IP3 Levels Research Articles

Systems Biology to Predict Platelet Function

AbstractAbstract SCI-38Systems Biology seeks to provide patient-specific prediction of dynamic cellular response to multiple stimuli, critical information toward predicting risk, disease progression, or response to therapy. We deployed two distinct approaches, bottom-up and top-down analyses, to gain insight into platelet signaling.The bottom-up approach required a definition of reaction network and kinetic equations (topology), kinetic parameters, and initial concentrations in order to simulate platelet signaling. We developed a computational platelet model – assembled from 24 peer-reviewed platelet studies to yield 132 measured kinetic rate constants – that accurately predicts resting levels of cytosolic calcium, IP3, diacylglycerol, phosphatidic acid, phosphoinositol, PIP, and PIP2. The model accurately predicts the full transient calcium dynamics in response to increasing levels of ADP. In the first full stochastic simulation of single platelet response to ADP, the model provides an extremely accurate prediction of the statistics of the asynchronous [Ca]i spikes observed in single platelets. Specifically, this is the first work to provide a quantitative molecular explanation of the asynchronous calcium spiking observed in ADP-activated human platelets. We show the asynchronous spiking is a result of the fundamentally stochastic nature of signal transduction in cells as small as human platelets. Specific testable predictions have emerged about the requirement of high SERCA/IP3R ratios in functional platelets, limits on the concentration of calcium in the DTS, and relative potencies of PAR peptides and ADP.For functional phenotyping platelets, a top-down approach linking multiple inputs to functional outputs was used to understand how human platelets integrate diverse signals encountered during thrombosis. We developed a high-throughput platform that measures the human platelet calcium mobilization in response to all pairwise combinations of six major agonists. Agonists tested in this study were: convulxin (CVX; GPVI activator), ADP, the thromboxane analog U46619, PAR1 agonist peptide (SFLLRN), PAR4 agonist peptide (AYPGKF), and PGE2 (activator of IP and EP receptor). The calcium responses to single agonists at 0.1, 1, 10′ EC50 and 135 pairwise combinations trained a neural network (NN) model to predict the entire 6-dimensional platelet response space. The NN model successfully predicted responses to sequential additions and 27 ternary combinations of [ADP], [convulxin], and [SFLLRN] (R=0.881). With 4077 NN simulations spanning the 6-dimensional agonist space, 45 combinations of 4–6 agonists (ranging from synergism to antagonism) were selected and confirmed experimentally (R=0.883), revealing a highly synergistic condition of high U46619/PGE2 ratio, consistent with the risk of COX-2 therapy. Furthermore, pairwise agonist scanning (PAS) provided a direct measurement of 135 synergy values, thus allowing a unique phenotypic scoring of 10 human donors.Patient-specific training of NNs represent a compact and robust approach for prediction of cellular integration of multiple signals in a complex disease milieu. Either bottom-up models or top-down NN models are ideal for incorporation into systems biology simulations of thrombotic pathways under flow conditions. Disclosures:No relevant conflicts of interest to declare.

GPCR Activation is Measured with Cisbio’s cAMP and IP1 HTRF HTplex Cell-Based Assay

BioTechniquesVol. 49, No. 4 Sponsored Paper - Application ForumOpen AccessGPCR activation is measured with Cisbio's cAMP and IP1 HTRF HTplex cell-based assayE.J. Dell, Franka Ganske & Laurence JacquemartE.J. DellBMG LABTECH GmbH, Offenburg, GermanySearch for more papers by this author, Franka GanskeBMG LABTECH GmbH, Offenburg, GermanySearch for more papers by this author & Laurence JacquemartCisbio Bioassays, Bagnols-sur-Cèze, FranceSearch for more papers by this authorPublished Online:28 Jun 2018https://doi.org/10.2144/000113517AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit IntroductionThe G protein–coupled receptor (GPCR) superfamily has almost 1000 members and thus represents the largest protein family in the human genome. GPCRs carry information within cells via two major signaling pathways: regulation of cAMP levels and increases in intracellular Ca2+ triggered by inositol 1,4,5-triphosphate (IP3). Activation of these pathways via Gs-or Gi-coupled receptors results in the increase or decrease of cAMP levels, respectively, while activation of a Gq-coupled receptor activates phospholipase C (PLC) and triggers the inositol phosphate (IP) cascade.Cisbio Bioassays' new generation Lumi4-Tb TR-FRET cryptate chemistry allows for the detection of cAMP and IP1 levels in one well by using two different acceptors, a green dye (λ = 520 nm) and a red dye (λ = 665 nm). The experiments described here where performed on the PHERAstar Plus HTS microplate reader using Simultaneous Dual Emission detection. This unique feature allows the plate to be read once for dual emission assays, thereby decreasing time and variability.The HTplex assay from CisbioThe HTplex assay uses two antibodies labeled with Lumi4-Tb (anti-cAMP Cryptate and anti-IP1 Cryptate as donors) and two acceptors (cAMP-green dye and IP1-red dye). In the inactivate state, a high TR-FRET signal is seen for both the red and green emissions (Figure 1). CHO cells were stimulated with 10 µM vasopression. Upon GPCR activation, cAMP and IP1 were produced, thereby decoupling the tracer green-cAMP and the tracer red-IP1 from the Tb-cryptate antibody. This leads to a decrease in the TR-FRET signal. Hence specific emission signals are inversely proportional to the concentration of cAMP and IP.Figure 1. Principle of the HTplex assay.Results & DiscussionFigure 2 shows an initial cAMP (Gs) response at lower concentrations of vasopressin presumably through activation of endogenous V1 receptors, and a delayed IP3 (Gq) response at higher concentrations through activation of the transfected V2 receptor (as measured by IP1 accumulation). EC50 for cAMP and IP1 were 15 × 10-6 M and 23 × 10-6 M, respectively, and both signals were well above the reliability range for an HTS assay, giving Z' values > 0.75. The HTPlex assay from Cisbio was successfully employed with BMG LABTECH's PHERAstar Plus HTS microplate reader.Figure 2. IP1 and cAMP Signaling in CHO cells upon stimulation with vasopressin.

Role of the second disulfide bridge (Cys18-Cys274) in stabilizing the inactive AT1 receptor

Previous research showed that disruption of the Cys(18)-Cys(274) bond in the angiotensin II (AngII) AT₁ receptor mutant (C18S), expressed in CHO cells, causes an increase in the basal activity and attenuation of the maximum response to AngII. In addition, this mutant was mostly intracellularly distributed. Our aim was to investigate whether the intracellular presence of the mutant was due to a constitutive internalization or to a defective maturation of the receptor. The first hypothesis was assessed by pretreating the cells with losartan or [Sar¹Leu⁸]-AngII, specific AT₁ receptor antagonists, a maneuver to revert the receptor internalization. The second hypothesis was tested using calnexin, an endoplasmic reticulum marker. We found that treatment with AT₁ receptor antagonists causes an increase in the binding ability of the mutant to AngII. Furthermore, whereas the maximum effect is increased, it reduces the enhanced basal levels of IP₃. The hypothesis for a lack of maturation of the mutant receptor was ruled out because calnexin was poorly colocalized with the intracellular C18S receptor. Our results suggest that the mutation of the AT₁ receptor leads to a conformational structure similar to that of the active mode of the AT₁ receptor, favoring its internalization in the absence of the agonist.

Chemical Inducers of Autophagy That Enhance the Clearance of Mutant Proteins in Neurodegenerative Diseases

Many of the neurodegenerative diseases that afflict people are caused by intracytoplasmic aggregate-prone proteins. These include Parkinson disease, tauopathies, and polyglutamine expansion diseases such as Huntington disease. In Mendelian forms of these diseases, the mutations generally confer toxic novel functions on the relevant proteins. Thus, one potential strategy for dealing with these mutant proteins is to enhance their degradation. This can be achieved by up-regulating macroautophagy, which we will henceforth call autophagy. In this minireview, we will consider the reasons why autophagy up-regulation may be a powerful strategy for these diseases. In addition, we will consider some of the drugs and associated signaling pathways that can be used to induce autophagy with these therapeutic aims in mind.

Histamine and specific histamine receptors increase normal cholangiocyte growth via differential mechanisms

Normal cholangiocytes proliferate after injury. Histamine (HA) interacts with four receptors (H1–H4). We have shown that histamine receptors (HRs) induce differential effects in cholangiocytes. We evaluated: (i) HR expression, (ii) chronic effects of HA or specific HRs in normal rats (NR) and (iii) signaling mechanisms. HR expression was evaluated in cholangiocytes and cholangiocyte cultures (NRC). In vivo, NR were treated with HA or selected HR agonists: H1, HTMT dimaleate; H2, amthamine dihydrobromide; RAMH; H4, clobenpropit or NaCl. Cholangiocyte growth was evaluated by: (i) immunohistochemistry in liver sections and (ii) PCNA expression. NRIC were treated with HA or selected HR agonists. We measured growth, IP3 and cAMP levels and phosphorylation of PKA and ERK1/2. HRs are found in NR and NRC. Administration of HA or H1 and H2 (but not H3 or H4) to NR increased cholangiocyte growth compared to NR + NaCl. In vitro, HA and H1HR increased growth (via Ca2+signaling), IP3 and ERK1/2 phosphorylation. HA and H2HR stimulation increased growth (via PKA signaling), cAMP and phosphorylation of PKA and ERK 1/2. H3 and H4 had no effect. HA induces normal cholangiocyte growth. Activation of specific HRs modulates growth via independent mechanisms. Stimulatory (H1/H2) and inhibitory (H3/H4) effects by HRs may be important in the regulation of cholangiocyte growth/loss related to cholangiopathies.

Suppression of the autooscillatory contractile activity of Physarum polycephalum plasmodium by the inhibitor of the IP3-Induced Ca2+ release, 2-aminoethoxydiphenyl borate

In order to elucidate the function of inositol 1,4,5-trisphosphate (IP3)-activated reticular Ca2+ channel (IP3R) in autooscillatory contractile activity of Physarum polycephalum plasmodium, we applied 2-aminoethoxydiphenyl borate (2-APB), a membrane-permeable inhibitor of IP3-induced Ca2+ release. Taking into account that for the type 1 IP3 R the inhibitory efficacy of 2-APB decreases with the rise of the IP3 level [Bilmen, J.G. and Michelangeli, F., Cell Signal., 2002, vol. 14, no. 11, pp. 955–960], 2-APB was applied to plasmodium in normal conditions and after the treatment with glucose or 3-O-methylglucose, the attractants capable to induce an elevation of the IP3 production. We found that 20–50 μM 2-APB induced a reversible cessation of contractile autooscillations, which occurred in two different modes: as a fast stop and a gradual damping. The damping of oscillations was accompanied by an increase in their period, a prolongation of the contraction phase, and, often, by an increase in the mean level of the contraction force. The number of species responding by the fast stop at a 2-APB concentration of 50 μM was two times greater than at 20 μM 2-APB. In the presence of the attractants in concentrations of 10 and 50 mM, the fast stop was never observed at 20 μM of 2-APB. Moreover, the damping of oscillations was preceded by a period of varying duration, when the regular oscillatory mode was maintained. We conclude that the fast stop results from the direct inter-action of 2-APB with IP3R of Physarum polycephalum plasmodium and that IP3R is indispensable for the plasmodial oscillator.

Purinoceptor Signaling in Arterial Smooth Muscle is Regulated by G-Protein-Coupled Recptor Kinase-2

The regulation of arterial smooth muscle cell (SMC) contraction by adenine and uridine nucleotides, plays a key role in controlling systemic blood pressure. In SMCs, UTP activates P2Y receptors (subtypes 2/4/6), which couple via Gαq/11-proteins to stimulate phospholipase C, increasing IP3/Ca2+ concentrations and leading to SMC contraction. Continuous or repeated receptor stimulation reduces responsiveness to further stimulation, a process termed desensitization. Receptor desensitization is often regulated by G protein-coupled receptor kinases (GRKs), which phosphorylate receptors, enhancing their interaction with β-arrestins and uncoupling them from G-proteins.We investigated the regulation of receptors responding to UTP, which mediates concentration-dependent contraction in rat mesenteric arteries. To characterize adaptations that occur on repeated UTP additions, changes in IP3 and [Ca2+]i were assessed using single-cell imaging. Receptor desensitization was assessed by challenging mesenteric SMCs with an EC50 concentration of UTP (10 μM) for 30 sec before (R1) and after (R2) the addition of a maximal UTP concentration (Rmax, 100 μM, 30 sec) with 5 min washout periods. The change in R2 relative to R1 was used to characterize P2Y receptor desensitization. By extending the washout period after Rmax a time-dependent recovery of IP3/Ca2+ responses were observed. To evaluate the involvement of individual GRKs in this process, cells were transfected with catalytically-inactive, dominant-negative GRK mutants.Using IP3 generation to indicate receptor recovery (R2/R1%), over-expression of D110A,K220RGRK3 (34±4%), K215RGRK5 (38±7%), or K215RGRK6 (29±8%) caused similar reductions in IP3 levels to those in empty-vector-transfected cells (23±5%). In contrast, expression of D110A,K220RGRK2 (58±7%) markedly attenuated receptor desensitization (n=10-19 cells, >3 animals). Furthermore, siRNA-mediated knockdown (>75%) of GRK2 protein also attenuated agonist-induced receptor desensitization compared to control (68±8% versus 29±6%, respectively, n=8-12). In conclusion, this work implicates GRK2 as the pre-eminent GRK isoenzyme regulating UTP signaling in SMCs.

Muscarinic Receptor 1 Agonist Activity of Novel Arecoline Derivatives in Alzheimer’s Dementia Models

T cholinergic deficit in Alzheimer’s disease (AD) patient’s brain has intensified research efforts to test cholinomimetic approaches for efficacy in AD therapy. Various therapies may be of potential clinical use in AD. Among these are cholinergic agents including muscarinic agonists, acetylcholinesterase inhibitors, and acetylcholine releasing agents. One of the muscarinic agonists tested in AD is arecoline and its bioisosters, which are widely, explored as muscarinic receptor 1 agonist (M1 receptor agonist) in AD research. In this regard, we have synthesized five and six membered heterocyclic ring system attached arecoline basic nucleus (N-methyl tetrahydropyridines) at 3rd position. Subsequently the synthesized arecolines derivatives were subjected to in vitro muscarinic receptor 1 binding affinity studies using male wistar rat brain synaptosomal membrane (cerebral cortex) and also cell line culture studies and extended this in vitro studies to in vivo pharmacological evaluation of memory and learning in male wistar rats (Rodent memory evaluation, plus and Y maze studies). Some of our synthesized molecules have shown very potent M1 receptor agonist activity and significantly elevated the basal IP3 levels in vitro and also have decreased beta-amyloid (Abeta40 and Abeta42) deposition in cell lines culture. These molecules have also shown very good antidementia activity in rat dementia model. Conclusions: Molecules with electron donating group as a substitute, has shown very good affinity towards the M1 receptor in vitro and has also elicited beneficial effects in vivo memory and learning models. Muscarinic Receptor 1 Agonist Activity of Novel Arecoline Derivatives in Alzheimer’s Dementia Models

H3 Histamine Receptor–Mediated Activation of Protein Kinase Cα Inhibits the Growth of Cholangiocarcinoma In vitro and In vivo

Histamine regulates functions via four receptors (HRH1, HRH2, HRH3, and HRH4). The d-myo-inositol 1,4,5-trisphosphate (IP(3))/Ca(2+)/protein kinase C (PKC)/mitogen-activated protein kinase pathway regulates cholangiocarcinoma growth. We evaluated the role of HRH3 in the regulation of cholangiocarcinoma growth. Expression of HRH3 in intrahepatic and extrahepatic cell lines, normal cholangiocytes, and human tissue arrays was measured. In Mz-ChA-1 cells stimulated with (R)-(alpha)-(-)-methylhistamine dihydrobromide (RAMH), we measured (a) cell growth, (b) IP(3) and cyclic AMP levels, and (c) phosphorylation of PKC and mitogen-activated protein kinase isoforms. Localization of PKCalpha was visualized by immunofluorescence in cell smears and immunoblotting for PKCalpha in cytosol and membrane fractions. Following knockdown of PKCalpha, Mz-ChA-1 cells were stimulated with RAMH before evaluating cell growth and extracellular signal-regulated kinase (ERK)-1/2 phosphorylation. In vivo experiments were done in BALB/c nude mice. Mice were treated with saline or RAMH for 44 days and tumor volume was measured. Tumors were excised and evaluated for proliferation, apoptosis, and expression of PKCalpha, vascular endothelial growth factor (VEGF)-A, VEGF-C, VEGF receptor 2, and VEGF receptor 3. HRH3 expression was found in all cells. RAMH inhibited the growth of cholangiocarcinoma cells. RAMH increased IP(3) levels and PKCalpha phosphorylation and decreased ERK1/2 phosphorylation. RAMH induced a shift in the localization of PKCalpha expression from the cytosolic domain into the membrane region of Mz-ChA-1 cells. Silencing of PKCalpha prevented RAMH inhibition of Mz-ChA-1 cell growth and ablated RAMH effects on ERK1/2 phosphorylation. In vivo, RAMH decreased tumor growth and expression of VEGF and its receptors; PKCalpha expression was increased. RAMH inhibits cholangiocarcinoma growth by PKCalpha-dependent ERK1/2 dephosphorylation. Modulation of PKCalpha by histamine receptors may be important in regulating cholangiocarcinoma growth.

LANCL2 Is Necessary for Abscisic Acid Binding and Signaling in Human Granulocytes and in Rat Insulinoma Cells

Abscisic acid (ABA) is a plant hormone regulating fundamental physiological functions in plants, such as response to abiotic stress. Recently, ABA was shown to be produced and released by human granulocytes, by insulin-producing rat insulinoma cells, and by human and murine pancreatic beta cells. ABA autocrinally stimulates the functional activities specific for each cell type through a receptor-operated signal transduction pathway, sequentially involving a pertussis toxin-sensitive receptor/G-protein complex, cAMP, CD38-produced cADP-ribose and intracellular calcium. Here we show that the lanthionine synthetase C-like protein LANCL2 is required for ABA binding on the membrane of human granulocytes and that LANCL2 is necessary for transduction of the ABA signal into the cell-specific functional responses in granulocytes and in rat insulinoma cells. Co-expression of LANCL2 and CD38 in the human HeLa cell line reproduces the ABA-signaling pathway. Results obtained with granulocytes and CD38(+)/LANCL2(+) HeLa transfected with a chimeric G-protein (G alpha(q/i)) suggest that the pertussis toxin-sensitive G-protein coupled to LANCL2 is a G(i). Identification of LANCL2 as a critical component of the ABA-sensing protein complex will enable the screening of synthetic ABA antagonists as prospective new anti-inflammatory and anti-diabetic agents.

Inhibition of Heterotrimeric G Protein Signaling by a Small Molecule Acting on Gα Subunit

The simultaneous activation of many distinct G protein-coupled receptors (GPCRs) and heterotrimeric G proteins play a major role in various pathological conditions. Pan-inhibition of GPCR signaling by small molecules thus represents a novel strategy to treat various diseases. To better understand such therapeutic approach, we have characterized the biomolecular target of BIM-46187, a small molecule pan-inhibitor of GPCR signaling. Combining bioluminescence and fluorescence resonance energy transfer techniques in living cells as well as in reconstituted receptor-G protein complexes, we observed that, by direct binding to the Galpha subunit, BIM-46187 prevents the conformational changes of the receptor-G protein complex associated with GPCR activation. Such a binding prevents the proper interaction of receptors with the G protein heterotrimer and inhibits the agonist-promoted GDP/GTP exchange. These observations bring further evidence that inhibiting G protein activation through direct binding to the Galpha subunit is feasible and should constitute a new strategy for therapeutic intervention.

Blue light induced changes in inositol 1,4,5-trisphosphate inCuscuta campestrisseedlings

Previous studies revealed that blue light stimulates and red light inhibits prehaustoria development in young seedlings of the parasitic weed Cuscuta campestris (field dodder). This study showed a positive correlation between blue light induced increases in inositol 1,4,5-trisphosphate (IP3) and blue light-mediation of prehaustoria development, prior to host attachment. Blue light induced a significant increase in the level of IP3, with a peak at about 30 min. Thereafter, the level of IP3 declined to the resting value after 2 h of blue light. Irradiation with 10 min red light pulse applied directly at the end of each 0.05, 0.1, 0.15, 0.2, 0.3, 1, 2 and 4 h blue light significantly reduced IP3, while high levels of IP3 were observed after 10 min far-red pulse. The G-protein inhibitor pertussis toxin inhibited prehaustoria developed under blue light, suggesting that receptor-coupled G-proteins are likely to be involved in prehaustoria development. These results are the first in vivo demonstration of a possible role for IP3 as a second messenger in the blue light signal transduction process in prehaustoria development in Cuscuta.

P2X7-mediated Increased Intracellular Calcium Causes Functional Derangement in Schwann Cells from Rats with CMT1A Neuropathy

Charcot-Marie-Tooth (CMT) is the most frequent inherited neuromuscular disorder, affecting 1 person in 2500. CMT1A, the most common form of CMT, is usually caused by a duplication of chromosome 17p11.2, containing the PMP22 (peripheral myelin protein-22) gene; overexpression of PMP22 in Schwann cells (SC) is believed to cause demyelination, although the underlying pathogenetic mechanisms remain unclear. Here we report an abnormally high basal concentration of intracellular calcium ([Ca(2+)](i)) in SC from CMT1A rats. By the use of specific pharmacological inhibitors and through down-regulation of expression by small interfering RNA, we demonstrate that the high [Ca(2+)](i) is caused by a PMP22-related overexpression of the P2X7 purinoceptor/channel leading to influx of extracellular Ca(2+) into CMT1A SC. Correction of the altered [Ca(2+)](i) in CMT1A SC by small interfering RNA or with pharmacological inhibitors of P2X7 restores functional parameters of SC (migration and release of ciliary neurotrophic factor), which are typically defective in CMT1A SC. More significantly, stable down-regulation of the expression of P2X7 restores myelination in co-cultures of CMT1A SC with dorsal root ganglion sensory neurons. These results establish a pathogenetic link between high [Ca(2+)](i) and impaired SC function in CMT1A and identify overexpression of P2X7 as the molecular mechanism underlying both abnormalities. The development of P2X7 inhibitors is expected to provide a new therapeutic strategy for treatment of CMT1A neuropathy.

Muscarinic receptor 1 agonist activity of novel arecoline derivatives in Alzheimer's dementia models

T cholinergic deficit in Alzheimer’s disease (AD) patient’s brain has intensified research efforts to test cholinomimetic approaches for efficacy in AD therapy. Various therapies may be of potential clinical use in AD. Among these are cholinergic agents including muscarinic agonists, acetylcholinesterase inhibitors, and acetylcholine releasing agents. One of the muscarinic agonists tested in AD is arecoline and its bioisosters, which are widely, explored as muscarinic receptor 1 agonist (M1 receptor agonist) in AD research. In this regard, we have synthesized five and six membered heterocyclic ring system attached arecoline basic nucleus (N-methyl tetrahydropyridines) at 3rd position. Subsequently the synthesized arecolines derivatives were subjected to in vitro muscarinic receptor 1 binding affinity studies using male wistar rat brain synaptosomal membrane (cerebral cortex) and also cell line culture studies and extended this in vitro studies to in vivo pharmacological evaluation of memory and learning in male wistar rats (Rodent memory evaluation, plus and Y maze studies). Some of our synthesized molecules have shown very potent M1 receptor agonist activity and significantly elevated the basal IP3 levels in vitro and also have decreased beta-amyloid (Abeta40 and Abeta42) deposition in cell lines culture. These molecules have also shown very good antidementia activity in rat dementia model. Conclusions: Molecules with electron donating group as a substitute, has shown very good affinity towards the M1 receptor in vitro and has also elicited beneficial effects in vivo memory and learning models. Muscarinic Receptor 1 Agonist Activity of Novel Arecoline Derivatives in Alzheimer’s Dementia Models

Phosphatidylinositol-4-phosphate 5-kinase 1α Mediates Extracellular Calcium-induced Keratinocyte Differentiation

Extracellular calcium (Cao) is a major regulator of keratinocyte differentiation, but the mechanism is unclear. Phosphatidylinositol-4-phosphate 5-kinase 1alpha (PIP5K1alpha) is critical in synthesizing phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. In this study, we sought to determine whether PIP5K1alpha plays a role in mediating the ability of Cao to induce keratinocyte differentiation. We found that treatment of human keratinocytes in culture with Cao resulted in increased PIP5K1alpha level and activity, as well as PI(4,5)P2 level, binding of phosphatidylinositol 3,4,5-triphosphate [PI(3,4,5)P3] to and activation of phospholipase C-gamma1 (PLC-gamma1), with the resultant increase in inositol 1,4,5-trisphosphate (IP3) and intracellular calcium (Cai). Knockdown of PIP5K1alpha in human keratinocytes blocked Cao-induced increases in the binding of PI(3,4,5)P3 to PLC-gamma1; PLC-gamma1 activity; levels of PI(4,5)P2, IP3, and Cai; and induction of keratinocyte differentiation markers. Coimmunoprecipitation and confocal studies revealed that Cao stimulated PIP5K1alpha recruitment to the E-cadherin-catenin complex in the plasma membrane. Knockdown of E-cadherin or beta-catenin blocked Cao-induced activation of PIP5K1alpha. These results indicate that after Cao stimulation PIP5K1alpha is recruited by the E-cadherin-catenin complex to the plasma membrane where it provides the substrate PI(4,5)P2 for both PI3K and PLC-gamma1. This signaling pathway is critical for Cao-induced generation of the second messengers IP3 and Cai and keratinocyte differentiation.

TRPCs, GPCRs and the Bayliss effect

An increase in pressure inside most small arteries unexpectedly results in a vasoconstriction. This phenomenon, known as the Bayliss effect, involves the stretch‐induced activation of non‐selective cation channels in the vascular smooth muscle cells. Recent work by Mederos y Schnitzler et al . now demonstrates that the stretch‐induced channel activity originates from a fascinating interplay between a TRP cation channel and the angiotensin receptor.

Mass Spectrometric Analysis of Type 1 Inositol 1,4,5-Trisphosphate Receptor Ubiquitination

Inositol 1,4,5-trisphosphate (IP(3)) receptors form tetrameric channels in endoplasmic reticulum membranes of mammalian cells and mediate IP(3)-induced calcium mobilization. In response to various extracellular stimuli that persistently elevate IP(3) levels, IP(3) receptors are also ubiquitinated and then degraded by the proteasome. Here, for endogenous type 1 IP(3) receptor (IP(3)R1) activated by endogenous signaling pathways and processed by endogenous enzymes, we sought to determine the sites of ubiquitination and the composition of attached ubiquitin conjugates. Our findings are (i) that at least 11 of the 167 lysines in IP(3)R1 can be ubiquitinated and that these are clustered in the regulatory domain and are found in surface regions, (ii) that at least approximately 40% of the IP(3)R1-associated ubiquitin is monoubiquitin, (iii) that both Lys(48) and Lys(63) linkages are abundant in attached ubiquitin chains, and (iv) that Lys(63) linkages accumulate most rapidly. Additionally, we find that not all IP(3)R1 subunits in a tetramer are ubiquitinated and that nontetrameric IP(3)R1 complexes form as degradation proceeds, suggesting that ubiquitinated subunits may be selectively extracted and degraded. Overall, these data show that endogenous IP(3)R1 is tagged with an array of ubiquitin conjugates at multiple sites and that both IP(3)R1 ubiquitination and degradation are highly complex processes.

Muscarinic receptor 1 agonist activity of novel N-arylthioureas substituted 3-morpholino arecoline derivatives in Alzheimer’s presenile dementia models

As part of our continuing effort aimed at the development of selective, efficacious and centrally active M1 muscarinic agonists for the treatment of Alzheimer’s presenile dementia, a series of N-arylthioureas substituted 3-morpholino arecoline derivatives 9(a–j) were synthesized by using N-benzyl amino ethanol coupling with α-bromo acetyl pyridine followed by reduction and cyclization to develop a new class of M1 receptor agonists. Subsequently the synthesized compounds were subjected to in vitro radioligand M1 receptor affinity studies, IP3 formation studies and also to in vivo pharmacological evaluation of memory and learning in male Wistar rats. Derivatives with chloro ( 9f) and methoxy ( 9c) groups on the para position of the benzene ring attached to the nitrogen of thiourea showed several fold high affinity for the M1 receptor (in vitro) among all the synthesized molecules 9( a–j), and also significantly elevated IP3 levels and as well elicited beneficial effects in vivo in memory and learning models in rats (rodent memory evaluation, plus and Y maze studies).

Lysophospholipid Receptor-Mediated Calcium Signaling in Human Keratinocytes

The lysophospholipids, sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA), stimulate chemotaxis and induce differentiation of human keratinocytes. As Ca(2+) plays an important role in keratinocyte differentiation, we studied Ca(2+) signaling by S1P and LPA in these cells, known to express mRNA transcripts of the S1P(1-5) and LPA(1-3) receptors, and the receptor subtypes involved in this process. S1P and LPA caused transient increases in intracellular free Ca(2+) concentration ([Ca(2+)](i)), with pEC(50) values of 8.5+/-0.11 and 7.5+/-0.23, respectively. The [Ca(2+)](i) increases are apparently mediated by stimulation of phospholipase C and involve Ca(2+) mobilization from thapsigargin-sensitive stores and subsequent Ca(2+) influx. The LPA-induced [Ca(2+)](i) increases were not inhibited by the LPA(1/3) receptor antagonist, dioctanoylglycerol pyrophosphate. The S1P-induced [Ca(2+)](i) increases were largely inhibited by the putative S1P(3) antagonist, BML-241, and the S1P(1/3) antagonist, VPC23019. The S1P(1)-specific agonist, SEW2871, did not increase [Ca(2+)](i) but stimulated chemotaxis of keratinocytes, which was fully blocked by S1P(1) antisense oligonucleotides. The data indicate that LPA and S1P potently increase [Ca(2+)](i) in human keratinocytes and that the effect of LPA is mediated by LPA(2), whereas that of S1P is mediated at least to a large part by S1P(3). The S1P(1) receptor, without stimulating [Ca(2+)](i) increases, mediates chemotaxis of keratinocytes.

Small mouse cholangiocytes proliferate in response to H1 histamine receptor stimulation by activation of the IP3/CaMK I/CREB pathway

Cholangiopathies are characterized by the heterogeneous proliferation of different-sized cholangiocytes. Large cholangiocytes proliferate by a cAMP-dependent mechanism. The function of small cholangiocytes may depend on the activation of inositol trisphosphate (IP(3))/Ca(2+)-dependent signaling pathways; however, data supporting this speculation are lacking. Four histamine receptors exist (HRH1, HRH2, HRH3, and HRH4). In several cells: 1) activation of HRH1 increases intracellular Ca(2+) concentration levels; and 2) increased [Ca(2+)](i) levels are coupled with calmodulin-dependent stimulation of calmodulin-dependent protein kinase (CaMK) and activation of cAMP-response element binding protein (CREB). HRH1 agonists modulate small cholangiocyte proliferation by activation of IP(3)/Ca(2+)-dependent CaMK/CREB. We evaluated HRH1 expression in cholangiocytes. Small and large cholangiocytes were stimulated with histamine trifluoromethyl toluidide (HTMT dimaleate; HRH1 agonist) for 24-48 h with/without terfenadine, BAPTA/AM, or W7 before measuring proliferation. Expression of CaMK I, II, and IV was evaluated in small and large cholangiocytes. We measured IP(3), Ca(2+) and cAMP levels, phosphorylation of CaMK I, and activation of CREB (in the absence/presence of W7) in small cholangiocytes treated with HTMT dimaleate. CaMK I knockdown was performed in small cholangiocytes stimulated with HTMT dimaleate before measurement of proliferation and CREB activity. Small and large cholangiocytes express HRH1, CaMK I, and CaMK II. Small (but not large) cholangiocytes proliferate in response to HTMT dimaleate and are blocked by terfenadine (HRH1 antagonist), BAPTA/AM, and W7. In small cholangiocytes, HTMT dimaleate increased IP(3)/Ca(2+) levels, CaMK I phosphorylation, and CREB activity. Gene knockdown of CaMK I ablated the effects of HTMT dimaleate on small cholangiocyte proliferation and CREB activation. The IP(3)/Ca(2+)/CaMK I/CREB pathway is important in the regulation of small cholangiocyte function.