P2Y2 receptor-Gq/11 signaling at lipid rafts is required for UTP-induced cell migration in NG 108-15 cells.

Lipid rafts, formed by sphingolipids and cholesterol within the membrane bilayer, are believed to have a critical role in signal transduction. P2Y(2) receptors are known to couple with G(q) family G proteins, causing the activation of phospholipase C (PLC) and an increase in intracellular Ca(2+) ([Ca(2+)](i)) levels. In the present study, we investigated the involvement of lipid rafts in P2Y(2) receptor-mediated signaling and cell migration in NG 108-15 cells.

Adenosine uptake-dependent C6 cell growth inhibition.

In C6 glioma cells, adenine nucleotides, especially AMP, and adenosine inhibited cell proliferation in time- and concentration-dependent manners. alpha,beta-methylene-ADP, an ecto-5'-nucleotidase inhibitor, suppressed the hydrolysis of AMP and reversed the inhibition of cell growth induced by AMP but not by adenosine. Adenosine deaminase eliminated both AMP- and adenosine-mediated growth inhibitions.

Cooperation of calcineurin and ERK for UTP-induced IL-6 production in HaCaT keratinocytes.

UTP causes IL-6 production in HaCaT keratinocytes, which is partially inhibited by PD98059, a mitogen-activated protein kinase kinase (MEK) inhibitor, suggesting that a pathway other than the extracellular signal-regulated kinase (ERK) pathway is involved in the production. In the present study, we examined the involvement of calcineurin in the UTP-induced interleukin (IL)-6 production in HaCaT keratinocytes. FK506 and cyclosporine A, calcineurin inhibitors, partially inhibited UTP-induced IL-6 mRNA expression and protein production.

Piperlongumine, a constituent of Piper longum L., inhibits rabbit platelet aggregation as a thromboxane A(2) receptor antagonist.

Piper longum L. has been used as a crude drug for the treatment of the disorder of peripherally poor blood circulation in Asia. In the present study, we examined the effect of piperlongumine, a constituent of P.

Low expression of cell-surface thromboxane A2 receptor beta-isoform through the negative regulation of its membrane traffic by proteasomes.

Human thromboxane A(2) receptor (TP) consists of two alternatively spliced isoforms, TP alpha and TP beta, which differ in their cytoplasmic tails. To examine the functional difference between TP alpha and TP beta, we searched proteins bound to C termini of TP isoforms by a yeast two-hybrid system, and found that proteasome subunit alpha 7 and proteasome activator PA28 gamma interacted potently with the C terminus of TP beta. The binding of TP beta with alpha 7 and PA28 gamma was confirmed by co-immunoprecipitation and pull-down assays.

[Role of lipid rafts in trimeric G protein-mediated signal transduction].

Lipid rafts and caveolae are microdomains in the cell membranes, which contain cholesterol, glycolipids, and sphingomyelin. While caveolae are relatively stable because caveolin, an integral protein, supports the structure, lipid rafts are considered to be unstable, being dynamically produced and degraded. Recent studies have reported that lipid rafts contain many signaling molecules, such as glycosylphosphatidylinositol-anchored proteins, acylated proteins, G-protein-coupled receptors (GPCRs), trimeric and small G-proteins and their effectors, suggesting that the lipid rafts have an important role in receptor-mediated signal transduction.

Distinct effects of z-335, a new thromboxane A2 receptor antagonist, on rabbit platelets and aortic smooth muscle.

The effect of a novel thromboxane A2 receptor (TP) antagonist, (+/-)-sodium[2-(4-chlorophenylsulfonylaminomethyl)- indan-5-yl]acetate monohydrate (Z-335), on the U46619-induced responses was compared between rabbit platelets and aorta. Z-335 inhibited platelet shape change induced by U46619 with higher efficacy than SQ29548, a common TP antagonist. The U46619-induced platelet aggregation was inhibited by Z-335 in a noncompetitive manner, while it was competitively inhibited by SQ29548.

Contribution of extracellular signal-regulated kinase to UTP-induced interleukin-6 biosynthesis in HaCaT keratinocytes.

UTP causes interleukin (IL)-6 production via mRNA expression through P2Y(2)/P2Y(4) receptors in human HaCaT keratinocytes. In the present study, we analyzed the mechanism of UTP-induced IL-6 production in these cells. UTP, an agonist of P2Y(2)/P2Y(4) receptors, induced phosphorylation of extracellular signal-regulated kinase (ERK) in a concentration- and time-dependent manner.

Thromboxane A2 receptor-mediated G12/13-dependent glial morphological change.

Glial cells express thromboxane A(2) receptor, but its physiological role remains unknown. The present study was performed to examine thromboxane A(2) receptor-mediated morphological change in 1321N1 human astrocytoma cells. Thromboxane A(2) receptor agonists U46619 and STA(2) caused a rapid morphological change to spindle shape from stellate form of the cells pretreated with dibutyryl cyclic AMP, but neither carbachol nor histamine caused the change, suggesting that G(q) pathway may not mainly contribute to the change.

ATP stimulates interleukin-6 production via P2Y receptors in human HaCaT keratinocytes.

We evaluated the role of ATP in functions of human HaCaT keratinocytes. ATP was released from HaCaT cells by changing the culture medium. Reverse transcription-polymerase chain reaction analysis revealed that HaCaT cells expressed multiple P2 purinergic receptor mRNAs.

Different pathways for activation of extracellular signal-regulated kinase through thromboxane A2 receptor isoforms.

Thromboxane A2 receptor (TP) consists of two alternatively spliced isoforms, TPalpha and TPbeta, which differ in their cytoplasmic tails. In the present study, we examined the difference in signal transduction of TPalpha and TPbeta, using stably expressing cells of TPalpha and TPbeta. The cells expressing TPalpha (TPalpha-SC2) and TPbeta (TPbeta-SC15) were selected based on the similar binding sites of [3H]-SQ29548, a TP antagonist.

Neurotrophic factor production in human astrocytoma cells by 2,5,6-tribromogramine via activation of epsilon isoform of protein kinase C.

It is known that astrocytes secrete several neurotrophic factors to promote the survival of neurons. For the treatment of neuronal disorders, low molecular weight compounds inducing neurotrophic factor synthesis are useful, because neurotrophic factors are polypeptides which cannot cross the blood brain barrier. When rat pheochromocytoma (PC-12) cells were cultivated in the medium of human astrocytoma cells (1321N1) treated with 2,5,6-tribromogramine, they differentiated to neuron-like cells possessing neurites, indicating that 2,5,6-tribromogramine released neurotrophic factors from 1321N1 cells.

Physiological significance of thromboxane A(2) receptor dimerization.

The thromboxane A(2) receptor (TP), one of the G protein-coupled receptors (GPCRs), consists of two splicing variants, TPalpha and TPbeta, which differ in their C-terminal regions. In the present study, we investigated whether TPalpha and TPbeta formed homo- or hetero-dimers and whether the dimerization changed the function of TP. The immunofluorescent analysis using human embryonic kidney (HEK) 293 cells expressing either FLAG-tagged TPalpha or TPbeta showed that TPalpha is mainly distributed on plasma membranes and TPbeta existed on plasma membranes and within the cells.

Mastoparan changes the cellular localization of Galphaq/11 and Gbeta through its binding to ganglioside in lipid rafts.

Although it is known that mastoparan, a wasp venom toxin, directly activates Gi/o, mastoparan-induced biological responses are not always explained by this mechanism. For instance, we have demonstrated previously that mastoparan suppressed phosphoinositide hydrolysis induced by carbachol in human astrocytoma cells (FEBS Lett 206:91-94, 1990). In the present study, we examined whether mastoparan affected phosphoinositide hydrolysis by interacting with lipid rafts in PC-12 cells.

A comparative study on contractile responses of rabbit and guinea pig vasa deferentia to electrical field stimulation.

Contractile responses of rabbit and guinea pig vasa deferentia to electrical field stimulation (EFS) are compared. A muscarinic receptor blocking agent, 1 microM atropine markedly reduced phasic and tonic contraction induced by EFS (20 Hz, 0.5 msec, 30 V, for 30 sec) in rabbit vas deferens, while it only slightly depressed those in guinea pig vas deferens.

ATP- and adenosine-mediated signaling in the central nervous system: adenosine receptor activation by ATP through rapid and localized generation of adenosine by ecto-nucleotidases.

Extracellular ATP is now recognized as a neurotransmitter or neuromodilator in the nervous system, producing diverse physiological effects by activating multiple P2 receptors. Although P2-receptor signaling is terminated by hydrolysis of ATP by the ecto-nucleotidase cascade, such a metabolic step leads to adenosine generation, thereby initiating adenosine (P1)-receptor activation. Because most cells and tissues co-express P1 and P2 receptors, ecto-nucleotidase on target tissues, especially enzymes catalyzing adenosine formation, are determinants of the cellular response to ATP.

[Lipid rafts and their analytical methods].

Microdomains in cell membranes consist of caveolae and lipid rafts, in which cholesterol, glycolipids, and sphingomyelin are concentrated. While caveolae are relatively stable because caveolin, an integral protein, supports the structure, lipid rafts are unstable, being dynamically produced and degraded. In lipid rafts, flotillin is assumed to be one of the specifically located proteins.

Theonezolide A, a novel marine macrolide, induces drastic shape change in rabbit platelets by reorganization of microtubules.

Theonezolide A, a marine macrolide, and thrombin caused a shape change followed by an aggregation in the rabbit platelets. Theonezolide A-induced platelet shape change, estimated by a decrease in light transmission, appeared to a greater extent than thrombin-induced one. Morphological studies using an electron microscope showed that theonezolide A changed platelet shape with various numbers of long pseudopods, loosing their discoid shape.

Baicalein inhibits Raf-1-mediated phosphorylation of MEK-1 in C6 rat glioma cells.

Baicalein is a flavonoid derived from the Scutellaria root. In investigations of the inhibitors of prostaglandin synthesis in C6 rat glioma cells, we found that baicalein had a potent inhibitory activity on prostaglandin synthesis induced by either histamine or A23187, a Ca(2+) ionophore. Baicalein inhibited histamine- or A23187-induced phosphorylation of p42/p44 extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK), which causes the phosphorylation of cytosolic phospholipase A(2) (PLA(2)).

Adenine nucleotide-induced activation of adenosine A(2B) receptors expressed in Xenopus laevis oocytes: involvement of a rapid and localized adenosine formation by ectonucleotidases.

We recently demonstrated that extracellular ATP effectively activates adenosine (Ade) A(2B) receptors indirectly through a localized rapid conversion to Ade by ectonucleotidases on the membrane surface of C6Bu-1 rat glioma cells. These responses were observed even in the presence of adenosine deaminase (ADA). Here, we demonstrate that such responses indeed occur in A(2B) receptor-expressing Xenopus laevis oocytes, which possess endogenous ectonucleotidase activity.