Inositol Trisphosphate Levels Research Articles

The effect of taurochenodeoxycholic acid on the glucocorticoid receptormediated PLC-IP3 -calcium pathway in synoviocytes derived from a rat adjuvant arthritis model

Taurochenodeoxycholic acid is one of the main active components of the bile acid pool and has important anti-inflammatory and immunomodulatory properties. This study aimed to explore the therapeutic effects and mechanism of action of taurochenodeoxycholic acid in inflammatory arthritis using a rat adjuvant arthritis model. Fibroblast-like synoviocytes were derived from rats with adjuvant arthritis and cultured using the adherent tissue explant method. Western blotting, enzyme-linked immunosorbent assay, and fluorescent probe-based methods were used to detect the effects of taurochenodeoxycholic acid on glucocorticoid receptor expression, phospholipase C protein levels, and inositol trisphosphate and intracellular free Ca2+ concentrations in primary rat fibroblast-like synoviocytes. Taurochenodeoxycholic acid significantly induced the expression of glucocorticoid receptor (P<0.05) and phospholipase C (P<0.05) and enhanced phospholipase C phosphorylation. Moreover, taurochenodeoxycholic acid significantly increased the levels of inositol trisphosphate (P<0.05) and intracellular free Ca2+ concentrations. Our results suggest that taurochenodeoxycholic acid activates the phospholipase C-inositol trisphosphate-Ca2+ signaling pathway by increasing glucocorticoid receptor expression. These findings provide a theoretical foundation for future studies on the molecular mechanisms underlying taurochenodeoxycholic acid-based treatment of adjuvant arthritis. In conclusion, taurochenodeoxycholic acid exerted anti-inflammatory and immunomodulatory effects by enhancing glucocorticoid receptor expression via non-genomic signalling.

Activation of canine neutrophils by platelet-activating factor

Neutrophils are essential for innate immunity as the first line of defence. Neutrophils act as phagocytic white blood cells to kill bacteria and other microorganisms. A strong respiratory burst of neutrophils, dependent on reactive oxygen species, is produced during phagocytosis. Platelet-activating factor (PAF) is a signalling molecule with several prominent roles in tissue injury, inflammation, and platelet aggregation. However, the detailed mechanisms and intracellular signalling pathways involved in PAF-mediated neutrophil activation remain unclear. Here, we investigated the effect of PAF on changes in calcium concentration ([Ca2+]i) and oxygen radical (O2−) generation in activating canine neutrophils. We further evaluated these effects of PAF with inhibition of G protein-coupled receptors using the specific inhibitor suramin. Blood samples were collected from a total of five dogs and neutrophils were isolated. PAF stimulation of canine neutrophils caused an increase in [Ca2+]i as well as the generation of O2−, and the PAF receptor was sensitive to suramin. The results suggested that PAF stimulation of canine neutrophils may cause Ca2+ influx from the endoplasmic reticulum into the cytoplasm (as the first wave) and then trigger store-operated Ca2+ entry (as the second wave), which is an important intracellular signal transduction pathway for neutrophil activation. Furthermore, O2− generation by PAF stimulation may depend on the intracellular signalling pathway, with increasing inositol trisphosphate levels and [Ca2+]i via G protein-coupled receptors. The finding that PAF-activating platelet aggregation is involved in canine neutrophil activation suggests a close relationship between haemostasis and neutrophil activation in dogs, offering new insight into the response to infection.

Severe Trauma Leads to a Fall in Intracellular Triphosphates and a Rise in cAMP in Platelets

Background: Severe trauma and hemorrhage leads to an acute coagulopathy as exemplified by a rise in prothrombin time, and a fall in clotting firmness and platelet aggregation. Because platelets contribute 70-80% of the clot strength, evaluating the intracellular mechanisms that regulate aggregation may lead to strategies to mitigate the development of coagulopathy. Platelet aggregation requires energy, and energy stores may be depleted after hemorrhagic shock and tissue trauma. Also, aggregation is initiated by elevations in inositol trisphosphate and calcium, and inhibited by elevations in cyclic adenosine and guanosine monophosphates. Platelet dysfunction after trauma may involve one or more of these intracellular intermediates.Objective: To measure intracellular levels of mono- and tri-phosphates in platelets from rats subjected to polytrauma and hemorrhagic shock.Methods: Polytrauma was induced in isoflurane anesthetized Sprague-Dawley rats (n=10 per group) by damage to the small intestines, right and medial lobes of the liver, the right leg skeletal muscle, and by fracturing the right femur. 40% hemorrhage was then performed immediately after trauma. Blood samples were taken before, immediately after trauma, and at 2 and 4hrs. Platelet rich plasma PRP was generated by centrifugation of whole blood at 200g for 10min, no brakes. Cyclic AMP and cGMP were extracted from 100μl of PRP after adding to 1ml of EtOH, 10mM ammonium formate, with 10μg/ml cGMP-Br as an internal control. Triphosphates were extracted from another 100μl PRP by addition of 50% EtOH, 500mM Formic Acid with 10μg/ml ATP-C10 as an internal control. Samples were centrifuged at 20K g for 10min, and supernatant dried. All Samples were brought up in 200ul of 0.1% formic acid for analysis by LC-MS/MS.Results: In platelets, the intracellular levels of Adenosine Triphosphate (ATP), Guanosine Triphosphate (GTP) and Inositol Trisphosphate (IP3) all fell significantly by 4hrs after polytrauma and hemorrhage (ATP: 5.8±0.9 vs. 0.9±0.7μg/ml, GTP: 1.23±0.22 vs 0.01±0.01 μg/ml and IP3: 625±310 vs 125±36 ng/ml). Cyclic AMP was significantly elevated over baseline (10.4±0.7 vs 15.1±1.63ng/ml), however cGMP was down (1.17±0.39 vs 0.10±0.12 ng/ml).Conclusion: The platelet dysfunction that accompanies polytrauma and hemorrhagic shock may be a result of falling energy stores (ATP, GTP), a loss of IP3 mediation of Ca dependent aggregation, and a rise in the inhibitory mediator cAMP. These results suggest that the addition of metabolic substrates to standard resuscitation fluids may help restore platelet function after trauma and hemorrhage. DisclosuresNo relevant conflicts of interest to declare.

Fear memory-induced alterations in the mRNA expression of G proteins in the mouse brain and the impact of immediate posttraining treatment with morphine

Disturbances in fear-evoked signal transduction in the hippocampus (HP), the nuclei of the amygdala (AMY), and the prefrontal cortex (PFC) underlie anxiety-related disorders. However, the molecular mechanisms underlying these effects remain elusive. Heterotrimeric G proteins (GPs) are divided into the following four families based on the intracellular activity of their alpha subunit (Gα): Gα(s) proteins stimulate cyclic AMP (cAMP) generation, Gα(i/o) proteins inhibit the cAMP pathway, Gα(q/11) proteins increase the intracellular Ca++ concentration and the inositol trisphosphate level, and Gα(12/13) proteins activate monomeric GP-Rho. In the present study, we assessed the effects of a fear memory procedure on the mRNA expression of the Gα subunits of all four GP families in the HP, AMY and PFC. C57BL/6 J mice were subjected to a fear conditioning (FC) procedure followed by a contextual or cued fear memory test (CTX-R and CS-R, respectively). Morphine (MOR, 1 mg/kg/ip) was injected immediately after FC to prevent the fear consolidation process. Real-time quantitative PCR was used to measure the mRNA expression levels of Gα subunits at 1 h after FC, 24 h after FC, and 1 h after the CTX-R or CS-R. In the HP, the mRNA levels of Gα(s), Gα(12) and Gα(11) were higher at 1 h after training. Gα(s) levels were slightly lower when consolidation was stabilized and after the CS-R. The mRNA levels of Gα(12) were increased at 1 h after FC, returned to control levels at 24 h after FC and increased again with the CTX-R. The increase in the Gα(11) level persisted at 24 h after FC and after CTX-R. In the AMY, no specific changes were induced by FC. In the PFC, CTX-R was accompanied by a decrease in Gα(i/o) mRNA levels; however, only Gα(i2) downregulation was prevented by MOR treatment. Hence, the FC-evoked changes in Gα mRNA expression were observed mainly in the HP and connected primarily to contextual learning. These results suggest that the activation of signaling pathways by Gα(s) and Gα(12) is required to begin the fear memory consolidation process in the HP, while signal transduction via Gα(11) is implicated in the maintenance of fear consolidation. In the PFC, the downregulation of Gα(i2) appears to be related to the contextual learning of fear.

Evidence for the existence of pyrimidinergic transmission in rat brain

The uridine nucleotides uridine-5′-triphosphate (UTP) and uridine-5′-diphosphate (UDP) have previously been identified in media from cultured cells. However, no study to date has demonstrated their presence in brain extracellular fluid (ECF) obtained in vivo. Using a novel method, we now show that UTP and UDP, as well as uridine, are detectable in dialysates of striatal ECF obtained from freely-moving rats. Intraperitoneal (i.p.) administration of uridine or exposure of striatum to depolarizing concentrations of potassium chloride increases extracellular uridine, UTP and UDP, while tetrodotoxin (TTX) decreases their ECF levels. Uridine administration also enhances cholinergic neurotransmission which is accompanied by enhanced brain levels of diacylglycerol (DAG) and inositol trisphosphate (IP3) and blocked by suramin, but not by PPADS (pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid) or MRS2578 suggesting a possible mediation of P2Y2 receptors activated by UTP. These observations suggest that uridine, UTP and UDP may function as pyrimidinergic neurotransmitters, and that enhancement of such neurotransmission underlies pharmacologic effects of exogenous uridine on the brain.

Do phosphoinositides regulate membrane water permeability of tobacco protoplasts by enhancing the aquaporin pathway?

Enhancing the membrane content of PtdInsP 2 , the already-recognized protein-regulating lipid, increased the osmotic water permeability of tobacco protoplasts, apparently by increasing the abundance of active aquaporins in their membranes. While phosphoinositides are implicated in cell volume changes and are known to regulate some ion channels, their modulation of aquaporins activity has not yet been reported for any organism. To examine this, we compared the osmotic water permeability (P f) of protoplasts isolated from tobacco (Nicotiana tabacum) cultured cells (NT1) with different (genetically lowered or elevated relative to controls) levels of inositol trisphosphate (InsP3) and phosphatidyl inositol [4,5] bisphosphate (PtdInsP2). To achieve this, the cells were transformed with, respectively, the human InsP3 5-phosphatase ('Ptase cells') or human phosphatidylinositol (4) phosphate 5-kinase ('PIPK cells'). The mean P f of the PIPK cells was several-fold higher relative to that of controls and Ptase cells. Three results favor aquaporins over the membrane matrix as underlying this excessive P f: (1) transient expression of the maize aquaporin ZmPIP2;4 in the PIPK cells increased P f by 12-30μms(-1), while in the controls only by 3-4μms(-1). (2) Cytosol acidification-known to inhibit aquaporins-lowered the P f in the PIPK cells down to control levels. (3) The transcript of at least one aquaporin was elevated in the PIPK cells. Together, the three results demonstrate the differences between the PIPK cells and their controls, and suggest a hitherto unobserved regulation of aquaporins by phosphoinositides, which could occur through direct interaction or indirect phosphoinositides-dependent cellular effects.

Modelling the transition from simple to complex Ca²⁺ oscillations in pancreatic acinar cells.

A mathematical model is proposed which systematically investigates complex calcium oscillations in pancreatic acinar cells. This model is based on calcium-induced calcium release via inositol trisphosphate receptors (IPR) and ryanodine receptors (RyR) and includes calcium modulation of inositol (1,4,5) trisphosphate (IP3) levels through feedback regulation of degradation and production. In our model, the apical and the basal regions are separated by a region containing mitochondria, which is capable of restricting Ca2+ responses to the apical region. We were able to reproduce the observed oscillatory patterns, from baseline spikes to sinusoidal oscillations. The model predicts that calcium-dependent production and degradation of IP3 is a key mechanism for complex calcium oscillations in pancreatic acinar cells. A partial bifurcation analysis is performed which explores the dynamic behaviour of the model in both apical and basal regions.

Regulation of Hepatic Glucose Production by Gq-Coupled Receptors: Potential New Targets for Treatment of Type 2 Diabetes

G protein coupled receptors (GPCRs) constitute the largest family of receptors in the human genome with more than 700 members divided into 5 subfamilies based on similarities of sequence (1). Most, but not all, GPCRs couple through a smaller family of heterotrimeric G proteins to an even smaller group of signaling systems. These include the well-characterized systems of adenylyl cyclase activated by Gs and inhibited by Gi to regulate intracellular cAMP levels, as well as phospholipase Cproteins activated by Gq/11 proteins to regulate intracellular inositol trisphosphate, diacylglycerol, and intracellular calcium levels. With few exceptions, cells express multiple GPCRs, and most GPCRs are expressed in more than one tissue, making for a complex array of responses to endogenous ligands that are not overly amenable to tissue-selective pharmacologic regulation. Pharmacologic approaches are therefore limited in their ability to inform about the specific functions of individual GPCRs or even individual signaling systems in specific cells in vivo. Genetic approaches using loss-of-function animal models have been extensively used to identify functions of individual GPCRs in both nonselective and tissue selective models (2–4). Nevertheless it would be helpful to more specifically assess the effect of activating a particular G protein-regulated signaling system rather than knocking it out. One approach to this question has been the use of so called “designer” GPCRs that do not respond to endogenous ligands but can be selectively stimulated with synthetic ligands. This approach was first introduced more than 20 years ago when a modified 2-adrenergic receptor that had significant reduction in catecholamine stimulation but response, albeit at low affinity, to foreign ligands was reported (5). Later introduction of modified receptors such as Ro1, a modified Gi-coupled -opioid receptor, activated by synthetic ligands, have been useful tools (6– 8). The utility of these systems is limited by the selectivity of the synthetic ligand for the modified receptor; ideally there should be no binding to native receptors and no background biological activity. An improved synthetic ligand, clozapine-N-oxide (CNO) that has minimal biological activity but reasonable potency on modified muscarinic acetylcholine receptors was developed in 2007 (9). The article by Li et al. (10) in this issue of Endocrinology reports the generation of a transgenic mouse line (Hep-Rq) selectively expressing a modified Gq-coupled muscarinic M3 receptor, Rq (11) in liver. Importantly, the mice express levels of Rq comparable to other endogenous GPCRs. They used the mice to determine what role Gqcoupled GPCRs might play in regulation of blood glucose. Between meals, glucagon regulation of hepatic glucose production (HGP) maintains blood glucose levels. Glucagon acts through its Gs-coupled receptor in liver to stimulate gluconeogenesis and glycogenolysis. The effect of hepatic Gq-coupled receptors in regulation of blood glucose is less understood. Fasting hyperglycemia in type 2 diabetes is a significant clinical issue and is one of the targets of biguanides, such as metformin, a mainstay of antidiabetic therapy (12). Thus, further understanding of the role of hepatic Gq-coupled receptors could reveal new targets for therapy. Li et al. show that the synthetic ligand CNO increases blood glucose in fasted Hep-Rq mice in a dose-dependent manner with no effect in wild-type mice. Serum insulin response after glucose injection and blood glucose re-

The effect of CD137–CD137 ligand interaction on phospholipase C signaling pathway in human endothelial cells

We previously reported the emerging role of CD137–CD137L interaction in inflammation and atherosclerosis. The mechanism of CD137–CD137L interaction may be related to a variety of signaling pathways. The most important signaling pathway involves the activation of phospholipase C(PLC) which induces the diacylglycerol–protein kinase C(DAG–PKC) and the inositol trisphosphate-intracellular free calcium (IP3-[Ca2+]i) pathway. In the current study, we investigated whether CD137–CD137L interaction can stimulate the PLC signaling pathway in human umbilical vein endothelial cells (HUVEC). The diacylglycerol (DAG) and inositol trisphosphate (IP3) levels in HUVEC were measured by radioenzymatic assay. The activity of protein kinase (PKC) was detected by its ability to transfer phosphate from [γ-32P]ATP to lysine-rich histone. The [Ca2+]i concentrations were measured by flow cytometric analysis. The DAG level and PKC activity were increased in a concentration-dependent, biphasic manner in HUVEC induced by anti-CD137. PKC activity was mainly in the cytosol at rest, and then translocated to the membrane when stimulated by anti-CD137. Similarly, rapid IP3 formation induced by anti-CD137 coincided with the peak of the DAG level. Moreover, anti-CD137 induced peak [Ca2+]i responses including the rapid transient phase and the sustained phase. However, anti-CD137L suppressed the activation of the DAG–PKC and IP3-[Ca2+]i signaling pathway, which was stimulated by anti-CD137 in HUVEC. In conclusion, the data suggested that CD137–CD137L interaction induces robust activation of the PLC signaling pathway in HUVEC.

Calcium Sensing Receptor Expression in Ovine Amniotic Fluid Mesenchymal Stem Cells and the Potential Role of R-568 during Osteogenic Differentiation

Amniotic fluid-derived stem (AFS) cells have been identified as a promising source for cell therapy applications in bone traumatic and degenerative damage. Calcium Sensing Receptor (CaSR), a G protein-coupled receptor able to bind calcium ions, plays a physiological role in regulating bone metabolism. It is expressed in different kinds of cells, as well as in some stem cells. The bone CaSR could potentially be targeted by allosteric modulators, in particular by agonists such as calcimimetic R-568, which may potentially be helpful for the treatment of bone disease. The aim of our study was first to investigate the presence of CaSR in ovine Amniotic Fluid Mesenchymal Stem Cells (oAFMSCs) and then the potential role of calcimimetics in in vitro osteogenesis. oAFMSCs were isolated, characterized and analyzed to examine the possible presence of CaSR by western blotting and flow cytometry analysis. Once we had demonstrated CaSR expression, we worked out that 1 µM R-568 was the optimal and effective concentration by cell viability test (MTT), cell number, Alkaline Phosphatase (ALP) and Alizarin Red S (ARS) assays. Interestingly, we observed that basal diffuse CaSR expression in oAFMSCs increased at the membrane when cells were treated with R-568 (1 µM), potentially resulting in activation of the receptor. This was associated with significantly increased cell mineralization (ALP and ARS staining) and augmented intracellular calcium and Inositol trisphosphate (IP3) levels, thus demonstrating a potential role for calcimimetics during osteogenic differentiation. Calhex-231, a CaSR allosteric inhibitor, totally reversed R-568 induced mineralization. Taken together, our results demonstrate for the first time that CaSR is expressed in oAFMSCs and that calcimimetic R-568, possibly through CaSR activation, can significantly improve the osteogenic process. Hence, our study may provide useful information on the mechanisms regulating osteogenesis in oAFMSCs, perhaps prompting the use of calcimimetics in bone regenerative medicine.

FluoQ: A Tool for Rapid Analysis of Multiparameter Fluorescence Imaging Data Applied to Oscillatory Events

The number of fluorescent sensors and their use in living cells has significantly increased in the past years. Yet, the analysis of data from single cells or cell populations usually remains a very time-consuming enterprise. Here, we introduce FluoQ, a new macro for the image analysis software ImageJ, which enables fast analysis of multiparameter time-lapse fluorescence microscopy data with minimal manual input. FluoQ provides statistical analysis of all measured parameters and delivers the results in multiple graphic and numeric displays. We demonstrate the power of FluoQ by applying the macro to data analysis in the development and optimization of novel FRET reporters for monitoring the performance of calcium/calmodulin-binding inositol trisphosphate kinases A and B (ITPKA and ITPKB) in HeLa cells. We find that conformational changes in the ITPKA-based sensor follow receptor-mediated calcium oscillations. This indicates that ITPKA contributes to the regulation of intracellular calcium transients by limiting inositol trisphosphate levels.

Characterization of an inositol 1,3,4-trisphosphate 5/6-kinase gene that is essential for drought and salt stress responses in rice

Drought and salt stresses are major limiting factors for crop production. To identify critical genes for stress resistance in rice (Oryza sativa L.), we screened T-DNA mutants and identified a drought- and salt-hypersensitive mutant dsm3. The mutant phenotype was caused by a T-DNA insertion in a gene encoding a putative inositol 1,3,4-trisphosphate 5/6-kinase previously named OsITPK2 with unknown function. Under drought stress conditions, the mutant had significantly less accumulation of osmolytes such as proline and soluble sugar and showed significantly reduced root volume, spikelet fertility, biomass, and grain yield; however, malondialdehyde level was increased in the mutant. Interestingly, overexpression of DSM3 (OsITPK2) in rice resulted in drought- and salt-hypersensitive phenotypes and physiological changes similar to those in the mutant. Inositol trisphosphate (IP3) level was decreased in the overexpressors under normal condition and drought stress. A few genes related to osmotic adjustment and reactive oxygen species scavenging were down-regulated in the mutant and overexpression lines. The expression level of DSM3 promoter-driven β-glucuronidase (GUS) reporter gene in rice was induced by drought, salt and abscisic acid. Protoplast transient expression assay indicated that DSM3 is an endoplasmic reticulum protein. Sequence analysis revealed six putative ITPKs in rice. Transcript level analysis of OsITPK genes revealed that they had different tempo-spatial expression patterns, and the responses of DSM3 to abiotic stresses, including drought, salinity, cold, and high temperature, were distinct from the other five members in rice. These results together suggest that DSM3/OsITPK2 is an important member of the OsITPK family for stress responses, and an optimal expression level is essential for drought and salt tolerance in rice.

Pharmacological Modulation of Diacylglycerol-Sensitive TRPC3/6/7 Channels

Members of the classic type of transient receptor potential channels (TRPC) represent important molecules involved in hormonal signal transduction. TRPC3/6/7 channels are of particular interest as they are components of phospholipase C driven signalling pathways. Upon receptor-activation, G-protein-mediated stimulation of phospholipase C results in breakdown of phosphatidylinositides leading to increased intracellular diacylglycerol and inositol-trisphosphate levels. Diacylglycerol activates protein kinase C, but more interestingly diacylglycerol directly activates TRPC2/3/6/7 channels. Molecular cloning, expression and characterization of TRP channels enabled reassignment of traditional inhibitors of receptor-dependent calcium entry such as SKF-96365 and 2-APB as blockers of TRPC3/6/7 and several members of non-classic TRP channels. Furthermore, several enzyme inhibitors have also been identified as TRP channel blockers, such as ACA, a phospholipase A2 inhibitor, and W-7, a calmodulin antagonist. Finally, the naturally occurring secondary plant compound hyperforin has been identified as TRPC6-selective drug, providing an exciting proof of concept that it is possible to generate TRPC-selective channel modulators. The description of Pyr3 as the first TRPC3-selective inhibitor shows that not only nature but also man is able to generate TRP-selective modulators. The review sheds lights on the current knowledge and historical development of pharmacological modulators of TRPC3/6/7. Our analysis indicates that Pyr3 and hyperforin provide promising core structures for the development of new, selective and more potent modulators of TRPC3/6/7 activity.

Phosphatidylinositol (4,5)bisphosphate inhibits K+-efflux channel activity in NT1 tobacco cultured cells.

In the animal world, the regulation of ion channels by phosphoinositides (PIs) has been investigated extensively, demonstrating a wide range of channels controlled by phosphatidylinositol (4,5)bisphosphate (PtdInsP2). To understand PI regulation of plant ion channels, we examined the in planta effect of PtdInsP2 on the K+-efflux channel of tobacco (Nicotiana tabacum), NtORK (outward-rectifying K channel). We applied a patch clamp in the whole-cell configuration (with fixed "cytosolic" Ca2+ concentration and pH) to protoplasts isolated from cultured tobacco cells with genetically manipulated plasma membrane levels of PtdInsP2 and cellular inositol (1,4,5)trisphosphate: "Low PIs" had depressed levels of these PIs, and "High PIs" had elevated levels relative to controls. In all of these cells, K channel activity, reflected in the net, steady-state outward K+ currents (IK), was inversely related to the plasma membrane PtdInsP2 level. Consistent with this, short-term manipulations decreasing PtdInsP2 levels in the High PIs, such as pretreatment with the phytohormone abscisic acid (25 microM) or neutralizing the bath solution from pH 5.6 to pH 7, increased IK (i.e. NtORK activity). Moreover, increasing PtdInsP2 levels in controls or in abscisic acid-treated high-PI cells, using the specific PI-phospholipase C inhibitor U73122 (2.5-4 microM), decreased NtORK activity. In all cases, IK decreases stemmed largely from decreased maximum attainable NtORK channel conductance and partly from shifted voltage dependence of channel gating to more positive potentials, making it more difficult to activate the channels. These results are consistent with NtORK inhibition by the negatively charged PtdInsP2 in the internal plasma membrane leaflet. Such effects are likely to underlie PI signaling in intact plant cells.

Effects of Different Periods of Lithium Pretreatment and Aminoglycoside Antibiotics on Apomorphine-Induced Yawning in Rats

Interactive effects of intracerebroventricular administration of the aminoglycoside antibiotics, amikacin and gentamicin, and different duration of lithium pretreatment on apomorphine-induced yawning were investigated in male rats. The study was designed to investigate whether the hypothesis that the aminoglycoside antibiotics, amikacin and gentamicin, via their effects on phosphoinositide pathways and calcium channel might influence dopaminergic mechanisms as manifested in the yawning effect. Lithium is known to interact with phosphoinositide metabolism and was also tested after chronic studies on the apomorphine yawning model. Subcutaneous administration of apomorphine (0.1, 0.2 and 0.4 mg/kg) to rats induced yawning in a biphasic manner. However the maximum response was obtained by 0.2 mg/kg of the drug. Intracerebroventricular administration of aminoglycoside antibiotics amikacin (25 μg/rat) increased and gentamicin (10 and 20 μg/rat) decreased apomorphine-induced yawning. Pretreatment of animals with lithium (600 mg/l) in drinking water for 7, 14 and 21 days reduced yawning induced by apomorphine. Administration of lithium for 28 days did not induce any significant effect on yawning response. Amikacin and gentamicin function via the same mechanism on phosphoinositide cascade. Since amikacin and gentamicin did not affect the yawning response similarly, they apparently do not involve inositol trisphosphate level in the alterations of dopaminergic-induced yawning. Probably, the effect of lithium pretreatment on the number of yawns is also time-dependent and some tolerance to the inhibitory effect of lithium might occur after 28 days' treatment.

Estradiol regulation of progesterone synthesis in the brain

Steroidogenesis is now recognized as a global phenomenon in the brain, but how it is regulated and its relationship to circulating steroids of peripheral origin have remained more elusive issues. Neurosteroids, steroids synthesized de novo in nervous tissue, have a large range of actions in the brain, but it is only recently that the role of neuroprogesterone in the regulation of arguably the quintessential steroid-dependent neural activity, regulation of the reproduction has been appreciated. Circuits involved in controlling the LH surge and sexual behaviors were thought to be influenced by estradiol and progesterone synthesized in the ovary and perhaps the adrenal. It is now apparent that estradiol of ovarian origin regulates the synthesis of neuroprogesterone, and it is the locally produced neuroprogesterone that is involved in the initiation of the LH surge and subsequent ovulation. In this model, estradiol induces the transcription of progesterone receptors while stimulating synthesis of neuroprogesterone. Although the complete signaling cascade has not been elucidated, many of the features have been characterized. The synthesis of neuroprogesterone occurs primarily in astrocytes and requires the interaction of membrane-associated estrogen receptor-alpha with metabotropic glutamate receptor-1a. This G protein-coupled receptor activates a phospholipase C that in turn increases inositol trisphosphate (IP3) levels mediating the release of intracellular stores of Ca2+ via an IP3 receptor gated Ca2+ channel. The large increase in free cytoplasmic Ca2+ ([Ca2+]i) stimulates the synthesis of progesterone, which can then diffuse out of the astrocyte and activate estradiol-induced progesterone receptors in local neurons to trigger the neural cascade to produce the LH surge. Thus, it is a cooperative action of astrocytes and neurons that is needed for estrogen positive feedback and stimulation of the LH surge.

Cyclothiazide selectively inhibits mGluR1 receptors interacting with a common allosteric site for non-competitive antagonists

Metabotropic glutamate receptors mGluR1 and mGluR5 stimulate phospholipase C, leading to an increased inositol trisphosphate level and to Ca 2+ release from intracellular stores. Cyclothiazide (CTZ), known as a blocker of AMPA receptor desensitization, produced a non-competitive inhibition of [Ca 2+] i increases induced by mGluR agonists in HEK 293 cells transfected with rat mGluR1a but had no effect on the [Ca 2+] i signals in cells expressing rat mGluR5a. In cells expressing mGluR1, CTZ also inhibited phosphoinositide hydrolysis, as well as cAMP accumulation and arachidonic acid release induced by mGluR1 agonists, indicating a direct inhibition of the receptor and not of a particular signal transduction system. However, CTZ failed to antagonize cAMP inhibition stimulated by rat mGluR2, -3, -4, -6, -7 and -8 receptors confirming its selectivity for mGluR1. The use of chimeric receptors with substituted N-terminal domains showed that CTZ did not interact with the N-terminal mGluR1a domain. Instead, mutation analysis revealed that CTZ interacts with the Thr-815 and Ala-818 residues, located at the 7th transmembrane domain, similarly as the mGluR1-selective antagonist CPCCOEt. In primary cultures of cerebellar granule neurons, expressing native metabotropic and ionotropic glutamate receptors, the final outcome of CTZ effects depended on its combined ability to potentiate AMPA receptors and inhibit mGluR1 receptors.

Gastroesophageal Reflux Disease–Associated Esophagitis Induces Endogenous Cytokine Production Leading to Motor Abnormalities

Gastroesophageal reflux disease is a condition frequently associated with esophagitis and motor abnormalities. Recent evidence suggests that proinflammatory cytokines, such as interleukin (IL)-1beta and IL-6, may be implicated because they reduce esophageal muscle contractility, but these results derive from in vitro or animal models of esophagitis. This study used human esophageal cells and tissues to identify the cellular source of cytokines in human esophagitis investigate whether cytokines can be induced by gastric refluxate, and examine whether esophageal tissue- or cell-derived mediators affect muscle contractility. Endoscopic mucosal biopsy specimens were obtained from patients with and without esophagitis, organ-cultured, and undernatants were assessed for cytokine content. The cytokine profile of esophageal epithelial, fibroblast, and muscle cells was analyzed, and esophageal mucosa and cell products were tested in an esophageal circular muscle contraction assay. The mucosa of esophagitis patients produced significantly greater amounts of IL-1beta and IL-6 compared with those of control patients. Cultured esophageal epithelial cells produced IL-6, as did fibroblasts and muscle cells. Epithelial cells exposed to buffered, but not denatured, gastric juice produced IL-6. Undernatants of mucosal biopsy cultures from esophagitis patients reduced esophageal muscle contraction, as did supernatants from esophageal epithelial cell cultures. The human esophagus produces cytokines capable of reducing contractility of esophageal muscle cells. Exposure to gastric juice is sufficient to stimulate esophageal epithelial cells to produce IL-6, a cytokine able to alter esophageal contractility. These results indicate that classic cytokines are important mediators of the motor disturbances associated with human esophageal inflammation.

Rhabdomeric phototransduction initiated by the vertebrate photopigment melanopsin

Melanopsin is the photopigment that confers light sensitivity on intrinsically photosensitive retinal ganglion cells. Mammalian intrinsically photosensitive retinal ganglion cells are involved in the photic synchronization of circadian rhythms to the day-night cycle. Here, we report molecular components of melanopsin signaling using the cultured Xenopus dermal melanophore system. Photo-activated melanopsin is shown to initiate a phosphoinositide signaling pathway similar to that found in invertebrate photo-transduction. In melanophores, light increases the intracellular level of inositol trisphosphate and causes the dispersion of melanosomes. Inhibition of phospholipase C and protein kinase C and chelation of intracellular calcium block the effect of light on melanophores. At least four proteins, 43, 74, 90, and 134 kDa, are phosphorylated by protein kinase C upon light stimulation. This provides evidence of an invertebrate-like light-activated signaling cascade within vertebrate cells.

Evidence for a new human CYP1A1 regulation pathway involving PPAR-α and 2 PPRE sites

Cytochrome P450 1A1 catalyzes the degradation of endobiotics (estradiol, fatty acids, and so on) and the bioactivation of numerous environmental procarcinogens, such as arylamines and polycyclic aromatic hydrocarbons, that are found in food. Several peroxisome proliferators and arachidonic acid derivatives enhance cytochrome P450 1A1 activity, but the mechanisms involved remain unknown. The aim of this work was to study the role of peroxisome proliferator-activated receptors in cytochrome P450 1A1 gene induction. The role of peroxisome proliferator-activated receptor transcription factors in cytochrome P450 1A1 induction was assessed by means of enzymatic activities, quantitative real-time polymerase chain reaction, gene reporter assays, mutagenesis, and electrophoretic mobility shift assay. We show that peroxisome proliferator-activated receptor-alpha agonists (WY-14643, bezafibrate, clofibrate, and phthalate) induce human cytochrome P450 1A1 gene expression, whereas 2,4-thiazolidinedione, a specific peroxisome proliferator-activated receptor-gamma agonist, represses it. The induction of cytochrome P450 1A1 transcripts by WY-14643 was associated with a marked increase of ethoxyresorufin O -deethylase activity (10-fold at 200 mumol/L). Transfection of peroxisome proliferator-activated receptor-alpha complementary DNA enhanced cytochrome P450 1A1 messenger RNA induction by WY-14643, although WY-14643 failed to activate xenobiotic responsive element sequences. Two peroxisome proliferator response element sites were located at positions -931/-919 and -531/-519 of the cytochrome P450 1A1 promoter. Their inactivation by directed mutagenesis suppressed the inductive effect of WY-14643 on cytochrome P450 1A1 promoter activation. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay experiments showed that the 2 cytochrome P450 1A1 peroxisome proliferator response element sites bind the peroxisome proliferator-activated receptor-alpha/retinoid X receptor-alpha heterodimer. We describe here a new cytochrome P450 1A1 induction pathway involving peroxisome proliferator-activated receptor-alpha and 2 peroxisome proliferator response element sites, indicating that peroxisome proliferator-activated receptor-alpha ligands, which are common environmental compounds, may be involved in carcinogenesis.