1321N1 Cells Research Articles

Functional role of JNK in neuritogenesis of PC12-N1 cells

JNKs, also known as SAPKs, are activated in response to a wide variety of factors including growth factors, cytokines, UV radiation, and heat shock. In the rat pheochromocytoma PC12 cells, the JNK signaling pathway mediates diverse functions such as differentiation and apoptosis. We have previously shown that activated JNK is required for later stages of neuritogeneis induced by NGF in a variant PC12 cell line (N1). Here, the functional role of JNK in N1 cells is further investigated. We show that NGF treatment, which induces extensive neurite branching and cell soma enlargement in the N1 cells, stimulates a biphasic activation of JNK. The first phase of activation is rapid and transient, beginning at 15 min after NGF exposure and lasting approximately 45 min. The second phase of activation is sustained, beginning at 9–12 h of NGF treatment and lasting for at least 24 h. Similar biphasic pattern of JNK activation is also observed in the parental PC12 cells. Using the specific JNK inhibitor SP600125, we show that the biphasic activation is necessary for neurite outgrowth and branching, and that inhibition of either phase suppresses neuritogenesis in the N1 cells. These results suggest that dynamic JNK activation may play a key role in neurite outgrowth during neuronal development.

The Synthetic Cannabinoid R(+)WIN 55,212-2 Inhibits the Interleukin-1 Signaling Pathway in Human Astrocytes in a Cannabinoid Receptor-independent Manner

R(+)WIN 55,212-2 is a synthetic cannabinoid that controls disease progression in models of multiple sclerosis. This is associated with its ability to reduce migration of leukocytes into the central nervous system. Because leukocyte migration is dependent on induction of adhesion molecules and chemokines by pro-inflammatory cytokines, we examined the effects of R(+)WIN 55,212-2 on their expression. Using 1321N1 astrocytoma and A-172 glioblastoma as cell models we show that R(+)WIN 55,212-2, but not its inactive chiral form S(-)WIN 55,212-2, strongly inhibits the interleukin-1 (IL-1) induction of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) and the chemokine IL-8. This inhibition is not mediated via the CB1 or CB2 cannabinoid receptors, because their selective antagonists and pertussis toxin failed to affect the inhibitory effects of R(+)WIN 55,212-2. Furthermore reverse transcription-PCR analysis did not detect the expression of either receptor in 1321N1 cells. R(+)WIN 55,212-2 was shown to inhibit adhesion molecule and chemokine expression at the level of transcription, because it strongly inhibited the IL-1 induction of ICAM-1, VCAM-1, and IL-8 mRNAs and blocked the IL-1 activation of their promoters. The NFkappaB pathway was then assessed as a lead target for R(+)WIN 55,212-2. NFkappaB was measured by expression of a transfected NFkappaB-regulated reporter gene. Using this assay, R(+)WIN 55,212-2 strongly inhibited IL-1 activation of NFkappaB. Furthermore R(+)WIN 55,212-2 inhibited the ability of overexpressed Myd88, Tak-1, and IKK-2 to induce the reporter gene suggesting that R(+)WIN 55,212-2 acts at or downstream of IKK-2 in the IL-1 pathway. However R(+)WIN 55,212-2 failed to inhibit IL-1-induced degradation of IkappaBalpha, excluding IKK-2 as a direct target. In addition electrophoretic mobility shift and chromatin immunoprecipitation assays showed that R(+)WIN 55,212-2 does not regulate the IL-1-induced nuclear translocation of NFkappaB or the ability of the latter to bind to promoters regulating expression of ICAM-1 and IL-8. These data suggest that R(+)WIN 55,212-2 blocks IL-1 signaling by inhibiting the transactivation potential of NFkappaB.

Endogenous opioid mechanisms partially mediate P2X3/P2X2/3‐related antinociception in rat models of inflammatory and chemogenic pain but not neuropathic pain

P2X3/P2X2/3 receptors have emerged as important components of nociception. However, there is limited information regarding the neurochemical systems that are affected by antagonism of the P2X3/P2X2/3 receptor and that ultimately contribute to the ensuing antinociception. In order to determine if the endogenous opioid system is involved in this antinociception, naloxone was administered just prior to the injection of a selective P2X3/P2X2/3 receptor antagonist, A-317491, in rat models of neuropathic, chemogenic, and inflammatory pain. Naloxone (1-10 mg kg(-1), i.p.), dose-dependently reduced the antinociceptive effects of A-317491 (1-300 micromol kg(-1), s.c.) in the CFA model of thermal hyperalgesia and the formalin model of chemogenic pain (2nd phase), but not in the L5-L6 spinal nerve ligation model of neuropathic allodynia. In comparison experiments, the same doses of naloxone blocked or attenuated the actions of morphine (2 or 8 mg kg(-1), s.c.) in each of these behavioral models. Injection of a peripheral opioid antagonist, naloxone methiodide (10 mg kg(-1), i.p.), did not affect A-317491-induced antinociception in the CFA and formalin assays, suggesting that the opioid component of this antinociception occurred within the CNS. Furthermore, this utilization of the central opioid system could be initiated by antagonism of spinal P2X3/P2X2/3 receptors since the antinociceptive actions of intrathecally delivered A-317491 (30 nmol) in the formalin model were reduced by both intrathecally (10-50 nmol) and systemically (10 mg kg(-1), i.p.) administered naloxone. This utilization of the opioid system was not specific to A-317491 since suramin-, a nonselective P2X receptor antagonist, induced antinociception was also attenuated by naloxone. In in vitro studies, A-317491 (3-100 microM) did not produce any agonist response at delta opioid receptors expressed in NG108-15 cells. A-317491 had been previously shown to be inactive at the kappa and mu opioid receptors. Furthermore, naloxone, at concentrations up to 1 mM, did not compete for [3H] A-317491 binding in 1321N1 cells expressing human P2X3 receptors. Taken together, these results indicate that antagonism of spinal P2X3/P2X2/3 receptors results in an indirect activation of the opioid system to alleviate inflammatory hyperalgesia and chemogenic nociception.

Effect of organophosphorus insecticides and their metabolites on astroglial cell proliferation

Though little attention has been given to the possibility that glial cells may represent a target for the developmental neurotoxicity of organophosphorus (OP) insecticides, recent evidence, obtained in particular with chlorpyrifos (CP), suggests that developmental exposure to this compound may indeed target astrocytes. To substantiate and expand these observations, we carried out a series of in vitro studies utilizing fetal rat astrocytes and a human astrocytoma cell line, 1321N1 cells, to investigate the effect of the OPs CP, diazinon (DZ) and parathion (P), their oxygen analogs chlorpyrifos oxon (CPO), diazoxon (DZO) and paraoxon (PO), and their metabolites 3,5,6-trichloro-2-pyridinol (TCP), 2-isopropyl-6-methyl-4-pyrimidol (IMP) and para-nitrophenol (PNP), on cell proliferation. In fetal rat astrocytes and astrocytoma cells maintained in serum, CP, DZ, P, CPO, DZO, and PO induced a concentration-dependent inhibition in [ 3H]thymidine incorporation with a very similar potency (IC 50 between 45 and 57 μM). Among the other metabolites, PNP was the most potent (IC 50 = 70–80 μM), while TCP and IMP were much less effective (IC 50 > 100 μM). Cytotoxicity appears to account only for a small part of the effect on DNA synthesis. OP insecticides and their oxons were three- to six-fold more potent in inhibiting [ 3H]thymidine incorporation when cells were synchronized in the G 0/G 1 phase of the cell cycle and re-stimulated by carbachol or epidermal growth factor. These results suggest that OP insecticides and their oxons affect astroglial cell proliferation and that the transition from the G 0/G 1 to the S/G 2 phase of the cell cycle may be particularly sensitive to the action of these compounds.

Thromboxane A2Promotes Interleukin-6 Biosynthesis Mediated by an Activation of Cyclic AMP-Response Element-Binding Protein in 1321N1 Human Astrocytoma Cells

1321N1 human astrocytoma cells express thromboxane A2 (TXA2) receptors (TP). However, physiological consequences of TXA2 signaling in glial cells remain unclear. Herein, we show that TXA2 promotes interleukin-6 (IL-6) biosynthesis in glial cells. A TP agonist, 9,11-dideoxy-9alpha,11alpha-methanoepoxy-prosta-5Z,13E-dien-1-oic acid (U46619), enhanced IL-6 production in both 1321N1 cells and cultured mouse astrocytes. It has been shown that IL-6 gene expression is regulated by various transcription factors. Among them, we found a significant increase in cyclic AMP-response element-binding protein (CREB) activity with its phosphorylation at Ser133 by U46619 in 1321N1 cells. Although U46619 increased IL-6 promoter activity, a mutation at cyclic AMP-response element (CRE) on the promoter clearly suppressed the effect, suggesting that CRE is involved in U46619-induced IL-6 expression. Furthermore, both CREB and IL-6 promoter activities were suppressed by SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole], a p38 mitogen-activated protein kinase (MAPK) inhibitor, and H89 [N-[2-(4-bromocinnamylamino)-ethyl]-5-isoquinoline], a protein kinase A (PKA) inhibitor, indicating involvements of p38 MAPK and PKA in CREB activation and IL-6 expression. To determine which G-proteins are implicated in the U46619-induced IL-6 synthesis, the interfering mutants of Galpha(q), Galpha12, or Galpha13 by were overexpressed in 1321N1 cells adenoviral approach. It is noteworthy that the Galpha(q) or Galpha13 mutant blocked the IL-6 production by U46619. The constitutively active mutant of Galpha(q), Galpha12, or Galpha13 enhanced IL-6 production, indicating that Galpha(q) and Galpha13 were involved in U46619-induced IL-6 production. In conclusion, TXA2 enhances the IL-6 biosynthesis via the PKA p38 MAPK/CREB pathway in 1321N1 cells. IL-6 induction depends on Galpha(q) and Galpha13 as well. This is the first report showing TP-mediated IL-6 production in glial cells.

P2Y1 and P2Y12 receptors for ADP desensitize by distinct kinase-dependent mechanisms

AbstractAdenosine 5′-diphosphate (ADP) plays a central role in regulating platelet function by the activation of the G protein–coupled receptors P2Y1 and P2Y12. Although it is well established that aggregation responses of platelets to ADP desensitize, the underlying mechanisms involved remain unclear. In this study we demonstrate that P2Y1- and P2Y12-mediated platelet responses desensitize rapidly. Furthermore, we have established that these receptors desensitize by different kinase-dependent mechanisms. G protein–coupled receptor kinase (GRK) 2 and GRK6 are both endogenously expressed in platelets. Transient overexpression of dominant-negative mutants of these kinases or reductions in endogenous GRK expression by the use of specific siRNAs in 1321N1 cells showed that P2Y12, but not P2Y1, desensitization is mediated by GRKs. In contrast, desensitization of P2Y1, but not P2Y12, is largely dependent on protein kinase C activity. This study is the first to show that both P2Y1 and P2Y12 desensitize in human platelets, and it reveals ways in which their sensitivity to ADP may be differentially and independently altered.

3H]BQ-123 Binding to Native Endothelin ETA Receptors in Human Astrocytoma 1321N1 Cells and Screening of Potential Ligands

Endothelin ET_A receptors on the human astrocytoma 1321N1 cell line were characterized in radioligand binding studies using the ET_A-selective antagonist radioligand [³H]BQ-123. A membrane preparation of the cell line showed a high expression level of ET_A receptors (B_max = 128 fmol/mg protein). The K_D value for [³H]BQ-123 (K_D = 2.29 nmol/l) was comparable to that previously determined at the human neuroblastoma cell line SK-N-MC. The specific labeling of ET_A receptors on astrocytoma 1321N1 cells by [³H]BQ-123 was confirmed by determining the rank order of potency of a series of agonists and antagonists. A competition assay for the screening of potential ET_A ligands was established and a series of new disubstituted imidazo[1,2-c]pyrimidine and pyrimido[1,6-a]pyrimidine derivatives was investigated. Moderate ET_A affinity was detected for several derivatives [30–40% inhibition of [³H]BQ-123 binding (3 nmol/l) at a concentration of 10 µmol/l]. Astrocytoma 1321N1 cells natively express relatively high levels of ET_A receptors. They are useful for the screening of potential ET_A ligands in radioligand binding assays with [³H]BQ-123.

The pyrimidinergic P2Y 6 receptor mediates a novel release of proinflammatory cytokines and chemokines in monocytic cells stimulated with UDP

The human P2Y 6 receptor (hP2Y 6) is a member of the G protein-coupled pyrimidinergic P2 receptor family that responds specifically to the extracellular nucleotide uridine diphosphate (UDP). Recently, the hP2Y 6 receptor has been reported to mediate monocyte IL-8 production in response to UDP or lipopolysaccharide (LPS), but the role of hP2Y 6 in regulating other pro-inflammatory cytokines or mediators is largely unknown. We demonstrate here that UDP specifically induces soluble TNF-α and IL-8 production in a promonocytic U937 cell line stably transfected with hP2Y 6. However, we did not detect IL-1α, IL-1β, IL-6, IL-10, IL-18, and PGE 2 in the conditioned media from the same cell line. These results distinguish UDP/P2Y 6 signaling from LPS signaling. Interestingly, UDP induces the production of IL-8, but not TNF-α, in human astrocytoma 1321N1 cell lines stably transfected with hP2Y 6. Therefore, the immune effect of UDP/P2Y 6 signaling on the production of proinflammatory cytokines is selective and dependent on cell types. We further identify that UDP can also induce the production of proinflammatory chemokines MCP-1 and IP-10 in hP2Y 6 transfected promonocytic U937 cell lines, but not astrocytoma 1321N1 cell lines stably transfected with hP2Y 6. From the Taqman analysis, UDP stimulation significantly upregulates the mRNA levels of IL-8, IP-10, and IL-1β, but not TNF-α. Taken together, these new findings expand the pro-inflammatory biology of UDP mediated by the P2Y 6 receptor.

Pharmacological characterization of P2X7 receptors in rat peritoneal cells

P2X(7) receptor activation by ATP results in the release of IL-1beta and IL-18. Prolonged stimulation can lead to pore formation and cell death. In this study we pharmacologically characterized P2X(7) receptors on rat peritoneal cells (RPC) and on 1321N1 cells transfected with rat P2X(7) receptor (1321rP2X(7)-11). RPC were isolated from rats by lavage. P2X(7) agonist induced pore formation in RPC was measured by EtBr uptake. P2X(7)-stimulated pore formation and Ca(++) influx in 1321rP2X(7)-11 cells were measured by a fluorometric imaging plate reader. The effects of pyridoxal phosphate-6-azo phenyl -2'-4'-disulfonic acid (PPADS) on pore formation and Ca(++) influx were examined in both RPC and 1321rP2X(7)-11. P2X(7)-mediated IL-1beta release in RPC and the effect of PPADS were determined. RPC express functional P2X(7) receptors that were activated by ATP analogs with a rank order of potency of 2'- 3'-O-(4-Benzoylbenzoyl) adenosine 5'-triphosphate (BzATP) > ATP > alpha,beta-methylene ATP. Activation of P2X(7) receptors by BzATP was inhibited by PPADS. Similar results were also obtained in 1321rP2X(7)-11 cells. Activation of P2X(7) receptors on RPC resulted in IL-1 beta secretion, which was inhibited by PPADS. RPC express functional P2X(7) receptors that form pores and mediate the release of IL-1beta.

Potentiation of P2Y Receptors by Physiological Elevations of Extracellular K+via a Mechanism Independent of Ca2+Influx

Many physiological and pathophysiological situations generate a significant increase in extracellular K+ concentration. This is known to influence a number of membrane conductances and exchangers, whereas direct effects of K+ on the activation of G protein-coupled receptors have not been reported. We now show that Ca2+ release evoked by P2Y1 receptors expressed in 1321-N1 astrocytoma cells is markedly potentiated by small increases in external K+ concentration. This effect was blocked by the phospholipase-C inhibitor U-73122 (1-[6-[[17 beta]-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione), but not by its analog U-73343 (1-[6-[[17 beta]-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidinedione), and not by nifedipine, Ni2+, Cd2+, or Gd3+. Thus, K+ enhances d-myo-inositol 1,4,5-trisphosphate-dependent Ca2+ release without a requirement for Ca2+ influx. The cation dependence of this effect displayed the order K+ > Rb+ > N-methyl-D-glucamine+, and Cs+ and choline+ were ineffective. The potentiation by K+ is half-maximal at an increase of 2.6 mM (total K+ of 7.6 mM). K+ caused a reduction in EC50 (2.7-fold for a 29 mM increase) without a change of slope; thus, the greatest effect was observed at near-threshold agonist levels. The response to K+ can be explained in part by depolarization-dependent potentiation of P2Y1 receptors [J Physiol (Lond) 555:61-70, 2004]. However, electrophysiological recordings of 1321-N1 cells and megakaryocytes demonstrated that K+ also amplifies ADP-evoked Ca2+ responses independently of changes in membrane potential. Elevated K+ also amplified endogenous UTP-dependent Ca2+ responses in human embryonic kidney 293 cells, suggesting that other P2Y receptors are K(+)-dependent. P2Y receptors display a widespread tissue distribution; therefore, their modulation by small changes in extracellular K+ may represent a novel means of autocrine and paracrine regulation of cellular activity.

The Involvement of Calcium in the Protective and Toxic (Nonlinear) Responses of Rodent and Human Astroglial Cells

The involvement of [Ca(2+)](i) in the reactive changes of astrocytes which accompany exposure to different chemicals were studied in cultures of C6 and 1321N1 cells. Cells were exposed to up to three serial pulses of the differentiating agent dBcAMP, which induces activation-type changes in the cells. Other cells, with or without the dBcAMP treatments, were treated with a range of concentrations of the antidepressants amitriptyline and fluoxetine and the glial toxicants acrylamide and chloroquine. In some experiments the L-type voltage calcium channel blocker Nifedipine was employed. [Ca(2+)](i) was measured in populations of the cells using Fura-2AM and a charge coupled device (CCD) camera attached to a fluorescence microscope. dBcAMP induced both dose- and time-dependent changes in [ Ca(2+)](i) with increases in both the [Ca(2+)](i) oscillations and mean [Ca(2+)](i) (e.g. in C6 cells at 18 min mean [Ca(2+)](i) was 318 +/- 20nM following the single differentiating dBcAMP pulses, 489 +/- 17nM (p < 0.001) following two serial pulses, and 275 +/- 30nM (not significant) following three pulses). Therapeutic doses of fluoxetine and amitriptyline caused increases in the calcium oscillations and the mean calcium concentrations ( maximum recorded mean increase was in the C6 cells at 10min by 0.02 muM fluoxetine when [Ca(2+)](i) was 411 +/- 35nM c.f. control 254 +/- 25nM, p = 0.01). Higher (non-therapeutic) doses of both antidepressants caused significant reductions. Chloroquine and acrylamide also caused dose-dependent bi-phasic types of alterations in [Ca(2+)](i), with significant reductions at lower, sub-cytotoxic doses followed by significant increases at higher concentrations, approaching those which cause cell damage. Nifedipine treatment caused some reductions in the dBcAMP, antidepressant or toxicant-induced calcium changes, but this substance also initiated cytotoxic alterations. The findings show that both the activation-type changes (which are frequently associated with increased protective capacities) and toxic responses of C6 and 1321N1 cells to different chemical agents are associated with dose-dependent alterations in [Ca(2+)](i).

Evidence for the Functional Expression and Pharmacological Characterization of Adenine Receptors in Native Cells and Tissues

An orphan G protein-coupled receptor from rat has recently been discovered to be activated by the nucleobase adenine (Proc Natl Acad Sci USA 99:8573-8578, 2002). In the present study, we show for the first time that the adenine receptor is expressed in membrane preparations of native tissues and cell lines in high density, including rat brain cortex, rat brain striatum, and the mouse neuroblastoma x rat glioma hybrid cell line NG108-15. Saturation analysis with [3H]adenine at rat brain cortical membranes exhibited a single high-affinity binding site with a KD value of 27.2 nM, and a binding capacity of 2.28 pmol/mg of protein. Kinetic studies revealed unusual binding kinetics of [3H]adenine with rapid association and slow dissociation. A series of compounds were investigated in [3H]adenine competition experiments at rat brain cortex. Only minor substitution of the adenine structure was tolerated, the most potent compounds of the present series being 2-fluoroadenine (Ki value of 620 nM), 8-thioadenine (Ki value of 2.77 microM), N6-methyladenine (Ki value of 3.64 microM), and 7-methyladenine (Ki value of 4.13 microM), all of which were partial agonists (40-60% intrinsic activity). Adenine dose dependently inhibited forskolin-stimulated adenylate cyclase in membrane preparations of NG108-15 cells as well as in intact cells, showing that the receptor is functional in NG108-15 cells. Reverse transcriptase-polymerase chain reaction experiments followed by sequencing indicate that the NG108-15 cells express the murine ortholog of the adenine receptor. Moreover, preliminary radioligand binding studies with [3H]adenine at membranes of human astrocytoma 1321N1 cells suggest that a human ortholog of the rat adenine receptor exists.

Rho-mediated cytoskeletal rearrangement in response to LPA is functionally antagonized by Rac1 and PIP2.

G-protein-coupled receptors signal through Rho to induce actin cytoskeletal rearrangement. We previously demonstrated that thrombin stimulates Rho-dependent process retraction and rounding of 1321N1 astrocytoma cells. Surprisingly, while lysophosphatidic acid (LPA) activated RhoA in 1321N1 cells, it failed to produce cell rounding. Thrombin, unlike LPA, decreased Rac1 activity, and activated (GTPase-deficient) Rac1 inhibited thrombin-stimulated cell rounding, while expression of dominant-negative Rac1 promoted LPA-induced rounding. LPA and thrombin receptors appear to differ in coupling to Gi, as LPA but not thrombin-stimulated 1321N1 cell proliferation was pertussis toxin-sensitive. Blocking Gi with pertussis toxin enabled LPA to induce cell rounding and to decrease activated Rac1. These data support the hypothesis that Rac1 and Gi activation antagonize cell rounding. Thrombin and LPA receptors also differentially activated Gq pathways as thrombin but not LPA increased InsP3 formation and reduced phosphatidylinositol 4,5-bisphosphate (PIP2) levels. Microinjection of the plekstrin homology domain of phospholipase C (PLC)delta1, which binds PIP2, enabled LPA to elicit cell rounding, consistent with a requirement for PIP2 reduction. We suggest that Rho-mediated cytoskeletal responses are enhanced by concomitant reductions in cellular levels of PIP2 and Rac1 activation and thus effected only by G-protein-coupled receptors with appropriate subsets of G protein activation.

Cloning, up-regulation, and mitogenic role of porcine P2Y2 receptor in coronary artery smooth muscle cells.

Previous work has shown up-regulation of a UTP-sensitive P2Y receptor in porcine coronary smooth muscle cells (CSMC) of organ-cultured arteries. However, the molecular identity and functional role of this putative receptor remained undefined. Here we report the cloning of the cDNA for this receptor that encodes an open reading frame for a protein of 373 amino acids with the highest homology to the human P2Y(2) receptor (84%). Heterologous expression of this receptor in 1321N1 cells revealed a novel pharmacology in that UTP and ITP were full agonists and UTP was more potent and efficacious than ATP for increasing intracellular [Ca(2+)] and extracellular signal-regulated kinase phosphorylation. Stimulation of subcultured CSMC with UTP, ITP, or ATP induced a concentration-dependent increase in cellular DNA content, protein synthesis, cell number, and proliferating cell nuclear antigen expression, indicating a mitogenic role for P2Y(2) receptors. This was supported by the finding that the treatment of CSMC with antisense oligonucleotides to the cloned cDNA sequence significantly inhibited UTP- and ATP-induced DNA and protein synthesis. In addition, reverse transcription-polymerase chain reaction analysis showed that P2Y(2) receptor mRNA was dramatically increased in cells of organ-cultured arteries compared with freshly harvested arteries, whereas the P2Y(6) receptor mRNA level was unchanged, and the P2Y(4) receptor mRNA was undetectable. This P2Y(2) subtype-specific up-regulation was confirmed in cells of coronary arteries stented in vivo. In conclusion, we have cloned the porcine P2Y(2) receptor with novel pharmacology and demonstrated that this receptor is up-regulated in CSMC of in vitro organ cultures or in vivo stented coronary arteries to mediate the mitogenic effects of nucleotides.

Gap junction channels coordinate the propagation of intercellular Ca2+ signals generated by P2Y receptor activation.

Astrocytes express gap junction proteins and multiple types of P2Y receptors (P2YRs) that contribute to the propagation of intercellular Ca(2+) waves (ICW). To gain access to the role played by gap junctional communication in ICW propagation generated by P2YR activation, we selectively expressed P2Y(1,2,4)R subtypes and Cx43 in the human 1321N1 astrocytoma cell line, which lacks endogenous P2 receptors. Fluorescence recovery after photobleaching revealed that 1321N1 cells are poorly dye-coupled and do not propagate ICW. Forced expression of Cx43 in 1321N1 cells (which did not show functional hemichannels) increased dye coupling and allowed short-range ICW transmission that was mainly mediated by intercellular diffusion of Ca(2+) generated in the stimulated cells. Astrocytoma clones expressing each of the P2YR subtypes were also able to propagate ICWs that were likely dependent on IP(3) generation. These waves exhibited properties particular to each P2YR subtype. Co-expression of eGFP-hCx43 and P2Y(1)R modified the properties of P2Y(1)R-generated ICW to those characteristics of P2Y(2)R. Increased coupling in P2Y(4)R clones induced by expression of eGFP-hCx43 abolished the ICWs observed in uncoupled P2Y(4)R clones. No changes in the behavior of ICWs generated in P2Y(2)R clones were observed after forced expression of Cx43. These data indicate that in 1321N1 cells gap junctional communication provides intercellular integration of Ca(2+) signals generated by P2YR activation, thus coordinating the propagation of intercellular calcium waves.

Cloning, pharmacological characterisation and distribution of the rat G-protein-coupled P2Y 13 receptor

The human P2Y 13 receptor is a new receptor characterized by coupling to Gi, responsiveness to adenine di-phospho-nucleotides and blockade by the P2Y antagonist AR-C69931MX. The mouse P2Y 13 ortholog has also been reported. Here we report, for the first time, the cloning of rat P2Y 13 receptor, its pharmacological analysis and tissue distribution. Rat P2Y 13 is 79% and 87% identical to human and mouse P2Y 13 receptors, respectively. Expression of rP2Y 13 receptor in 1321N1 cells induced the appearance of responses to the typical P2Y 13 receptor agonists ADP and 2MeSADP, as detected by stimulation of [ 35S]GTPγS binding. Agonist activities were higher in cells transfected with rP2Y 13 receptor in the presence of the Gα 16 subunit; in all cases agonist effects were abolished by pertussis toxin pre-treatment. At variance from both human and mouse receptors, ADP was more potent than 2MeSADP. Other nucleotides and sugar-nucleotides were ineffective. Both in the absence and presence of Gα 16, activation of rP2Y 13 receptor by ADP and 2MeSADP was completely inhibited by nM concentrations of AR-C69931MX. In contrast, no inhibition of rP2Y 13 receptor was induced by the selective P2Y 1 receptor antagonist MRS2179. rP2Y 13 receptor showed highest expression levels in spleen, followed by liver and brain (with particularly high levels in cortex and striatum as reported in man), suggesting important roles in the nervous and immune systems. Expression levels comparable to those of the other cloned P2Y receptors were found in primary rat astrocytes, indicating a possible role in reactive astrogliosis. Hence, rat P2Y 13 receptor displays several similarities but also interesting differences with its human and mouse orthologs, that will have to be taken into account when characterizing the pathophysiological roles of this receptor in the rat animal models.

3H]A-317491, a novel high-affinity non-nucleotide antagonist that specifically labels human P2X2/3 and P2X3 receptors.

A-317491 is a potent and selective antagonist of P2X3 and P2X(2/3) receptors. In the present studies, the ability of [3H]A-317491 to label recombinant human P2X(2/3) and P2X(3) receptors was characterized. Using membranes prepared from 1321N1 cells expressing P2X(2/3) receptors, [3H]A-317491 specifically labeled high-affinity (Kd = 0.9 nM) recognition sites. High-affinity [3H]A-317491 binding was not detected in membrane preparations from native 1321N1 cells or cells expressing homomeric P2X1, P2X2, or P2X3 receptors. Specific [3H]A-317491 P2X3 receptors could only be reliably detected following treatment of intact P2X3 receptor-expressing cells with apyrase (1 U/ml) both before and during membrane preparation. Under these conditions, [3H]A-317491 also labeled high-affinity (Kd = 9 nM) binding sites. Lower affinity binding components (Kd values of 87-790 nM) were detected in both assays using higher ligand concentrations that likely represent nonfunctional recognition sites. [3H]A-317491 binding to both P2X(2/3) and P2X3 receptors was reversible, and ligand kinetic studies provided similar estimates of the high-affinity binding constants. Potent P2X3 receptor agonists 2-methylthio-ATP, 2,3-O-(4-benzoylbenzoyl)-ATP, and alpha,beta-methylene adenosine triphosphate also potently inhibited specific [3H]A-317491 binding to both P2X(2/3) and P2X3 receptors. The pharmacological profile for P2X receptor antagonists to inhibit [3H]A-317491 binding to P2X(2/3) and P2X3 receptors was highly correlated (r = 0.98, P < 0.05), and a similar rank order of potency was observed for blockade of P2X(2/3) receptor-mediated calcium influx. These data demonstrate that [3H]A-317491 is the first useful radioligand for the specific labeling of P2X3-containing channels.

ADP-ribosylation Factor-dependent Phospholipase D Activation by the M3 Muscarinic Receptor

G protein-coupled receptors can potentially activate phospholipase D (PLD) by a number of routes. We show here that the native M3 muscarinic receptor in 1321N1 cells and an epitope-tagged M3 receptor expressed in COS7 cells substantially utilize an ADP-ribosylation factor (ARF)-dependent route of PLD activation. This pathway is activated at the plasma membrane but appears to be largely independent of G, phospholipase C, Ca2+ q/11, protein kinase C, tyrosine kinases, and phosphatidyl inositol 3-kinase. We report instead that it involves physical association of ARF with the M3 receptor as demonstrated by co-immunoprecipitation and by in vitro interaction with a glutathione S-transferase fusion protein of the receptor's third intracellular loop domain. Experiments with mutant constructs of ARF1/6 and PLD1/2 indicate that the M3 receptor displays a major ARF1-dependent route of PLD1 activation with an additional ARF6-dependent pathway to PLD1 or PLD2. Examples of other G protein-coupled receptors assessed in comparison display alternative pathways of protein kinase C- or ARF6-dependent activation of PLD2.

Calmodulin Is a Phospholipase C-β Interacting Protein

Phospholipase C-beta 3 (PLC beta 3) is an important effector enzyme in G protein-coupled signaling pathways. Activation of PLC beta 3 by G alpha and G beta gamma subunits has been fairly well characterized, but little is known about other protein interactions that may also regulate PLC beta 3 function. A yeast two-hybrid screen of a mouse brain cDNA library with the amino terminus of PLC beta 3 has yielded potential PLC beta 3 interacting proteins including calmodulin (CaM). Physical interaction between CaM and PLC beta 3 is supported by a positive secondary screen in yeast and the identification of a CaM binding site in the amino terminus of PLC beta 3. Co-precipitation of in vitro translated and transcribed amino- and carboxyl-terminal PLC beta 3 revealed CaM binding at a putative amino-terminal binding site. Direct physical interaction of PLC beta 3 and PLC beta 1 isoforms with CaM is supported by pull-down of both isoenzymes with CaM-Sepharose beads from 1321N1 cell lysates. CaM inhibitors reduced M1-muscarinic receptor stimulation of inositol phospholipid hydrolysis in 1321N1 astrocytoma cells consistent with a physiologic role for CaM in modulation of PLC beta activity. There was no effect of CaM kinase II inhibitors, KN-93 and KN-62, on M1-muscarinic receptor stimulation of inositol phosphate hydrolysis, consistent with a direct interaction between PLC beta isoforms and CaM.

Muscarinic receptor-mediated activation of p70 S6 kinase 1 (S6K1) in 1321N1 astrocytoma cells: permissive role of phosphoinositide 3-kinase.

In 1321N1 astrocytoma cells, carbachol stimulation of M3 muscarinic cholinergic receptors, coupled to phospholipase C, evoked a persistent 10-20-fold activation of p70 S6 kinase (S6K1). This response was abolished by chelation of cytosolic Ca2+ and reproduced by the Ca2+ ionophore ionomycin, but was not prevented by down-regulation or inhibition of protein kinase C. Carbachol-stimulated activation and phosphorylation of S6K1 at Thr389 were prevented by rapamycin, an inhibitor of mTOR (mammalian target of rapamycin), or by wortmannin, a phosphoinositide 3-kinase (PI3K) inhibitor. Carbachol also stimulated the phosphorylation of eukaryotic initiation factor 4E-binding protein-1 (4E-BP1), a second mTOR-dependent event, with similar potency to its effect on S6K1. This response was blocked by rapamycin, but was not markedly affected by 100 nM wortmannin, implying separate roles for mTOR and PI3K in S6K1 activation. Wortmannin abolished the carbachol-stimulated rise in PtdIns(3,4,5)P3 and greatly reduced unstimulated levels of this lipid. By contrast, an inhibitor of epidermal growth factor receptor kinase, AG1478, which prevents carbachol-stimulated ErbB3 transactivation, PI3K recruitment and protein kinase B activation in 1321N1 cells, reduced activation of S6K1 by no more than 30%. This effect was overcome by 10 nM insulin, which on its own did not stimulate S6K1, but increased cellular PtdIns(3,4,5)P3 concentrations comparably with carbachol alone. These observations distinguish obligatory roles for mTOR and PI3K in regulating S6K1, but imply that minimal PI3K activity is sufficient to permit stimulation of S6K1 by other activating factors such as increased cytosolic Ca2+ concentrations, which are essential to the muscarinic receptor-mediated response. Moreover, 4E-BP1 and hence, presumably, mTOR can be regulated independently of PI3K activation through these mechanisms.