganglion (DRG) neuronal hyperexcitability and behaviorally expressed hyperalgesia. CCI is a model of neuropathic pain. To investigate the association between the expression of protease activated receptor 2 (PAR2), TMEM16A and neuropathic pain, the expression of PAR2 and TMEM16A proteins in the DRG neurons of rats following CCI of the sciatic nerve was investigated. Following the creation of the CCI model, the thermal withdrawal latency (TWL) was examined by a hot plate test. An immunofluorescence assay and western blot assay were performed to determine the expression of PAR2 and TMEM16A proteins in the ipsilateral L4­6 DRG neurons. The concentration of inositol 1,4,5­triphosphate (IP3) in the L4­6 DRG was determined by ELISA. In the CCI­D7 (7 days after CCI) and CCI­D14 (14 days after CCI) treatment groups, the TWL of rats was significantly shorter than that in the sham operated group (P<0.01; n=12). The expression of PAR2 and TMEM16A proteins in the CCI­D7 and CCI­D14 groups were significantly upregulated compared with the sham operated group (P<0.05; n=12). Additionally, it was revealed that PAR2 and TMEM16A were co­expressed in DRG neurons. It was also observed that IP3 significantly increased in the CCI­D7 and CCI­D14 groups compared with the sham operation group (P<0.05; n=6) as PAR2 and TMEM16A also increased. These findings suggest that the upregulation of PAR2 and TMEM16A in DRG neurons, the co­expression of the two proteins and increasing IP3 are critical to the development of neuropathic pain.

Highly Sensitive Measurement of Inositol 1,4,5-Trisphosphate by Using a New Fluorescent Ligand and Ligand Binding Domain Combination.

Based on the results of our previous adenophostin A structure-activity relationship studies, two fluorescent inositol 1,4,5-trisphosphate (IP3 ) receptor ligands, fluorescent adenophostin A (FADA) and fluorescent low-affinity ligand (FLL), were designed. Binding of the fluorescent ligands to the fluorescent IP3 sensor in protein-expressing cells caused FRET. This principle was extended to a cell-free assay system by using the fluorescent IP3 sensor bound to agarose beads. The effect of FLL on the FRET signal was reduced by subsequent addition of IP3 . The IC50 values of IP3 on the FRET signals were 139.7 and 352.1 nm for 30 and 100 nm FLL, respectively. This method allowed quantitative measurement of IP3 concentrations below 10 nm and was applied to measure cytosolic IP3 concentrations in COS-7 cells and to examine the potency of synthesized adenophostin A analogues.

Profile and bioavailability analysis of myo-inositol phosphates in rye bread supplemented with phytases: a study using an in vitro method and Caco-2 monolayers.

Commercial preparations of 6-phytase A alone and in combination with phytase B were used in rye breadmaking. Determination of bioavailability of myo-inositol phosphates from bread was performed by an in vitro digestion method followed by the measurement of an uptake by Caco-2 cells in culture. In bread supplemented with a combination of 6-phytase A and phytase B, a significant reduction in phytate content was observed from 3.62 µmol/g in the control to 0.7 µmol/g. Bioavailability of phytate estimated by an in vitro method simulating digestion in the human alimentary tract was 9% in the bread supplemented with phytase B, 7% (6-phytase A) and 50% in the control bread. In cell culture, the bioaccessibilities of inositol triphosphates from bread baked with the addition of 6-phytase A was higher by 36% as compared to the samples baked with phytase B and by 32% in breads baked with combination of both phytases.

Picomolar sensitivity to inositol trisphosphate in Xenopus oocytes.

Ca(2+) liberation from the endoplasmic reticulum mediated by inositol trisphosphate receptor/channels (IP3Rs) in response to production of the second messenger IP3 regulates numerous signaling pathways. However, estimates of resting and physiologically relevant cytosolic concentrations of IP3 vary appreciably. Here we directly address this question, taking advantage of the large size of Xenopus oocytes to image Ca(2+) liberation evoked by bolus intracellular injections of known concentrations of IP3. Our principal finding is that IP3 evokes both global and local Ca(2+) signals in freshly isolated oocytes at concentrations as low as a few pM. A corollary is that basal, resting [IP3] must be even lower, given the absence of detectable Ca(2+) signals before injection. The dose/response curve for IP3-activation of Ca(2+) liberation suggests that freshly isolated oocytes express two distinct functional populations of IP3 receptors with EC50 values around 200 pM and tens of nM, whereas the high-affinity receptors are not apparent in oocytes examined later than about 3 days after isolation from the ovary.

Imaging local Ca2+ signals in cultured mammalian cells.

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of >500 frames sec(-1) (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Hypersensitive mAChRs are involved in the epiphora of transplanted glands.

Autologous transplantation of the submandibular gland is an effective treatment for severe dry eye syndrome. However, more than 40% of patients experience epiphora 3 to 6 months after transplantation. The underlying mechanism of epiphora remains to be elucidated. To investigate the potential roles of muscarinic acetylcholine receptors (mAChRs) in the induction of epiphora in transplanted glands, we assessed and found elevated mRNA and protein expression of M1- and M3-mAChR in transplanted glands from epiphora patients. The content of inositol 1, 4, 5-trisphosphate was also elevated. Moreover, carbachol (5 and 10 µM) induced greater increase of [Ca(2+)]i in isolated epiphora submandibular cells than in controls. Although aquaporin-5 (AQP5) content and distribution in the apical and lateral plasma of epiphora glands did not change, AQP5 content was reduced in lipid microdomains (lipid rafts and caveolae) but increased in non-lipid microdomains compared with controls. Carbachol (10 µM) increased the ratio of non-lipid microdomain to total AQP5 in the cultured control submandibular gland tissue. Taken together, these results indicated that hypersensitive mAChRs might be involved in the epiphora of transplanted submandibular glands by modulating AQP5 trafficking.

Studying the activation of epithelial ion channels using global whole-field photolysis.

The production of saliva by parotid acinar cells is stimulated by Ca(2+) activation of Cl(-) and K(+) channels located in the apical plasma membrane of these polarized cells. Here we provide a detailed description of a flash photolysis experiment designed to give a global and relatively uniform photorelease of inositol 1,4,5-trisphosphate (InsP(3)) or Ca(2+) from caged precursors (NPE-InsP(3) or NP-EGTA) combined with the simultaneous measurement of whole-cell Ca(2+)-activated currents. The photolysis light source can be either an ultraviolet (UV) flash lamp or alternatively the output from a 375-nm diode laser, which is defocused to illuminate the entire field.

Analyzing Ca(2+) dynamics in intact epithelial cells using spatially limited flash photolysis.

The production of saliva by parotid acinar cells is stimulated by Ca(2+) activation of Cl(-) and K(+) channels located in the apical plasma membrane of these polarized cells. Here we describe a paradigm for the focal photorelease of either Ca(2+) or an inositol 1,4,5 trisphosphate (InsP(3)) analog. The protocol is designed to be useful for investigating subcellular Ca(2+) dynamics in polarized cells with minimal experimental intervention. Parotid acinar cells are loaded with cell-permeable versions of the caged precursors (NP-EGTA-AM or Ci-InsP(3)/PM). Photolysis is accomplished using a spatially limited, focused diode laser, but the experiment can be readily modified to whole-field photolysis using a xenon flash lamp.

New insights into structural determinants for prostanoid thromboxane A2 receptor- and prostacyclin receptor-G protein coupling.

G protein-coupled receptors (GPCRs) interact with heterotrimeric G proteins and initiate a wide variety of signaling pathways. The molecular nature of GPCR-G protein interactions in the clinically important thromboxane A2 (TxA(2)) receptor (TP) and prostacyclin (PGI(2)) receptor (IP) is poorly understood. The TP activates its cognate G protein (Gαq) in response to the binding of thromboxane, while the IP signals through Gαs in response to the binding of prostacyclin. Here, we utilized a combination of approaches consisting of chimeric receptors, molecular modeling, and site-directed mutagenesis to precisely study the specificity of G protein coupling. Multiple chimeric receptors were constructed by replacing the TP intracellular loops (ICLs) with the ICL regions of the IP. Our results demonstrate that both the sequences and lengths of ICL2 and ICL3 influenced G protein specificity. Importantly, we identified a precise ICL region on the prostanoid receptors TP and IP that can switch G protein specificities. The validities of the chimeric technique and the derived molecular model were confirmed by introducing clinically relevant naturally occurring mutations (R60L in the TP and R212C in the IP). Our findings provide new molecular insights into prostanoid receptor-G protein interactions, which are of general significance for understanding the structural basis of G protein activation by GPCRs in basic health and cardiovascular disease.

Reduction of inositol (1,4,5)-trisphosphate affects the overall phosphoinositol pathway and leads to modifications in light signalling and secondary metabolism in tomato plants.

The phosphoinositol pathway is one of the major eukaryotic signalling pathways. The metabolite of the phosphoinositol pathway, inositol- (1,4,5) trisphosphate (InsP(3)), is a regulator of plant responses to a wide variety of stresses, including light, drought, cold, and salinity. It was found that the expression of InsP 5-ptase, the enzyme that hydrolyses InsP(3), also dramatically affects the levels of inositol phosphate metabolites and the secondary metabolites in transgenic tomato plants. Tomato plants expressing InsP 5-ptase exhibited a reduction in the levels of several important inositol phosphates, including InsP(1), InsP(2), InsP(3), and InsP(4). Reduced levels of inositol phosphates accompanied an increase in the accumulation of phenylpropanoids (rutin, chlorogenic acid) and ascorbic acid (vitamin C) in the transgenic fruits of tomato plants. The enhanced accumulation of these metabolites in transgenic tomato plants was in direct correspondence with the observed up-regulation of the genes that express the key enzymes of ascorbic acid metabolism (myo-inositol oxygenase, MIOX; L-galactono-γ-lactone dehydrogenase, GLDH) and phenylpropanoid metabolism (chalcone synthase, CHS1; cinnamoyl-CoA shikimate/quinate transferase, HCT). To understand the molecular links between the activation of different branches of plant metabolism and InsP(3) reduction in tomato fruits, the expression of transcription factors known to be involved in light signalling was analysed by real-time RT-PCR. The expression of LeHY5, SIMYB12, and LeELIP was found to be higher in fruits expressing InsP 5-ptase. These results suggest possible interconnections between phosphoinositol metabolism, light signalling, and secondary metabolism in plants. Our study also revealed the biotechnological potential for the genetic improvement of crop plants by the manipulation of the phosphoinositol pathway.

OX40-OX40 ligand interaction may activate phospholipase C signal transduction pathway in human umbilical vein endothelial cells.

Studies have recently supported the emerging role of OX40/OX40L interaction in atherosclerosis. The mechanism of OX40/OX40L interaction may be related to a variety of signal pathways. The most important signal pathway involves the activation of phospholipase C (PLC) which induces diacylglycerol-protein kinase C (DAG-PKC) and the inositol trisphosphate (IP(3))-intracellular free calcium ([Ca(2+)](i)) pathway. The aim of this work was to investigate whether OX40-OX40L interaction can stimulate the PLC signal pathway in human umbilical vein endothelial cells (HUVEC). The DAG and IP(3) level in HUVEC were measured by radio-enzymatic assay. The activity of PKC was detected by its ability to transfer phosphate from [gamma-(32)P]ATP to lysine-rich histone. [Ca(2+)](i) concentrations were measured by flow cytometric analysis. Results showed that the DAG level was markedly increased in a concentration-dependent, biphasic manner in HUVEC induced by OX40. The early phase was rapid, peaking at 30 s. The late phase reached the maximum level at 15 min and decayed slowly. OX40 increased PKC activity in a dose-dependent manner with two peaks at 40-50 s and 12-16 min, then decreased slowly, yet maintained a high level for at least 30 min. PKC activity was mainly in cytosol at rest and translocated from cytosol to membrane when stimulated by OX40. Similarly, OX40-induced rapid IP(3) formation coincided with the peak of DAG level. Moreover, OX40 also induced peak [Ca(2+)](i) responses including the rapid transient phase and the sustained phase. Anti-OX40L antibody significantly suppressed OX40-induced DAG-PKC and IP(3)-[Ca(2+)](i) signal pathway activation in HUVEC. In conclusion, the data suggested that OX40-OX40L interaction induced a robust stimulation of phospholipase C signal transduction pathway in HUVEC.

Hyponatremia in hepatic encephalopathy: an accomplice or innocent bystander?

Hyponatremia, a common complication inpatients with advanced liver disease and impaired free water clearance, has been shown to be an important predictor of short-term mortality. Hepatic encephalopathy, also a late complication of end-stage liver disease, has been associated with low-grade cerebral edema as a result of swelling of astrocytes. Guevara et al. hypothesized that hyponatremia and the resultant depletion of organic osmolytes (e.g.,myo-inositol) from brain cells contribute to brain edema, playing an important role in the pathogenesis of hepatic encephalopathy. Using a multivariable analysis, they demonstrated that hyponatremia increased the risk of hepatic encephalopathy more than eightfold, after adjustment for serum bilirubin and creatinine concentrations and previous history of encephalopathy. Their magnetic resonance spectroscopy data correlated low brain concentrations of myoinositol with hepatic encephalopathy. As both hyponatremia and encephalopathy occur in patients with advanced liver disease, it has been difficult to implicate hyponatremia independently in the pathogenesis of hepatic encephalopathy. Guevara's data do suggest that hyponatremia is more likely an accomplice than an innocent bystander.

Use of Fluorescence Resonance Energy Transfer-based Biosensors for the Quantitative Analysis of Inositol 1,4,5-Trisphosphate Dynamics in Calcium Oscillations.

Inositol 1,4,5-trisphosphate (IP(3)) is an intracellular messenger that elicits a wide range of spatial and temporal Ca(2+) signals, and this signaling versatility is exploited to regulate diverse cellular responses. In this study, we have developed a series of IP(3) biosensors that exhibit strong pH stability and varying affinities for IP(3), as well as a method for the quantitative measurement of cytosolic concentrations of IP(3) ([IP(3)](i)) in single living cells. We applied this method to elucidate IP(3) dynamics during agonist-induced Ca(2+) oscillations, and we demonstrated cell type-dependent differences in IP(3) dynamics, a nonfluctuating rise in [IP(3)](i) and repetitive IP(3) spikes during Ca(2+) oscillations in COS-7 cells and HSY-EA1 cells, respectively. The size of the IP(3) spikes in HSY-EA1 cells varied from 10 to 100 nm, and the [IP(3)](i) spike peak was preceded by a Ca(2+) spike peak. These results suggest that repetitive IP(3) spikes in HSY-EA1 cells are passive reflections of Ca(2+) oscillations, and are unlikely to be essential for driving Ca(2+) oscillations. In addition, the interspike periods of Ca(2+) oscillations that occurred during the slow rise in [IP(3)](i) were not shortened by the rise in [IP(3)](i), indicating that IP(3)-dependent and -independent mechanisms may regulate the frequency of Ca(2+) oscillations. The novel method described herein as well as the quantitative information obtained by using this method should provide a valuable and sound basis for future studies on the spatial and temporal regulations of IP(3) and Ca(2+).

Cellular response induced by a galactose-specific adhesin of enteroaggregative Escherichia coli in INT-407 cells.

In the present study, the role of a fimbrial galactose-specific adhesin of the T7 strain of enteroaggregative Escherichia coli (EAEC-T7) in the signal transduction pathways in human small intestinal epithelial cells (INT-407) was explored. The adhesin was purified by anion exchange chromatography using a Mono Q HR5/5 column in the AKTA purifier system. The characteristic stacked brick pattern of aggregative adherence of EAEC-T7 to INT-407 cells was found to be inhibited in the presence of immunoglobulin G against the purified adhesin as well as d-galactose. The adhesin induced a significant increase in the intracellular calcium concentration [Ca(2+)](i) in INT-407 cells, which was reduced in the presence of dantrolene (inhibitor of intracellular calcium stores), verapamil, calciseptin (calcium channel blockers) as well as neomycin [inhibitor of phospholipase C (PLC)]. Further, an increased level of PLCgamma1 and inositol 1,4,5-tri phosphate as well as enhanced activity of protein kinase C (PKC) in the adhesin-stimulated cells were found to be downregulated in the presence of neomycin and U73122 (inhibitors of PLC) and H-7 (inhibitor of PKC), respectively. The adhesin could also induce interleukin-8 secretion from INT-407 cells, which was inhibited in the presence of dantrolene as well as staurosporin (inhibitor of PKC). Collectively, our results have suggested that the galactose-specific adhesin-induced signal transduction pathway might play a crucial role in the EAEC-induced pathogenesis.

Fluorescent sensing of IP3 with a Trifurcate Zn(II)-containing chemosensing ensemble system.

A trifurcate receptor containing Zn(II)-dipicolylamine ligands is developed for the fluorescent sensing of IP 3, myo-inositol 1,4,5-tris(phosphate), through an indicator displacement approach. The chemosensing ensemble containing the Zn(II) complex and eosin Y as indicator shows the maximum fluorescence restoration for IP 3 among various other anions including phosphate derivatives in water buffered at pH = 7.

D4 dopamine receptor enhances angiotensin II-stimulated aldosterone secretion through PKC-epsilon and calcium signaling.

Aldosterone secretion is subjected to dopaminergic regulation. Our previous study showed that both human D2 and D4 dopamine receptors (D2R and D4R) modulate aldosterone secretion, but in opposing directions. The inhibitory effect of D2R is mediated by attenuating protein kinase C-micro (PKC-micro) and calcium-dependent signaling. The mechanism of D4R effect on angiotensin II (AII)-stimulated aldosterone secretion is explored in this study. Experiments were done with primary human adrenal cortical cells and human adrenocarcinoma (NCI-H295R) cells. Activation of different PKC isoforms was detected by specific phospho-PKC antibodies and PKC translocation. The role of calcium-dependent signaling was examined by measuring the cytoplasmic inositol 1,4,5-triphosphate (IP(3)) and calcium ([Ca(2+)](i)). The D4R agonist PD-168,077 enhanced AII-stimulated aldosterone synthesis and secretion as early as 30 min following exposure independently of the modulation of aldosterone synthase (CYP11B2) transcription. CYP11B2 mRNA level elevated by AII was augmented by D4R in the later period. These effects were reversed by the D4R antagonist L-745,870. AII activated PKC-alpha/betaII, -epsilon, and -micro but not PKC-delta, -theta, or -zeta/lambda of H295R cells. The D4R agonist selectively enhanced AII-stimulated PKC-epsilon phosphorylation and its translocation to the cell membrane. Furthermore, the D4R agonist enhanced the AII-stimulated elevation of intracellular IP(3) and [Ca(2+)](i). Inhibition of PKC-epsilon translocation by the PKC-epsilon-specific inhibitory peptide attenuated AII-stimulated aldosterone secretion, CYP11B2 mRNA expression, and elevation of intracellular IP(3) and [Ca(2+)](i). We conclude that D4R augmented aldosterone synthesis/secretion induced by AII. The mechanisms responsible for this augmentation are mediated through enhancing PKC-epsilon phosphorylation and [Ca(2+)](i) elevation.

Roles of calcium and IP3 in impaired colon contractility of rats following multiple organ dysfunction syndrome.

The purpose of the present study was to explore changes in rat colon motility, and determine the roles of calcium and inositol (1,4,5)-triphosphate (IP3) in colon dysmotility induced by multiple organ dysfunction syndrome (MODS) caused by bacteria peritonitis. The number of stools, the contractility of the muscle strips and the length of smooth muscle cells (SMC) in the colon, the concentration of calcium and IP3 in SMC, and serum nitric oxide were measured. Number of stools, fecal weight, IP3 concentration in SMC and serum nitric oxide concentration were 0.77 +/- 0.52 pellets, 2.51 +/- 0.39 g, 4.14 +/- 2.07 pmol/tube, and 113.95 +/- 37.89 micromol/L, respectively, for the MODS group (N = 11) vs 1.54 +/- 0.64 pellets, 4.32 +/- 0.57 g, 8.19 +/- 3.11 pmol/tube, and 37.42 +/- 19.56 micromol/L for the control group (N = 20; P < 0.05). After treatment with 0.1 mM acetylcholine and 0.1 M potassium chloride, the maximum contraction stress of smooth muscle strips, the length of SMC and the changes of calcium concentration were 593 +/- 81 and 458 +/- 69 g/cm(3), 48.1 +/- 11.8 and 69.2 +/- 15.7 microM, 250 +/- 70 and 167 +/- 48%, respectively, for the control group vs 321 +/- 53 and 284 +/- 56 g/cm(3), 65.1 +/- 18.5 and 87.2 +/- 23.7 microM, 127 +/- 35 and 112 +/- 35% for the MODS group (P < 0.05). Thus, colon contractility was decreased in MODS, a result possibly related to reduced calcium concentration and IP3 in SMC.