Interaction and interrelation of P2X7 and P2X4 receptor complexes in mouse lung epithelial cells

P2X4 and P2X7 receptors are ATP-gated ion channels that are co-expressed in alveolar epithelial type I cells. Both receptors are localized to the plasma membrane and partly associated with lipid rafts. Here we report on our study in an alveolar epithelial cell line of the molecular organization of P2X7R and P2X4R receptors and the effect of their knockdown. Native gel electrophoresis reveals three P2X7R complexes of ~430, ~580 and ~760 kDa. The latter two correspond exactly in size to signals of Cav-1, the structural protein of caveolae. Interestingly knockdown of P2rx7 affects protein levels, the intracellular distribution and the supramolecular organization of Cav-1 as well as of P2X4R, which is mainly detected in a complex of ~430 kDa. Our data suggest upregulation of P2X4R as a compensatory mechanism of P2X7R depletion.     

P2Y<Subscript>2</Subscript> receptor modulates shear stress-induced cell alignment and actin stress fibers in human umbilical vein endothelial cells

Endothelial cells release ATP in response to fluid shear stress, which activates purinergic (P2) receptor-mediated signaling molecules including endothelial nitric oxide (eNOS), a regulator of vascular tone. While P2 receptor-mediated signaling in the vasculature is well studied, the role of P2Y₂ receptors in shear stress-associated endothelial cell alignment, cytoskeletal alterations, and wound repair remains ill defined. To address these aspects, human umbilical vein endothelial cell (HUVEC) monolayers were cultured on gelatin-coated dishes and subjected to a shear stress of 1 Pa. HUVECs exposed to either P2Y₂ receptor antagonists or siRNA showed impaired fluid shear stress-induced cell alignment, and actin stress fiber formation as early as 6 h. Similarly, when compared to cells expressing the P2Y₂ Arg-Gly-Asp (RGD) wild-type receptors, HUVECs transiently expressing the P2Y₂ Arg-Gly-Glu (RGE) mutant receptors showed reduced cell alignment and actin stress fiber formation in response to shear stress as well as to P2Y₂ receptor agonists in static cultures. Additionally, we observed reduced shear stress-induced phosphorylation of focal adhesion kinase (Y397), and cofilin-1 (S3) with receptor knockdown as well as in cells expressing the P2Y₂ RGE mutant receptors. Consistent with the role of P2Y₂ receptors in vasodilation, receptor knockdown and overexpression of P2Y₂ RGE mutant receptors reduced shear stress-induced phosphorylation of AKT (S473), and eNOS (S1177). Furthermore, in a scratched wound assay, shear stress-induced cell migration was reduced by both pharmacological inhibition and receptor knockdown. Together, our results suggest a novel role for P2Y₂ receptor in shear stress-induced cytoskeletal alterations in HUVECs.     

Purinergic systems in microglia

Accumulating findings indicate that nucleotides play an important role in microglia through P2 purinoceptors. P2 purinoceptors are divided into two families, ionotropic receptors (P2X) and metabotropic receptors (P2Y). P2X receptors (7 types; P2X₁ – P2X₇) contain intrinsic pores that open by binding with ATP. P2Y receptors (8 types; P2Y_{1, 2, 4, 6, 11, 12, 13 and 14}) are activated by nucleotides and couple to intracellular second-messenger systems through heteromeric G-proteins. Nucleotides are released or leaked from non-excitable cells as well as neurons in physiological and pathophysiological conditions. Microglia express many types of P2 purinoceptors and are known as resident macrophages in the CNS. ATP and other nucleotides work as ‘warning molecules’ especially through activating microglia in pathophysiological conditions. Microglia play a key role in neuropathic pain, chemotaxis and phagocytosis through nucleotide-evoked activation of P2X₄, P2Y₁₂ and P2Y₆ receptors, respectively. These findings indicate that extracellular nucleotides are important players in the central stage of microglial function. Received 19 April 2008; received after revision 20 May 2008; accepted 23 May 2008     

Endocytosis mechanism of P2Y2 nucleotide receptor tagged with green fluorescent protein: clathrin and actin cytoskeleton dependence

Extracellular nucleotides exert a large number of physiological effects through activation of P2Y receptors. We expressed rat P2Y₂ (rP2Y₂) receptor, tagged with green fluorescent protein (GFP) in HEK-293 cells and visualized receptor translocation in live cells by confocal microscopy. Functional receptor expression was confirmed by determining [Ca²⁺]_i responses. Agonist stimulation caused a time-dependent translocation of the receptor from the plasma membrane to the cytoplasm. Rearrangement of the actin cytoskeleton was observed during agonist-mediated rP2Y₂-GFP receptor internalization. Colocalization of the internalized receptor with early endosomes, clathrin and lysosomes was detected by confocal microscopy. The inhibition of receptor endocytosis by either high-density medium or chlorpromazine in the presence of UTP indicates that the receptor was internalized by the clathrin-mediated pathway. The caveolin- mediated pathway was not involved. Targeting of the receptor from endosomes to lysosomes seems to involve the proteasome pathway, because proteasomal inhibition increased receptor recycling back to the plasma membrane.Received 8 February 2005; received after revision 18 March 2005; accepted 11 April 2005     

ADP receptor P2Y13 induce apoptosis in pancreatic β-cells

Pancreatic β-cell loss represents a key factor in the pathogenesis of diabetes. Since the influence of purinergic signaling in β-cell apoptosis has not been much investigated, we examined the role of the ADP receptor P2Y₁₃ using the pancreatic insulinoma-cell line MIN6c4 as a model system. Real time-PCR revealed high expression of the ADP receptors P2Y₁ and P2Y₁₃. Adding the ADP analogue, 2MeSADP, to MIN6c4 cells induced calcium influx/mobilization and inhibition of cAMP production by activation of P2Y₁ and P2Y₁₃, respectively. 2MeSADP reduced cell proliferation and increased Caspase-3 activity; both these effects could be fully reversed by the P2Y₁₃ receptor antagonist MRS2211. We further discovered that blocking the P2Y₁₃ receptor results in enhanced ERK1/2, Akt/PKB and CREB phosphorylation mechanisms involved in β-cell survival. These results indicate that P2Y₁₃ is a proapoptotic receptor in β-cells as the P2Y₁₃ receptor antagonist MRS2211 is able to protect the cells from ADP induced apoptosis.     

One lipid, multiple functions: how various pools of PI(4,5)P2 are created in the plasma membrane

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] is a minor lipid of the inner leaflet of the plasma membrane that controls the activity of numerous proteins and serves as a source of second messengers. This multifunctionality of PI(4,5)P₂ relies on mechanisms ensuring transient appearance of PI(4,5)P₂ clusters in the plasma membrane. One such mechanism involves phosphorylation of PI(4)P to PI(4,5)P₂ by the type I phosphatidylinositol-4-phosphate 5-kinases (PIP5KI) at discrete membrane locations coupled with PI(4)P delivery/synthesis at the plasma membrane. Simultaneously, both PI(4)P and PI(4,5)P₂ participate in anchoring PIP5KI at the plasma membrane via electrostatic bonds. PIP5KI isoforms are also selectively recruited and activated at the plasma membrane by Rac1, talin, or AP-2 to generate PI(4,5)P₂ in ruffles and lamellipodia, focal contacts, and clathrin-coated pits. In addition, PI(4,5)P₂ can accumulate at sphingolipid/cholesterol-based rafts following activation of distinct membrane receptors or be sequestered in a reversible manner due to electrostatic constrains posed by proteins like MARCKS.     

ATP-induced Ca<Superscript>2+</Superscript>-signalling mechanisms in the regulation of mesenchymal stem cell migration

The ability of cells to migrate to the destined tissues or lesions is crucial for physiological processes from tissue morphogenesis, homeostasis and immune responses, and also for stem cell-based regenerative medicines. Cytosolic Ca²⁺ is a primary second messenger in the control and regulation of a wide range of cell functions including cell migration. Extracellular ATP, together with the cognate receptors on the cell surface, ligand-gated ion channel P2X receptors and a subset of G-protein-coupled P2Y receptors, represents common autocrine and/or paracrine Ca²⁺ signalling mechanisms. The P2X receptor ion channels mediate extracellular Ca²⁺ influx, whereas stimulation of the P2Y receptors triggers intracellular Ca²⁺ release from the endoplasmic reticulum (ER), and activation of both type of receptors thus can elevate the cytosolic Ca²⁺ concentration ([Ca²⁺]_c), albeit with different kinetics and capacity. Reduction in the ER Ca²⁺ level following the P2Y receptor activation can further induce store-operated Ca²⁺ entry as a distinct Ca²⁺ influx pathway that contributes in ATP-induced increase in the [Ca²⁺]_c. Mesenchymal stem cells (MSC) are a group of multipotent stem cells that grow from adult tissues and hold promising applications in tissue engineering and cell-based therapies treating a great and diverse number of diseases. There is increasing evidence to show constitutive or evoked ATP release from stem cells themselves or mature cells in the close vicinity. In this review, we discuss the mechanisms for ATP release and clearance, the receptors and ion channels participating in ATP-induced Ca²⁺ signalling and the roles of such signalling mechanisms in mediating ATP-induced regulation of MSC migration.     

The nucleotide receptor P2Y13 is a key regulator of hepatic High-Density Lipoprotein (HDL) endocytosis

Cell surface receptors for high-density lipoprotein (HDL) on hepatocytes are major partners in the regulation of cholesterol homeostasis. We recently identified a cell surface ATP synthase as a high-affinity receptor for HDL apolipoprotein A-I (apoA-I) on human hepatocytes. Stimulation of this ectopic ATP synthase by apoA-I triggered a low-affinity-receptor-dependent HDL endocytosis by a mechanism strictly related to the generation of ADP. This suggests that nucleotide G-protein- coupled receptors of the P2Y family are molecular components in this pathway. Only P2Y₁ and P2Y₁₃ are present on the membrane of hepatocytes. Using both a pharmacological approach and small interference RNA, we identified P2Y₁₃ as the main partner in hepatic HDL endocytosis, in cultured cells as well as in situ in perfused mouse livers. We also found a new important action of the antithrombotic agent AR-C69931MX as a strong activator of P2Y₁₃-mediated HDL endocytosis. Received 9 May 2005; received after revision 24 June 2005; accepted 1 September 2005     

Role of the ubiquitin–proteasome system in the regulation of P2Y13 receptor expression: impact on hepatic HDL uptake

The protective effect of high density lipoproteins (HDL) against atherosclerosis is mainly attributed to their capacity to transport excess cholesterol from peripheral tissues back to the liver for further elimination into the bile, a process called reverse cholesterol transport (RCT). Recently, the importance of the P2Y₁₃ receptor (P2Y₁₃-R) was highlighted in HDL metabolism since HDL uptake by the liver was decreased in P2Y₁₃-R deficient mice, which translated into impaired RCT. Here, we investigated for the first time the molecular mechanisms regulating cell surface expression of P2Y₁₃-R. When transiently expressed, P2Y₁₃-R was mainly detected in the endoplasmic reticulum (ER) and strongly subjected to proteasome degradation while its homologous P2Y₁₂ receptor (P2Y₁₂-R) was efficiently targeted to the plasma membrane. We observed an inverse correlation between cell surface expression and ubiquitination level of P2Y₁₃-R in the ER, suggesting a close link between ubiquitination of P2Y₁₃-R and its efficient targeting to the plasma membrane. The C-terminus tail exchange between P2Y₁₃-R and P2Y₁₂-R strongly restored plasma membrane expression of P2Y₁₃-R, suggesting the involvement of the intra-cytoplasmic tail of P2Y₁₃-R in expression defect. Accordingly, proteasomal inhibition increased plasma membrane expression of functionally active P2Y₁₃-R in hepatocytes, and consequently stimulated P2Y₁₃-R-mediated HDL endocytosis. Importantly, proteasomal inhibition strongly potentiated HDL hepatic uptake (>200 %) in wild-type but not in P2Y₁₃-R-deficient mice, thus reinforcing the role of P2Y₁₃-R expression in regulating HDL metabolism. Therefore, specific inhibition of the ubiquitin–proteasome system might be a novel powerful HDL therapy to enhance P2Y₁₃-R expression and consequently promote the overall RCT.     

Interactions of the cell adhesion molecule nectin with transmembrane and peripheral membrane proteins for pleiotropic functions

Cell adhesion molecules (CAMs) have been implicated in the control of a wide variety of cellular processes, such as cell adhesion, polarization, survival, movement, and proliferation. Nectins have emerged as immunoglobulin-like CAMs that participate in calcium-independent cell-cell adhesion by homophilic and heterophilic trans-interactions with nectins and nectin-like molecules. Nectin-based cell-cell adhesion exerts its function independently or in cooperation with other CAMs including cadherins and is essential for the formation of intercellular junctions, including adherens junctions, tight junctions, and puncta adherentia junctions. Nectins cis-interact with integrin α_vβ₃ and platelet-derived growth factor receptor and facilitate their signals to regulate the formation and integrity of intercellular junctions and cell survival. Nectins intracellularly associate with peripheral membrane proteins, including afadin and Par-3. This review focuses on recent progress in understanding the interactions of nectins with other transmembrane and peripheral membrane proteins to exert pleiotropic functions. Received 27 June 2007; received after revision 14 August 2007; accepted 12 September 2007     

Scarites buparius,a caraboid beetle with an X1X2Y sex-chromosomes system

Summary Scarites buparius has 2n=37 and n=17+X₁X₂Y. This multiple sex-chromosomes system seems to be the result of a reciprocal translocation between the primitive X-chromosome and an autosome. This is the first caraboid beetle, exceptingCicindela spp., that has multiple sex-chromosomes.This work has been supported by a grant of the Comisión Asesora de Investigación Científica y Técnica, No. 1.552.     

Purine and pyrimidine receptors

Adenosine 5′-triphosphate (ATP), in addition to its intracellular roles, acts as an extracellular signalling molecule via a rich array of receptors, which have been cloned and characterised. P1 receptors are selective for adenosine, a breakdown product of ATP, produced after degradation by ectonucleotidases. Four subtypes have been identified, A₁, A_2A, A_2B and A₃ receptors. P2 receptors are activated by purines and some subtypes also by pyrimidines. P2X receptors are ligand-gated ion channel receptors and seven subunits have been identified, which form both homomultimers and heteromultimers. P2Y receptors are G protein-coupled receptors, and eight subtypes have been cloned and characterised to date. Received 22 November 2006; received after revision 11 January 2007; accepted 27 February 2007     

EP4 receptor stimulation down-regulates human eosinophil function

Accumulation of eosinophils in tissue is a hallmark of allergic inflammation. Here we observed that a selective agonist of the PGE₂ receptor EP4, ONO AE1-329, potently attenuated the chemotaxis of human peripheral blood eosinophils, upregulation of the adhesion molecule CD11b and the production of reactive oxygen species. These effects were accompanied by the inhibition of cytoskeletal rearrangement and Ca²⁺ mobilization. The involvement of the EP4 receptor was substantiated by a selective EP4 antagonist, which reversed the inhibitory effects of PGE₂ and the EP4 agonist. Selective kinase inhibitors revealed that the inhibitory effect of EP4 stimulation on eosinophil migration depended upon activation of PI 3-kinase and PKC, but not cAMP. Finally, we found that EP4 receptors are expressed by human eosinophils, and are also present on infiltrating leukocytes in inflamed human nasal mucosa. These data indicate that EP4 agonists might be a novel therapeutic option in eosinophilic diseases.     

Signalling roles of mammalian phospholipase D1 and D2

Phospholipase D (PLD) catalyses the hydrolysis of phosphatidylcholine to generate the lipid second messenger, phosphatidate (PA) and choline. PLD activity in mammalian cells is low and is transiently stimulated upon activation by G-protein-coupled and receptor tyrosine kinase cell surface receptors. Two mammalian PLD enzymes (PLD1 and PLD2) have been cloned and their intracellular regulators identified as ARF and Rho proteins, protein kinase Cα as well as the lipid, phosphatidylinositol [4, 5] bisphosphate (PIP₂). I discuss the regulation of these enzymes by cell surface receptors, their cellular localisation and the potential function of PA as a second messenger. Evidence is presented for a role of PA in regulating the lipid kinase activity of PIP 5-kinase, an enzyme that synthesises PIP₂. A signalling role of phospholipase D via PA and indirectly via PIP₂ in regulating membrane traffic and actin dynamics is indicated by the available data. Received 25 April 2001; received after revision 15 June 2001; accepted 15 June 2001     

Effect of maternal under-nutrition on pup body weight and hypothalamic endocannabinoid levels

Dietary long-chain polyunsaturated fatty acids are known to influence brain levels of the endocannabinoid anandamide in newborn pigs and mice. Furthermore, endocannabinoids were shown to control pup suckling and body weight in mice, and food intake in adult rodents. Here we determined the effect of maternal under-nutrition during gestation, lactation, or both, on body weight, and on the levels of endocannabinoids and expression of cannabinoid CB₁ receptors and fatty acid amide hydrolase in the hypothalamus of rat pups at weaning (21 days old) or adult rats (4 months old). Maternal under-nutrition resulted in a striking decrease in body weight of weaning rats, paralleled by a decrease in the hypothalamic levels of the endocannabinoid anandamide, but not of 2-arachidonoylglycerol. No significant change in the hypothalamic expression of either cannabinoid CB₁ receptors or fatty acid amide hydrolase mRNA was detected in any of the three groups of weaned pups. The decrease in pup body weight and hypothalamic anandamide levels was not observable in 4-month-old rats from any of the three groups. These data suggest that maternal under-nutrition causes a decrease in hypothalamic anandamide levels and loss of body weight, and confirm a crucial role for endocannabinoid signalling in neonatal development. Received 4 November 2002; received after revision 29 November 2002; accepted 16 December 2002 RID="*" ID="*"Corresponding author.     

Expression of membrane and nuclear melatonin receptor mRNA and protein in the mouse immune system

The neurohormone melatonin plays a fundamental role in neuroimmunomodulation of several mammalian species, including mice. This effect is supported by the existence of specific melatonin-binding sites in murine immunocompetent organs. Moreover, using melatonin receptor analogues, several effects of the neurohormone on mice physiology through its membrane and nuclear receptors have been described. The expression of these receptors has never been studied, despite indirect evidence showing the presence of melatonin receptor in the murine immune system. At present, the MT₁ and MT₂ membrane receptors, and nuclear receptors belonging to the RZR/ROR family have been related to the immunomodulator effect of melatonin. Here, we show the presence of membrane and nuclear melatonin-binding sites in mouse thymus and spleen, using the specific melatonin membrane (S 20098) and nuclear (CGP 52608) receptor agonist. To confirm the presence of melatonin receptors, we analyzed the presence of membrane and nuclear receptor mRNA and protein by RT-PCR, Southern blot, and Western blot. Thus, we show that MT₁ and ROR receptor mRNA and protein are expressed in both thymus and spleen, while MT₂ receptor mRNA is only detected in the thymus. This expression of melatonin receptors strongly supports the idea of an immunomodulatory role of melatonin through its receptors.Received 2 June 2003; received after revision 6 August 2003; accepted 14 August 2003     

Proinsulin C-peptide and its analogues induce intracellular Ca2+ increases in human renal tubular cells

Based on the findings that proinsulin C-peptide binds specifically to cell membranes, we investigated the effects of C-peptide and related molecules on the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in human renal tubular cells using the indicator fura-2/AM. The results show that human C-peptide and its C-terminal pentapeptide (positions 27–31, EGSLQ), but not the des (27–31) C-peptide or randomly scrambled C-peptide, elicit a transient increase in [Ca²⁺]_i. Rat C-peptide and rat C-terminal pentapeptide also induce a [Ca²⁺]_i response in human tubular cells, while a human pentapeptide analogue with Ala at position 1 gives no [Ca²⁺]_i response, and those with Ala at positions 2–5 induce responses with different amplitudes. These results define a species cross-reactivity for C-peptide and demonstrate the importance of Glu at position 1 of the pentapeptide. Preincubation of cells with pertussis toxin abolishes the effect on [Ca²⁺]_i by both C-peptide and the pentapeptide. These results are compatible with previous data on C-peptide binding to cells and activation of Na⁺,K⁺ATPase. Combined, all data show that C-peptide is a bioactive peptide and suggest that it elicits changes in [Ca²⁺]_i via G-protein-coupled pathways, giving downstream enzyme effects. Received 13 May 2002; accepted 16 May 2002     

Intracellular trafficking of AMPA receptors in synaptic plasticity

Modification of ligand-gated receptor function at the postsynaptic domain is one of the most important mechanisms by which the efficacy of synaptic transmission in the nervous system is regulated. Traditionally, these types of modifications have been thought to be achieved mainly by altering the channel-gating properties or conductance of the receptors. However, recent evidence suggests that AMPA (α-amino-3-hydroxyl-5-methyl-4-isoxayolepropionic acid)-type ligand-gated glutamate receptors are continuously recycling between the plasma membrane and the intracellular compartments via vesicle-mediated plasma membrane insertion and clathrin-dependent endocytosis. Regulation of either receptor insertion or endocytosis results in a rapid change in the number of these receptors expressed on the plasma membrane surface and in the receptor-mediated responses, thereby playing an important role in mediating certain forms of synaptic plasticity. Thus, controlling the number of postsynaptic receptors by regulating the intracellular trafficking and plasma membrane expression of the postsynaptic receptors may be a common and important mechanism of synaptic plasticity in the mammalian central nervous system.     

Role of adenosine A1 and A2 receptors in the regulation of aldosterone production in rat adrenal glands

Summary To investigate the roles of adenosine A₁ and A₂ receptors in the regulation of aldosterone production, we examined the effects of adenosine and adenosine agonists (N⁶-cyclohexyl adenosine; selective adenosine A₁ receptor agonist and 5-N-ethylcarboxamine adenosine; selective adenosine A₂ receptor agonist) on aldosterone and cyclic AMP production in rat adrenal capsular cells. Neither adenosine nor 5-N-ethylcarboxamine adenosine caused significant effects on basal aldosterone or cyclic AMP production. Also, adenosine (10^–3M) showed no consistent effects on aldosterone and cyclic AMP production induced by ACTH. On the other hand, N⁶-cyclohexyl adenosine exhibited a significant inhibition of basal aldosterone and cyclic AMP production at doses of 10^–4 M and 10^–3 M; furthermore, 10^–3 M N⁶-cyclohexyl adenosine inhibited aldosterone and cyclic AMP production stimulated by ACTH. These results suggest that adenosine A₁ receptors are coupled to and inhibit adenylate cyclase and may be involved in the inhibition of aldosterone production.     

Differential effects of the serotonin receptors on cultured rat cerebral cortical neurons

Effects of serotonin (5-HT) on cerebral cortical neurons were examined by patch clamp techniques. 5-HT produced a variety of responses such as outward (19/73 patches/neurons), slow inward (15/73 patches/neurons), fast inward (8/73 patches/neurons), and mixed currents (initially fast inward deflection followed by an outward response: 2/73 patches/neurons), with a latency of 12 sec, 15 sec, 0 sec, and 0 sec respectively, at a holding potential of −60 mV in whole-cell patches. The fast inward currents were again evoked by a selective 5-HT3 receptor agonist, 1-(m-chlorophenyl)-biguanide hydrochloride (CPBG). In the cell-attached patch clamp configuration, 5-HT inside the patch pipette elicited single channel currents with slope conductances of 42 pS and 132 pS (4/42 patches/neurons). CPBG inside the patch pipette evoked inward single channel currents with a lower slope conductance of 41 pS (3/23 patches/neurons). In contrast, application of 5-HT or a 5-HT₂ receptor agonist, α-methyl-5-hydroxytryptamine-maleate, outside the patch pipette induced outward single channel currents with a major slope conductance of 140 pS (8/30 patches/neurons) or 135 pS (6/20 patches/neurons), respectively. These results indicate that the outward and fast inward currents may be mediated respectively by the 5-HT₂ receptor, which is coupled to a G-protein, and by the 5-HT₃ receptor, which contains the non-selective cation channel, and that the mixed type may be caused by both the 5-HT₂ and 5-HT₃ receptors. Received 27 September 1996; received after revision 4 November 1996; accepted 7 November 1996