Adenosine diphosphate (ADP) plays a crucial role in hemostasis and thrombosis by activating platelets. In platelets, the classical P2T receptor is now resolved into three P2 receptor subtypes: the P2Y1, the P2Y12, and the P2X1 receptors. Both pharmacological and molecular biological approaches have confirmed the role of the P2Y1 and P2Y12 receptors in the ADP-induced platelet fibrinogen receptor activation. The P2Y1 and the P2X1 receptors independently contribute to platelet shape change. Whereas the P2Y12 receptor mediates the potentiation of dense granule release reaction, both the P2Y1 and P2Y12 receptors play an important role in the ADP-induced phospholipase A2 activation. The signaling events downstream of these receptors leading to the physiological effects remain elusive, and they are yet to be delineated. KEY WORDS: platelets, P2X1 receptor, P2Y1 receptor, P2T receptor, P2Y12 receptor, ADP receptor, platelet aggregation DOMAINS: thrombosis, signaling, hematology INTRODUCTIONPlatelets aggregate at the site of vascular damage to form a hemostatic plug. Abnormal activation of platelets leads to thrombosis, which in turn leads to stroke and myocardial infarction [1]. Adhesion of platelets to subendothelium results in the release, generation, or exposure of agonists that can activate platelets in a positive-feedback loop. Among these agonists are collagen (exposed), thrombin and thromboxane A 2 (generated), and ADP (adenosine diphosphate), epinephrine, and serotonin (released) [2]. ADP was identified as a platelet agonist [3], which is released upon activation of platelets along with ATP (adenosine triphosphate) and serotonin [4]. The importance of ADP in platelet activation is substantiated by bleeding diathesis in patients with deficiencies in storage of ADP, in mechanisms of secretion, or in ADP receptors [5,6,7,8,9]. Kunapuli: Platelet P2 Receptor Subtypes TheScientificWorldJOURNAL (2002) 2, 424-433 425 NOMENCLATURE OF P2 RECEPTORSReceptors for nucleotides, designated P2 receptors, are divided into two main classes: ligandgated ion channels (P2X) and G protein-coupled receptors (P2Y) [10]. Initially, all the physiological and intracellular signaling events triggered by ADP in platelets were attributed to a single cell surface receptor, designated P2T (thrombocyte P2 receptors) [11]. The historic studies and theories on the nature of the P2T receptor have been dealt in recent review articles [12,13,14]. ADP RECEPTOR-COUPLED EFFECTOR SYSTEMS IN PLATELETSADP has several effects on the receptor-coupled effector systems in platelets:• ADP regulates several second messenger systems in platelets by acting on cell surface P2 receptors [12,13,14,15].• ADP causes rapid calcium influx into platelets in the presence of physiological extracellular calcium ion concentrations [16,17].• ADP activates platelet phospholipase C (PLC), resulting in inositol 1,4,5-trisphosphate formation that leads to mobilization of calcium from intracellular stores [18,19,20].• ADP inhibits stimulated platelet adenylyl cyclase throug...
Abstract:Drugs that influence tubulin function were used to investigate the role of microtubules in hexose uptake by C6 glioma cells. In C6 cells, colchicine and vinblastine (which inhibit tubulin polymerization) inhibited radioactive ['H]2-deoxy-D-glucose uptake by about 30%". Paclitaxel (which promotes tubulin polymerization) stimulated hexose uptake by about 25%. To further demonstrate that microtubules play a role in hexose uptake, C6 cells were transfected with GLUT1 cDNA and then challenged with 100 nM paclitaxel. In GLUTI-transfected cells paclitaxel stimulated 2-deoxy-D-glucose uptake by about 35%. To study the role of tubulin in agonist-stimulated hexose uptake, the effect of colchicine on carbachol-induced uptake was next examined. Hexose uptake was increased with carbachol in concentration-dependent manner which was abolished by pretreatment with colchicine. To examine the specificity of the inhibitory effect of colchicine on G protein-mediated signal transduction pathway, the influence of colchicine on insulin (which acts via tyrosine kinase pathway) stimulation of 2-deoxy-D-glucose uptake was investigated. Hexose uptake was increased by insulin in a concentration-dependent manner which was unaffected by pretreatment with colchicine. These results suggest that microtubules are involved in basal and carbachol-stimulated glucose uptake by C6 cells.Astrocytes are non-neuronal cells. Astrocytes are stellate or star-shaped cells bearing long cytoplasmic processes. Astrocytes play structural and supporting roles in the CNS. It is estimated that astrocytes comprise 25-50% of the total volume in some brain areas. Cell bodies or processes of neurones, individual or clusters of synapses are surrounded by astrocytic processes. These relationships suggest a structural as well as a functional role for astrocytes in the CNS (Kimelberg 1995). Some of the functions of astrocytes are: guidance of neuronal migration during development, production and excretion of extracellular matrix proteins and adhesion molecules, production of neurotrophic and neurite promoting factors, cerebral angiogenesis, induction and maintenance of blood-brain barrier characteristics, neurotransmission, regulation of pH, ion concentration, osmolarity, detoxification, phagocytosis, immune functions, macromolecular translocation, and neuroendocrine functions. Among the cells in the CNS astrocytes are the only ones containing glycogen. This system is important for providing glucose for neuronal metabolism (Montgomery 1994). Rat astrocytes as well as C6 glioma cells (derived from rat tumors induced by N-nitrosomethylurea, and similar to the rat astrocytes) are well established models to study glucose metabolism (Singh et al. 1990;Wei & Yeh 1991). Although the physiological modulator of glucose uptake by brain cells is not identified, it was demonstrated that insulin and carbachol stimulated glucose uptake by astrocytes and C6 glioma cells, respectively (Singh et al.
In order to study Gq-tubulin interaction in the cytosol, GH3 and AtT-20 cells (stably expressing TRH receptor) were transiently transfected with Gq alpha cDNA. Forty-eight hours after transfection, thyrotropin-releasing hormone (TRH)-stimulated prolactin (PRL) secretion by Gq alpha-transfected GH3 cells increased by 90% compared to mock-transfected cells. In addition, using immunocytochemistry it was observed that Gq alpha-specific staining was much more prominent in Gq alpha-transfected GH3 and AtT-20 cells (also transfected with Gq alpha) compared to mock-transfected cells. Thus, transfection resulted in successful overexpression of functional Gq alpha. Forty-eight hours after transfection, cells were processed to obtain soluble and polymerized tubulin fractions. Tubulin levels were determined in these fractions by immunoblotting using polyclonal anti-tubulin antibodies. Compared to mock-transfected cells soluble tubulin levels decreased in Gq alpha-transfected GH3 and AtT-20 cells, by 33 and 52%, respectively. Moreover, compared to mock-transfected cells a 50% reduction in the ratio (an index of the flux between tubulin pools) of soluble and polymerized tubulin levels was observed in Gq alpha-transfected GH3 and AtT-20 cells. To determine whether these effects on tubulin were mediated by Gq directly, we examined the influence of purified Gq on tubulin polymerization. Gq (0.5 microM) inhibited polymerization of crude tubulin (present in GH3 cell cytosol) by 53%. In contrast to its effects on GH3 cell cytosol tubulin, Gq stimulated purified tubulin polymerization by 160%. These results suggest that Gq modulates the polymerization and depolymerization cycles of tubulin and that this modulation is in turn influenced by other unknown cellular components.
SummaryHuman high molecular weight kininogen (HK), a single chain plasma glycoprotein, serves as a cofactor in the contact system of blood coagulation. After cleavage by human plasma kallikrein, the nonapeptide bradykinin is released. The HK light chain (LC) contains coagulant activity, which requires both the ability to bind the contact system zymogens, prekallikrein and factor XI, and the ability to interact with negatively charged surfaces. Since bacterial expression might not be successful if carbohydrate was required for activity, we evaluated that possibility by incubating plasma HK with endoglycosydase F. Although the procedure removed detectable N-linked carbohydrate, no change in specific activity occurred. We then developed a bacterial expression system to produce recombinant HK LC. The cDNA coding for the HK LC was prepared by polymerase chain reaction (PCR), digested with restriction enzymes EcoRI and PstI, and introduced into the bacterial expression vector pKK223-3. E. coli harboring this recombinant plasmid (pSKl) expressed HK LC upon induction with isopropylthio-galactoside (IPTG). The recombinant protein (27 kDa), when transferred onto a PVDF membrane, was recognized by monospecific polyclonal anti-HK LC-antibodies. The recombinant HK LC was purified by heparin agarose affinity chromatography to homogeneity and found to have a specific activity of 28 coagulant units per mg protein, similar to the specific activity of the LC derived by proteolytic digestion of human plasma HK. We conclude: 1) The HK LC synthesized in bacteria is biologically active, and 2) the 40% carbohydrate content of the HK LC is not required for its cofactor activity.
In order to study Gq-tubulin interaction in the cytosol, GH3 and AtT-20 cells (stably expressing TRH receptor) were transiently transfected with Gq alpha cDNA. Forty-eight hours after transfection, thyrotropin-releasing hormone (TRH)-stimulated prolactin (PRL) secretion by Gq alpha-transfected GH3 cells increased by 90% compared to mock-transfected cells. In addition, using immunocytochemistry it was observed that Gq alpha-specific staining was much more prominent in Gq alpha-transfected GH3 and AtT-20 cells (also transfected with Gq alpha) compared to mock-transfected cells. Thus, transfection resulted in successful overexpression of functional Gq alpha. Forty-eight hours after transfection, cells were processed to obtain soluble and polymerized tubulin fractions. Tubulin levels were determined in these fractions by immunoblotting using polyclonal anti-tubulin antibodies. Compared to mock-transfected cells soluble tubulin levels decreased in Gq alpha-transfected GH3 and AtT-20 cells, by 33 and 52%, respectively. Moreover, compared to mock-transfected cells a 50% reduction in the ratio (an index of the flux between tubulin pools) of soluble and polymerized tubulin levels was observed in Gq alpha-transfected GH3 and AtT-20 cells. To determine whether these effects on tubulin were mediated by Gq directly, we examined the influence of purified Gq on tubulin polymerization. Gq (0.5 microM) inhibited polymerization of crude tubulin (present in GH3 cell cytosol) by 53%. In contrast to its effects on GH3 cell cytosol tubulin, Gq stimulated purified tubulin polymerization by 160%. These results suggest that Gq modulates the polymerization and depolymerization cycles of tubulin and that this modulation is in turn influenced by other unknown cellular components.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.