Hydrolysis Of Phosphatidylinositol Bisphosphate Research Articles

Regulation of the two-pore domain potassium channel, THIK-1 and THIK-2, by G protein coupled receptors.

A member of THIK (two pore domain halothane-inhibited K+) channels, THIK-1, was reported as a target of Gi/o-coupled receptors (Gi/o-Rs) in neurons and microglia. We confirmed that in HEK293T cells the THIK-1 channel is activated by Gi/o-Rs and found that Gq-coupled receptors (Gq-Rs) also activates the channel. The effects of Gi/o-Rs and Gq-Rs were inhibited by the Gi/o inhibitor pertussis toxin and phospholipase C (PLC) inhibitor, respectively. The effects of Gi/o-Rs were attenuated when consensus Gβγ binding motif at the C-tail of the THIK-1 channel was mutated, suggesting that Gβγ serves as a THIK-1 channel activator upon the stimulation of Gi/o-Rs. As to the effects of Gq-Rs on the THIK-1 channel, a protein kinase C inhibitor and calcium chelators failed to inhibit the effect of a Gq coupled muscarinic M1R. Neither the hydrolysis of phosphatidyl inositol bisphosphate induced by voltage sensitive phosphatase nor the application of a diacylglycerol analogue, OAG, increased the channel current. The mediator of Gq-dependent activation of the THIK-1 channel remained unsolved. The effects of Gi/o- and Gq-Rs on the THIK-2 channel were also investigated, by using a THIK-2 mutant channel whose N-terminal domain is deleted to improve the surface membrane expression. We observed that Gi/o- and Gq-Rs activate the mutated THIK-2 channel, similarly to the THIK-1 channel. Interestingly, heterodimeric channels of THIK-1 and THIK-2 responded to Gi/o-R and Gq-R stimulation. Taken together, Gi/o- or Gq-Rs activates the THIK-1 and THIK-2 channels in a Gβγ or PLC dependent manner, respectively.

Divergent transcriptomic profiles in skeletal muscle of diabetics with and without heart failure

Abstract Funding Acknowledgements Type of funding sources: None. Background Patients with type 2 diabetes mellitus (DM) that have coexistent heart failure (HF) have exacerbated symptoms and prognosis, however beside cardiac dysfunction the mechanisms governing these features are incompletely understood. Evidence indicates abnormalities in the periphery could contribute to this worse clinical phenotype, including a role for skeletal muscle whereby disturbances in the transcriptome could disrupt muscle homeostasis/repair to offer a novel therapeutic approach. Purpose Is the skeletal muscle transcriptome distinguishable between DM patients with and without HF? Methods DM patients without (n = 11) or with HF with reduced left ventricular ejection fraction (LVEF) (n = 16) were included. Muscle biopsies were collected from the pectoralis major during pacemaker implantation. Following RNA extraction and cDNA synthesis, non-bias RNA sequencing (RNAseq) was performed (Cambridge Genomic Services, UK) followed by targeted RT-PCR gene expression of relevant targets. DESeq2 identified differentially expressed genes (DEGs) with a false discovery rate (p < 0.05). Gene enrichment analysis was performed with clusterProfiler v3.16.0 to interrogate the gene ontology database, while pathway analysis was conducted using ReactomePA v1.32.0 to interrogate the Reactome database, using an adjusted p value. Values of p < 0.05 were accepted as significant. Results Groups were not different (p > 0.05) for age (74 ± 11 vs. 66 ± 10 years), BMI (31 ± 7 vs 29 ± 6), sex (n = 2 females per group), or HbA1c (56 ± 10 vs. 57 ± 8 mmol/mol), although LVEF was lower in the group with HF (27 ± 8 vs. 54 ± 2%; p < 0.05). Of the 19,544 genes analysed, RNAseq identified 53 DEGs between DM patients with and without HF, with several relevant targets related to myofiber homeostasis such as autophagy (RUBCN), protein synthesis (DGKζ), and inflammation/apoptosis (TLE1). Follow-up RT-PCR analysis confirmed a trend towards upregulation of the autophagy-related machinery p62 (p = 0.043) and BNIP3 (p = 0.085) in the HF group, but not ubiquitin-proteasome (MuRF1, MAFbx; p > 0.05). Gene-enrichment analysis of DEGs identified 7 overrepresented terms (P < 0.05), including lipid metabolism/signalling alongside epigenetic modifications related to histone deacetylases (HDAC6/10). Furthermore, pathway analysis identified 4 terms (p < 0.05) related to NOTCH signalling and phosphatidyl inositol-bisphosphate (PIP2) hydrolysis thus indicating alterations to muscle repair and lipid signalling respectively. Conclusion(s): This study confirms that DM patients with and without HF demonstrate distinct skeletal muscle transcriptome profiles. Key differences related to skeletal muscle myogenesis, autophagy, epigenetic regulation, and lipid signalling were identified that could form part of important therapeutic targets. Whether these underlying muscle transcriptome differences contribute to poorer clinical outcomes in DM patients with HF remains to be determined.

GhPIPLC2D promotes cotton fiber elongation by enhancing ethylene biosynthesis

SummaryInositol-1,4,5-trisphosphate (IP3) is an important second messenger and one of the products of phosphoinositide-specific phospholipase C (PIPLC)-mediated phosphatidylinositol (4,5) bisphosphate (PIP2) hydrolysis. However, the function of IP3 in cotton is unknown. Here, we characterized the function of GhPIPLC2D in cotton fiber elongation. GhPIPLC2D was preferentially expressed in elongating fibers. Suppression of GhPIPLC2D transcripts resulted in shorter fibers and decreased IP3 accumulation and ethylene biosynthesis. Exogenous application of linolenic acid (C18:3) and phosphatidylinositol (PI), the precursor of IP3, improved IP3 and myo-inositol-1,2,3,4,5,6-hexakisphosphate (IP6) accumulation, as well as ethylene biosynthesis. Moreover, fiber length in GhPIPLC2D-silenced plant was reduced after exogenous application of IP6 and ethylene. These results indicate that GhPIPLC2D positively regulates fiber elongation and IP3 promotes fiber elongation by enhancing ethylene biosynthesis. Our study broadens our understanding of the function of IP3 in cotton fiber elongation and highlights the possibility of cultivating better cotton varieties by manipulating GhPIPLC2D in the future.

Running the hypothesis of a bio molecular approach to psychiatric disorder characterization and fatty acids therapeutical choices

Beyond the conviction that Major Depression can found its origin in genetics [1-3] a bio molecular mechanism could be hypothesized from what emerged from the studies on platelets fatty acid composition in human (normal and depressive subjects) which allowed classifying the depressive disorder [4] using an Artificial Neural Network (Self Organizing Map-SOM) [5], as mathematical tool, because of the complexity of the membrane dynamics. Rapid changes in membrane lipid composition or in the cytoskeleton could modify neuronal signalling. In the knowledge to have found something that could have implications in the understanding of some aspects of psychiatric disorders and a very suggestive hypothesis was build as summarized in Figure ​Figure1.1. In figure ​figure11 is described the molecular depression hypothesis made according to Cocchi and collegues [4], Donati and collegues [6], Hameroff and Penrose [7]. The membrane viscosity can modify the Gsα protein status. The Gsα protein is connected with Tubulin. Tubulin, depending on local membrane lipid fase concentration, may serve as a positive or negative regulator of phosphatidylinositol bisphosphate (PIP2) hydrolysis, such as Gsα protein does. Tubulin is known to form high-affinity complexes with certain G proteins. The formation of such complexes allows tubulin to activate Gsα, which, in turn, can activate the Protein Kinase C and fosters a system whereby elements of the cytoskeleton can influence G-protein signalling. Rapid changes in membrane lipid composition or in the cytoskeleton might modify neuronal signalling. We have hypothesized that through this mechanism is possible to modify the consciousness state and that it is mesurable through gamma syncrony EEG. Figure 1 The molecular depression hypothesis. There are strong reasons to think that each fatty acid combination of Palmitic Acid (PA), Linoleic Acid (LA) and Arachidonic Acid (AA), in platelet, is responsible of the membrane viscosity and, therefore, of the molecular conditioning of the cellular stuctures (Gsα and Tubulin) and that the main therapeutic target is the reduction of the Arachidonic Acid.

Serotonin 5-Hydroxytryptamine2CReceptor Signaling in Hypothalamic Proopiomelanocortin Neurons: Role in Energy Homeostasis in Females

Hypothalamic proopiomelanocortin (POMC) neurons play a critical role in the regulation of energy balance, and there is a convergence of critical synaptic input including GABA and serotonin on POMC neurons to regulate their output. We found previously that 17beta-estradiol (E(2)) reduced the potency of the GABA(B) receptor agonist baclofen to activate G protein-coupled inwardly rectifying potassium (GIRK) channels in hypothalamic POMC neurons through a membrane estrogen receptor (mER) via a Galpha(q) phospholipase C (PLC)-protein kinase Cdelta-protein kinase A pathway. We hypothesized that the mER and neurotransmitter receptor signaling pathways converge to control energy homeostasis. Because 5-HT(2C) receptors mediate many of the effects of serotonin in POMC neurons, we elucidated the common signaling pathways of E(2) and 5-HT in guinea pigs using single-cell reverse transcription-polymerase chain reaction (RT-PCR), real time RT-PCR, and whole-cell patch recording. Both 5-hydroxytryptamine(2C) (5-HT(2C)) and 5-HT(2A) receptors were coexpressed in POMC neurons. The 5-HT(2A/C) agonist (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) desensitized the GABA(B) response in a dose-dependent manner, which was antagonized by the selective 5-HT(2C) receptor antagonists 8-[5-(2,4-dimethoxy-5-(4-trifluoromethylphenylsulphonamido) phenyl-5-oxopentyl]1,3,8-triazaspiro[4.5] decane-2,4-dione hydrochloride (RS102221) and 1,2,3, 4,10,14b-hexahydro-2-methyldibenzo [c,f]pyrazino[1,2-a]-azepine hydrochloride (ORG 3363). The 5-HT(2C) receptor was Galpha(q)-coupled to PLC activation and hydrolysis of plasma membrane phosphatidylinositol bisphosphate to directly inhibit GIRK channel activity. Coapplication of the two agonists at their EC(50) concentrations (DOI, 20 muM, and E(2), 50 nM) produced additive effects. Although there was a significant gender difference in the effects of E(2) on baclofen responses, there was no gender difference in 5-HT(2C) receptor-mediated effects. Finally, both DOI and estrogen (intracerebroventricular) inhibited feeding in ovariectomized female mice. Therefore, the Galpha(q) signaling pathways of the mER and 5-HT(2C) receptors may converge to enhance synaptic efficacy in brain circuits that are critical for maintaining homeostatic functions.

DCC-dependent Phospholipase C Signaling in Netrin-1-induced Neurite Elongation

Netrins, a family of secreted molecules, play important roles in axon pathfinding during nervous system development. Although phosphatidylinositol signaling has been implicated in this event, how netrin-1 regulates phosphatidylinositol signaling remains poorly understood. Here we provide evidence that netrin-1 stimulates phosphatidylinositol bisphosphate hydrolysis in cortical neurons. This event appears to be mediated by DCC (deleted in colorectal cancer), but not neogenin or Unc5h2. Netrin-1 induces phospholipase Cgamma (PLCgamma) tyrosine phosphorylation. Inhibition of PLC activity attenuates netrin-1-induced cortical neurite outgrowth. These results suggest that netrin-1 regulates phosphatidylinositol turnover and demonstrate a crucial role of PLC signaling in netrin-1-induced neurite elongation.

Phosphatidylinositol transfer protein-α in netrin-1-induced PLC signalling and neurite outgrowth

Neurite extension is essential for wiring the nervous system during development. Although several factors are known to regulate neurite outgrowth, the underlying mechanisms remain unclear. Here, we provide evidence for a role of phosphatidylinositol transfer protein-alpha (PlTPalpha) in neurite extension in response to netrin-1, an extracellular guidance cue. PlTPalpha interacts with the netrin receptor DCC (deleted in colorectal cancer) and neogenin. Netrin-1 stimulates PlTPalpha binding to DCC and to phosphatidylinositol (5) phosphate [Pl(5)P], increases its lipid-transfer activity and elevates hydrolysis of phosphatidylinositol bisphosphate (PlP2). In addition, the stimulated PIP2 hydrolysis requires PlTPalpha. Furthermore, cortical explants of PlTPalpha mutant mice are defective in extending neurites in response to netrin-1. Commissural neurons from chicken embryos expressing a dominant-negative PlTPalpha mutant show reduced axon outgrowth. Morpholino-mediated knockdown of PlTPalpha expression in zebrafish embryos leads to dose-dependent defects in motor-neuron axons and reduced numbers of spinal-cord neurons. Taken together, these results identify a crucial role for PlTPalpha in netrin-1-induced neurite outgrowth, revealing a signalling mechanism for DCC/neogenin and PlTPalpha regulation.

Regulation of Phospholipase C-δ1 through Direct Interactions with the Small GTPase Ral and Calmodulin

Second messengers generated from membrane lipids play a critical role in signaling and control diverse cellular processes. Despite being one of the most evolutionarily conserved of all the phosphoinositide-specific phospholipase C (PLC) isoforms, a family of enzymes responsible for hydrolysis of the membrane lipid phosphatidylinositol bisphosphate, the mechanism of PLC-delta1 activation is still poorly understood. Here we report a novel regulatory mechanism for PLC-delta1 activation that involves direct interaction of the small GTPase Ral and the universal calcium-signaling molecule calmodulin (CaM) with PLC-delta1. In addition, we have identified a novel IQ type CaM binding motif within the catalytic region of PLC-delta1 that is not found in other PLC isoforms. Binding of CaM at the IQ motif inhibits PLC-delta1 activity, while addition of Ral reverses the inhibition. The overexpression of various Ral mutants in cells potentiates PLC-delta1 activity. Thus, the Ral-CaM complex defines a multifaceted regulatory mechanism for PLC-delta1 activation.

Role of cumulus cells during maturation of porcine oocytes in the rise in intracellular Ca 2+ induced by inositol 1,4,5-trisphosphate

At the time of fertilization, release of inositol 1,4,5-trisphosphate (IP3) into the cytoplasm of oocytes is said to be induced by hydrolysis of phosphatidylinositol bis phosphate (PI2) via activation of phospholipase C and is responsible for the Ca 2+ oscillation in oocytes immediately after sperm penetration. On the other hand, cumulus cells have been reported to play an important role in cytoplasmic maturation of mammalian oocytes and to affect embryonic development after fertilization. To obtain more information on the role of cumulus cells in cytoplasmic maturation of oocytes, the effects of cumulus cells on the rise in [Ca 2+]i and the rates of activation and development of porcine mature oocytes induced by IP3 injection were investigated. Mature porcine oocytes that had been denuded of their cumulus cells in the early stage of the maturation period had a depressed rise in [Ca 2+]i (4.0–6.0) and reduced rates of activation (31.4–36.8%) and development (10.0–24.4%) induced by IP3 injection compared with those of their cumulus-enclosed counterparts (7.3, 69.1% and 43.8%; P < 0.05). The [Ca 2+]i rise and the rates of activation and development depressed by the removal of cumulus cells were restored by adding pyruvate to the maturation medium. Furthermore, the IP3 injection-induced depression of [Ca 2+]i rise in mature oocytes derived from cumulus-denuded oocytes (DOs) was restored when they were cultured in a medium with pyruvate (3.9–6.3, P < 0.05). Also, mature oocytes from cumulus–oocyte complexes (COCs) cultured in a medium without glucose had a lower rise in [Ca 2+]i than that in mature oocytes from COCs cultured with glucose (7.4–6.0, P < 0.05). Cumulus cells supported porcine oocytes during maturation in the rise in [Ca 2+]i induced by IP3 and the following activation and development of porcine oocytes after injection of IP3. Moreover, we inferred that a function of cumulus cells is to produce pyruvate by metabolizing glucose and to provide oocytes with pyruvate during maturation, thereby promoting oocyte sensitivity to IP3.

HIV drug nevirapine (Viramune): risk of severe hepatotoxicity.

Platelet-activating factor (PAF) has recently been demonstrated to be metabolized by B lymphocytes and to cause enhancement of Ig synthesis by Ig-secreting B lymphoblastoid cell lines. We have now examined some of the early activation events triggered by PAF binding to three Ig-secreting B cell lines, LA350 (IgM secreting), HSCE- (IgG secreting), and U266 (IgE secreting). After addition of 10(-7) to 10(-11) M PAF, but not equimolar concentrations of the inactive metabolite lyso-PAF, all three cell lines demonstrated rapid dose-dependent increases in free cytosolic Ca2+ concentrations ([Ca2+]i). The increases in [Ca2+]i resulted from both the release of Ca2+ from internal stores as well as transmembrane Ca2+ uptake. Addition of PAF triggered the rapid hydrolysis of phosphatidylinositol bisphosphate and accumulation of inositol phosphates. PAF also increased expression of the cell cycle-active genes c-fos and EGR2 in a dose-dependent fashion. The stimulated increases in [Ca2+]i and phosphatidylinositol bisphosphate hydrolysis and the increases in gene expression were all inhibited by the specific PAF receptor antagonist Web 2086. The LA350 cell line (which expresses surface IgM) was also shown to increase [Ca2+]i after addition of anti-IgM antibodies. Sequential addition of PAF or anti-IgM antibody in either order failed to reveal any evidence for heterologous desensitization. Furthermore, the PAF receptor antagonist did not affect anti-IgM induced changes in [Ca2+]i. These data provide evidence for the presence of functional PAF receptors on B lymphoblastoid cells and indicate a potential role for PAF in the regulation of B cell activation.

Squamous cell carcinomas fail to respond to the prodifferentiating actions of 1,25(OH)2D: why?

1,25(OH)2D regulates a number of cellular events which contribute to its ability to stimulate differentiation of the keratinocyte. 1,25(OH)2D raises the intracellular calcium (Cai) level in part by increasing the expression of the calcium receptor (CaR). This sensitizes the cell to extracellular calcium, triggering the signaling pathway coupled to the CaR, which results in a rise in Cai. 1,25(OH)2D induces the family of phospholipases C (PLC). These enzymes mediate the hydrolysis of phosphatidyl inositol bisphosphate (PIP2) to form inositol tris phosphate (IP3) and diacylglycerol (DG), which stimulate calcium release from intracellular stores and activate protein kinases C (PKC), respectively. The CaR and other G protein coupled receptors signal through PLC-beta, whereas tyrosine kinase growth factor receptors such as the EGF receptor signal through PLC-gamma. Calcium and PKC regulate the expression of genes in part by controlling the levels and activity of AP-1 transcription factors. 1,25(OH)2D also directly induces structural genes such as involucrin, a substrate for transglutaminase, which crosslinks it to other substrates to form the cornified envelope. 1,25(OH)2D regulates gene expression by activating the vitamin D receptor (VDR), a transcription factor, which, in combination with the retinoid X receptor (RXR) or retinoid A receptor (RAR), binds to its vitamin D response elements (VDRE) in the promoters of genes whose expression it regulates. The VDR also binds to one of two coactivator complexes, Mediator/DRIP (VDR interacting proteins) or p160/SRC (steroid hormone receptor complex), complexes which link the VDR to the RNA polymerase complex. We have recently discovered that the binding of VDR to these complexes is sequential. Binding to Mediator/DRIP occurs in the undifferentiated keratinocyte, but as the cell differentiates, DRIP(205) (the key protein of the DRIP complex binding to the VDR) levels fall, and p160/SRC binding takes over. We hypothesize that this sequential replacement of Mediator/DRIP by p160/SRC is critical for differentiation. Squamous cell carcinomas (SCC) fail to respond to the prodifferentiating actions of 1,25(OH)2D. These cells have normal levels of VDR and normal binding of VDR to VDREs. However, they fail to down-regulate DRIP(205) such that the p160/SRC complex fails to bind to VDR. This lack of sequential binding of these coactivator complexes to the VDR, we believe, maintains the cell in a state of continued proliferation and blocks the ability of 1,25(OH)2D to induce the expression of genes required for the differentiation process.

Simulations of Inositol Phosphate Metabolism and Its Interaction with InsP 3-Mediated Calcium Release

Inositol phosphates function as second messengers for a variety of extracellular signals. Ins(1,4,5)P 3 generated by phospholipase C-mediated hydrolysis of phosphatidylinositol bisphosphate, triggers numerous cellular processes by regulating calcium release from internal stores. The Ins(1,4,5)P 3 signal is coupled to a complex metabolic cascade involving a series of phosphatases and kinases. These enzymes generate a range of inositol phosphate derivatives, many of which have signaling roles of their own. We have integrated published biochemical data to build a mass action model for InsP 3 metabolism. The model includes most inositol phosphates that are currently known to interact with each other. We have used this model to study the effects of a G-protein coupled receptor stimulus that activates phospholipase C on the inositol phosphates. We have also monitored how the metabolic cascade interacts with Ins(1,4,5)P 3-mediated calcium release. We find temporal dynamics of most inositol phosphates to be strongly influenced by the elaborate networking. We also show that Ins(1,3,4,5)P 4 plays a key role in InsP 3 dynamics and allows for paired pulse facilitation of calcium release. Calcium oscillations produce oscillatory responses in parts of the metabolic network and are in turn temporally modulated by the metabolism of InsP 3.

RGS-containing RhoGEFs: the missing link between transforming G proteins and Rho?

Many mitogens promote cell proliferation by acting on cell surface receptors that either possess an intrinsic protein-tyrosine kinase activity (Yarden et al., 1986) or interact with heterotrimeric GTPbinding proteins (G proteins). The latter are collectively known as G protein-coupled receptors (GPCRs) and comprise the largest group of cell surface molecules involved in signal transmission. With more than 1000 members, they are encoded by the biggest gene family in the human genome (Flower, 1999), and the importance and diversity of their physiological roles are directly supported by their link to a number of hereditary and acquired diseases (Spiegel, 1996; Stadel et al., 1997). Furthermore, these receptors and their ligands are the target of over 50% of all current therapeutic agents (Flower, 1999). GPCRs exhibit a common structural motif consisting of seven membrane-spanning regions (Dohlman et al., 1987), and can be activated by a diverse array of external stimuli, including growth factors, vasoactive polypeptides, chemoattractants, neurotransmitters, hormones, phospholipids, photons, odorants, and taste ligands. In general, agonist binding provokes rapid conformational changes in the transmembrane a helices, which result in the exposure of previously masked G protein binding sites in the intracellular loops (Altenbach et al., 1996; Bourne, 1997; Wess, 1997). This causes the exchange of GDP for GTP bound to the G protein a subunit, and GTP-bound a subunits or bg complexes then initiate intracellular signaling responses by acting on a variety of e€ector molecules. To date, sixteen distinct mammalian G protein a subunits have been identi®ed and divided into four families based on their sequence similarity: as, ai, aq, and a12 (Wilkie et al., 1992). The aq family of heterotrimeric G protein a subunits is coupled to phospholipase-C b to induce the hydrolysis of phosphatidylinositol bis phosphate, the consequent rise in the intracellular concentration of Ca, and the activation of protein kinase C, whereas the as and ai families are known to activate and inhibit adenylyl cyclase to increase and decrease the level of cyclic AMP, respectively. ai proteins also regulate ion channels, certain phospholipases and phosphodiesterases (Hamm, 1998). The targets for both members of the a12 family, a12 and a13, were unknown until recently. In addition, eleven-G protein g subunits and ®ve G protein b subunits have been cloned so far and they regulate certain ion channels, phospholipase-C, and phosphatidylinositol-3 kinases (Clapham and Neer, 1997). Through these G proteins subunits, GPCRs regulate a diverse array of physiological functions such as neurotransmission, chemoreception, photoreception, metabolism, growth, and di€erentiation. In addition to these physiological responses, there is also emerging evidence of the involvement of aberrant GPCR function in cellular transformation and oncogenesis (see Gutkind, 1998 for a recent review). In this regard, recent work suggests that the Rho family of small GTP-binding proteins plays a central role in the regulation of cell proliferation by GPCRs, and that the persistent activation of Rhomediated pathways by heterotrimeric G proteins can lead to cellular transformation. In this review, we will focus on recent e€orts addressing the molecular mechanisms by which GPCRs and G protein a subunits activate Rho, with emphasis on their contribution to the understanding on how this signal transducing system regulates normal and aberrant cell growth.

Monitoring Agonist-induced Phospholipase C Activation in Live Cells by Fluorescence Resonance Energy Transfer

Agonist-induced intracellular Ca(2+) signals following phospholipase C (PLC) activation display a variety of patterns, including transient, sustained, and oscillatory behavior. These Ca(2+) changes have been well characterized, but detailed kinetic analyses of PLC activation in single living cells is lacking, due to the absence of suitable indicators for use in vivo. Recently, green fluorescent protein-tagged pleckstrin homology domains have been employed to monitor PLC activation in single cells, based on (confocal) imaging of their fluorescence translocation from the membrane to the cytosol that occurs upon hydrolysis of phosphatidylinositol bisphosphate. Here we describe fluorescence resonance energy transfer between pleckstrin homology domains of PLCdelta1 tagged with cyan and yellow fluorescent proteins as a sensitive readout of phosphatidylinositol bisphosphate metabolism for use both in cell populations and in single cells. Fluorescence resonance energy transfer requires significantly less excitation intensity, enabling prolonged and fast data acquisition without the cell damage that limits confocal experiments. It also allows experiments on motile or extremely flat cells, and can be scaled to record from cell populations as well as single neurites. Characterization of responses to various agonists by this method reveals that stimuli that elicit very similar Ca(2+) mobilization responses can exhibit widely different kinetics of PLC activation, and that the latter appears to follow receptor activation more faithfully than the cytosolic Ca(2+) transient.

Protein Kinase C Activators Inhibit the Visual Cascade inLimulusVentral Photoreceptors at an Early Stage

The phosphoinositide cascade mediates visual transduction in invertebrate photoreceptors. Phospholipase C (PLC) catalyzes the hydrolysis of phosphatidylinositol bisphosphate, producing inositol trisphosphate (InsP(3)) and diacylglycerol (DAG). Protein kinase C (PKC) is a major target of DAG in many cell types. We have used PKC activators to investigate the function of the kinase in the phototransduction cascade in Limulus polyphemus ventral photoreceptors. Extracellular application of (-)-indolactam V (0. 03-30 microM) or phorbol-12,13-dibutyrate (10 microM) reversibly reduced the sensitivity of the electrical response of the photoreceptors to light by up to 1000-fold. The inert stereoisomer (+)-indolactam V and 4alpha-phorbol had no effect. The effect of (-)-indolactam V was antagonized by the PKC inhibitors bisindolylmaleimide I and Gö 6976. Coapplication of bisindolylmaleimide V, used as a negative control compound for PKC inhibition, did not reduce the effectiveness of (-)-indolactam V. These findings are consistent with (-)-indolactam V activating PKC and desensitizing the light response. Furthermore, our pharmacological results indicate that PKC activation does not appear to play a role in light adaptation. We localized the position of the target of PKC in the visual cascade. We chemically excited the cascade at various stages to determine the kinase's target. PKC activation by (-)-indolactam V decreased the light-induced elevation of intracellular calcium but had no effect on the photoreceptor's excitatory response to intracellular injection of InsP(3). However, the PKC activator greatly reduced the excitation caused by GTP-gamma-S injection. We propose that PKC inhibits the visual transduction cascade at the G-protein and/or PLC stage.

Protein kinases in mammalian sperm capacitation and the acrosome reaction.

Binding to the zona pellucida of an egg stimulates the spermatozoon to undergo the acrosome reaction, a process that enables it to penetrate the egg. Before this binding, the spermatozoon undergoes a series of biochemical transformations in the female reproductive tract, collectively called capacitation. Only capacitated spermatozoa can bind to the zona pellucida and undergo the acrosome reaction. Protein kinases may be involved in the regulation of intracellular Ca2+ during capacitation and the acrosome reaction. The first event in capacitation is the increase in intracellular calcium, bicarbonate and hydrogen peroxide, which collectively activate adenylyl cyclase to produce cyclic AMP, which activates protein kinase A to phosphorylate certain proteins. During capacitation, there is an increase in membrane-bound phospholipase C, and this binding is highly stimulated by the addition of epidermal growth factor to the cells. The capacitated spermatozoon binds to the zona pellucida of the egg via specific receptors and it is suggested that the zona pellucida binds to at least two different receptors in the sperm head plasma membrane. One is a Gi-coupled receptor that can activate phospholipase Cbeta1 and may regulate adenylyl cyclase to further increase cyclic AMP concentrations. The cyclic AMP activates protein kinase A to open a calcium channel in the outer acrosomal membrane, resulting in a relatively small increase in cytosolic calcium. This increase in Ca2+ leads to activation of phospholipase Cgamma, which is coupled to the second tyrosine kinase receptor. The products of phosphatidyl-inositol bisphosphate hydrolysis by phospholipase C, diacylglycerol and inositol-trisphosphate, induce the activation of protein kinase C and a calcium channel in the outer acrosomal membrane, respectively. Protein kinase C opens a calcium channel in the plasma membrane and, together with the inositol-trisphosphate-activated calcium channel, leads to a second and higher increase in cytosolic calcium. In addition, the depletion of calcium in the acrosome activates a capacitative calcium entry mechanism in the plasma membrane, leading to a rapid increase in cytosolic calcium (300-500 nmol l(-1)). This increase in intracellular calcium concentration (and pH) leads to membrane fusion and the acrosome reaction.

Measurement of inositol (poly)phosphate formation using [3H]inositol labeling protocols in permeabilized cells.

AbstractHormones, neurotransmitters, chemoattractants, and growth factors all elicit intracellular responses, on binding to cell surface receptors, by activating inositol phospholipid-specific phospholipase C (PLC). Activated PLC catalyzes the hydrolysis of phosphatidylinositol bisphosphate (PIP2), a minor membrane phospholipid, to form two second messengers, diacylglycerol (DAG) and inositol (1,4,5)trisphosphate [Ins(1,4,5,)P3]. DAG is a direct activator of protein kinase C isozymes, Ins(1,4,5)P3 mobilizes intracellular Ca2+. G protein-coupled receptors couple to the PLC-β family via G proteins, and tyrosine kinase receptors activate PLCγ isozymes (1). Regardless of the PLC isozyme activated, the product is invariantly Ins(1,4,5)P3.KeywordsHL60 CellPermeabilized CellInositol PhosphateInositol TrisphosphateAssay TubeThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Membrane Current Induced by Protein Kinase C Activators in Rhabdomeric Photoreceptors: Implications for Visual Excitation

Visual excitation in rhabdomeric photoreceptors is thought to be mediated by activation of a light-regulated phospholipase C (PLC) and the consequent hydrolysis of phosphatidylinositol bisphosphate. Whereas much attention has been devoted to inositol trisphosphate (IP3) production and intracellular Ca2+ release, little is known about the possible role of the DAG branch in the generation of the light response. We have tested the effect of chemically distinct surrogates of DAG on isolated Lima photoreceptors. Application of the phorbol ester PMA (0.5-10 microM) or the alkaloid (-)-indolactam (20-100 microM) from a holding potential of -50 mV elicited an inward current, several hundred picoamperes in amplitude, accompanied by a pronounced increase in membrane conductance. The stereoisomers 4alpha-PMA and (+)-indolactam were both inactive, arguing for the specificity of the effects. Elevation of cytosolic Ca2+ by intracellular dialysis accelerated this current, whereas chelerythrine antagonized it, suggesting the involvement of PKC. The reversal potential of the membrane current induced by PKC activators was approximately +10 mV; replacement of extracellular Na with impermeant N-methyl-D-glucamine decreased its amplitude and shifted the reversal potential in the negative direction. Stimulation by PMA and (-)-indolactam was accompanied by a pronounced depression of light responsiveness; conversely, steady illumination reduced the size of the current elicited by these PKC activators. Taken together, these results support the notion that the DAG branch of the PLC cascade, in addition to its suggested participation in visual adaptation, may play a role in the activation of the photoresponse or a component thereof, probably in synergy with IP3-mediated Ca2+ release.

Age-dependent decreases of NK cell phosphoinositide turnover during spontaneous but not Fc-mediated cytolytic activity.

The progressive increase in the number of peripheral NK cells found in the elderly does not correlate with a corresponding increase in lytic activity. On the contrary, a decreased function of circulating NK cells purified from old subjects was observed on a per cell basis. Most of the studies on NK cells have focused on late events such as lytic activity. In view of this, little is currently known about the modification of the early signalling pathways of NK cells in elderly people. This study investigated whether the modification of NK lytic activity could be related to differences in the metabolic pattern of activation of these cells in the elderly. NK cells were negatively purified by immunomagnetic depletion from the peripheral blood of selected old and young healthy subjects. Hydrolysis of inositol phospholipids was measured following incubation with K562 target cells and/or CD16 mAb for different times. Our data show that there is a pronounced age-related decrease in the ability to generate total inositol monophosphates and, particularly, inositol trisphosphates by NK cells following K562 stimulation (spontaneous cytolytic activity) together with an attenuated and delayed hydrolysis of phosphatidylinositol bisphosphate, while phosphoinositide turnover is preserved following Fc triggering (antibody-dependent cell-mediated cytotoxicity). These results confirm that, also in old subjects, different biochemical pathways of activation are involved in NK cells when target or antibody-mediated triggering occurs and may aid the development of experimental and therapeutic strategies to counteract declines in cell mediated immune functions associated to advancing age.

Leukotriene B4 Activates the NADPH Oxidase in Eosinophils by a Pertussis Toxin-Sensitive Mechanism That Is Largely Independent of Arachidonic Acid Mobilization

Experiments were designed to investigate whether leukotriene (LTB4) receptors can couple directly to phospholipase A2 (PLA2) in guinea pig eosinophils and the role of endogenous arachidonic acid (AA) in LTB4-induced activation of the NADPH oxidase. LTB4 (EC50 approximately 16 nM) and AA (EC50 approximately 6 microM) generated hydrogen peroxide (H2O2) in a concentration-dependent manner and at an equivalent maximum rate (5-6 nmol/min/10(6) cells). LTB4 stimulated PLA2 over a similar concentration range that activated the NADPH oxidase, although kinetic studies revealed that the release of [3H]AA (t1/2 approximately 2 s) preceded H2O2 generation (t1/2 > 30 s). Pretreatment of eosinophils with pertussis toxin abolished the increase in inositol(1,4,5)trisphosphate mass, [Ca2+]c, [3H]AA release, and H2O2 generation evoked by LTB4. Qualitatively identical results were obtained in eosinophils in which phospholipase C (PLC) was desensitized by 4beta-phorbol 12,13-dibutyrate with the exception that [3H]AA release was largely unaffected. Additional studies performed with the protein kinase C inhibitor, Ro 31-8220, and under conditions in which Ca2+ mobilization was abolished, provided further evidence that LTB4 released [3H]AA independently of signal molecules derived from the hydrolysis of phosphatidylinositol(4,5)bisphosphate by PLC. Pretreatment of eosinophils with the PLA2 inhibitor, mepacrine, abolished LTB4-induced [3H]AA release at a concentration that inhibited H2O2 by only 36%. Collectively, the results of this study indicate that agonism of LTB4 receptors on guinea pig eosinophils mobilizes AA by a mechanism that does not involve the activation of PLC. In addition, although LTB4 effectively stimulated PLA2, a central role for AA in the activation of the NADPH oxidase was excluded.