Human platelet alpha 2-adrenergic receptors: effect of Na+ on interaction with the adenylate cyclase system and on epinephrine-stimulated platelet secretion.
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Adenylyl Cyclase Inhibitors↗
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Differential activation of MAP kinase family members triggered by CD99 engagement.
The molecular basis for the modulatory properties of CD99 is not well understood. Treatment of human Jurkat T lymphocytes with anti-CD99 antibody led to activation of three mitogen-activated protein kinase (MAPK) members, ERK, JNK, and p38 MAPK, along with homotypic aggregation. While phosphorylation of ERK and JNK was inhibited by the pretreatment of a PKC inhibitor, bisindolylmaleimide I, activation of p38 MAPK was upregulated by the same pretreatment. The signaling pathways to MAPKs by CD99 engagement were independent of PI-3 kinase, distinguishing from those by CD3 engagement. Among MAPKs, ERK pathway was essential for homotypic aggregation together with intracytoplasmic Ca(2+).
Antibodies, Monoclonal↗
Identification of alpha-adrenergic receptors in human platelets by [3H]dihydroergocryptine binding.
Binding of [(3)H]dihydroergocryptine to platelet lysates appears to have all the characteristics of binding to alpha-adrenergic receptors. At 25 degrees C binding reaches equilibrium within 20 min and is reversible upon addition of excess phentolamine. Binding is saturable with 183+/-22 fmol of [(3)H]dihydroergocryptine bound per mg of protein at saturation, corresponding to 220+/-26 sites per platelet. Kinetic and equilibrium studies indicate the dissociation constant of [(3)H]dihydroergocryptine for the receptors is 1-3 nM. The specificity of the binding sites is typical of an alpha-adrenergic receptor. Catecholamine agonists compete for occupancy of the [(3)H]dihydroergocryptine binding sites with an order of potency (-)epinephrine> (-)norepinephrine>> (-)isoproterenol. Stereospecificity was demonstrated inasmuch as the (+)isomers of epinephrine and norepinephrine were 10-20-fold less potent than the (-)isomers. The potent alpha-adrenergic antagonists phentolamine, phenoxybenzamine, and yohimbine competed potently for the sites, whereas beta-antagonists such as propranolol and dichlorisoproterenol were quite weak. Dopamine and serotonin competed only at high concentrations (0.1 mM). The [(3)H]dihydroergocryptine binding sites could also be demonstrated in intact platelets where they displayed comparable specificity, stereospecificity, and saturability. Saturation binding studies with the intact platelets indicated 220+/-45 receptors per platelet, in good agreement with the value derived from studies with platelet lysates. Ability of alpha-adrenergic agonists to inhibit adenylate cyclase and of alpha-adrenergic antagonists to antagonize this inhibitory effect directly paralleled ability to interact with the [(3)H]dihydroergocryptine binding sites. These data demonstrate the feasibility of directly studying alpha-adrenergic receptor binding sites in human platelets with [(3)H]dihydroergocryptine.
Adenylyl Cyclases↗
I-BOP, the most potent radiolabelled agonist for the TXA2/PGH2 receptor.
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Binding, Competitive↗
Acrosome reaction induced by immunoaggregation of a proteinase inhibitor bound to the murine sperm head.
ZP3, a glycoprotein of the murine zona pellucida, functions both to bind acrosome intact sperm and to induce the acrosome reaction. Solubilized whole zonae as well as purified ZP3 are able to induce acrosome reactions in capacitated sperm. Pronase digests of whole zonae yield glycopeptides that bind to sperm but are unable to induce acrosome reactions. However, immunoaggregation of these glycopeptides results in the exocytosis of the acrosome in the majority of treated sperm. The data suggest that ZP3 triggers the acrosome reaction by the aggregation of ZP3 binding sites on the sperm head. If aggregation of ZP3 binding sites is important in the induction of the acrosome reaction, then it may be possible to induce the acrosome reaction in the absence of zona by immunoaggregation of the sites. This presentation deals with the immunoaggregation of a proteinase inhibitor of seminal vesicle origin (SVI) that binds to a site on the sperm head known to participate in zona binding. We show that capacitated murine sperm, pretreated with the SVI, will acrosome react, as determined by Coomassie brilliant blue staining, when incubated with rabbit antiinhibitor antiserum (anti-SVI). The percentage of SVI-treated sperm displaying an acrosome reaction is dependent on the concentration of the immune serum. Sperm stain positive for intact acrosomes when anti-SVI Fab fragments or normal rabbit serum is substituted for the immune serum. However, when capacitated sperm, treated with both SVI and anti-SVI Fab fragments, are incubated with goat antirabbit IgG, the majority of sperm acrosome react. The data suggest that the aggregation of SVI bound to the sperm surface, in the absence of zona glycoproteins, is sufficient to induce the acrosome reaction.
Acrosome↗
Relative importance of diacylglycerol, phosphatidate, lysophosphatidate, inositol trisphosphate and arachidonate metabolism in platelet receptor signalling.
Activation of platelets is correlated with phospholipase C-induced degradation of phosphatidylinositol 4,5-bisphosphate and the rapid formation of 1,2-diacylglycerol and myo-inositol 1,4,5-trisphosphate. Both products are considered second messengers and they, respectively, stimulate protein kinase C and Ca2+ mobilization. Mobilization of Ca2+ leads to activation of a Ca2+/calmodulin-dependent myosin light chain kinase and phospholipases A2 which liberate arachidonic acid from phospholipids. Arachidonate is then immediately converted to active endoperoxides and thromboxanes which are released and activate further platelets again through phospholipase C. The levels of phosphatidic acid and lysophosphatidic acid are also increased following receptor-stimulated hydrolysis of the inositol phospholipids. Lysophosphatidic acid might have a direct action on the opening of Ca2+-channels.
Arachidonic Acid↗
Glycoprotein IIb/IIIa antagonists--from bench to practice.
The central role played by the alphaIIb beta3 receptor in platelet aggregation, and hence in platelet thrombosis, has led to the development of a number of parenteral and oral glycoprotein (GP) IIb/IIIa inhibitors for use in cardiovascular disease states, such as acute coronary syndromes and stroke. The predominant effect of these agents is to inhibit platelet aggregation, although studies of alphaIIb beta3 receptor function and various GP IIb/IIIa inhibitors have demonstrated the potential for these agents to produce effects on other aspects of platelet function, in addition to non-platelet effects. Overall, clinical studies have demonstrated an impressive beneficial effect for parenteral agents in reducing ischemic complications following percutaneous intervention, and a more modest beneficial effect in the treatment of patients with acute coronary syndromes. Trials with oral GP IIb/IIIa inhibitors in similar patient populations have demonstrated toxicity, manifested by an increased mortality in treated patients. Increased understanding of molecular aspects of both alphaIIb beta3 receptor function and the effects of GP IIb/IIIa inhibition may help explain some of the inconsistency in recently reported clinical studies with parenteral agents, and the frank toxicity of oral agents. Such studies may also hold the key to the development of newer agents with enhanced therapeutic benefit.
Angina, Unstable↗
Immune complexes and IgG-Fc receptors on human platelets in essential mixed cryoglobulinemia.
An enzyme-linked immunosorbent assay (ELISA) using wells coated with normal platelets has been tested in order to quantitate cell-bound immune complexes (ICs) by measuring the uptake of alkaline phosphatase-conjugated antibodies to IgG in the immune complexes. All patients with essential mixed cryoglobulinemia had levels of serum IgG ICs above control values; a poor accordance was found between levels of ICs detected by this method and those obtained using the conglutinin method. The presence of these complexes was correlated with human platelet aggregation: platelets aggregated in vitro when cryoglobulins with higher IgG/IgM ratio were present. This type of ICs could be critical also for platelet activation and aggregation in vivo.
Antigen-Antibody Complex↗
Genetic studies at the receptor level: investigations in human twins and experimental animals.
In receptors, as in enzymes, quantitative as well as qualitative genetic variation may exist. Studies in inbred strains of mice have shown for various receptors that the receptor density as determined by Bmax values is under genetic control. In healthy adult twins we have shown that the density of alpha-adrenoceptors on platelets is also influenced by genetic factors, since monozygotic twins were much more similar to one another than dizygotic twins. However, Bmax values are up-regulated and down-regulated by endogenous neurotransmitters and pharmacologically active agents. Thus, receptor densities are under considerable regulatory influences. Bmax values therefore reflect regulatory mechanisms rather than innate characteristics of the receptor protein. In another twin study we failed to find evidence for a genetic influence on the density of imipramine-binding sites on platelets. Since qualitative variation (polymorphism) is well known in enzymes, it may also apply to receptors. Qualitative differences in the receptor protein within one species would be of particular interest because of possible functional implications. As a first approach we examined central benzodiazepine receptors by photoaffinity labelling and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. A comparison of fish, frog, chicken, mouse, rat and calf led to the detection of variation between species. Investigations in five inbred mouse and rat strains have not so far revealed genetic variation in benzodiazepine receptors. Nevertheless variation may be detectable by more sensitive methods such as peptide mapping after limited proteolysis or two-dimensional electrophoresis.
Affinity Labels↗
Changes in the structure and function of the human thrombin receptor during receptor activation, internalization, and recycling.
According to current models, human thrombin receptors are activated when thrombin cleaves the receptor's N terminus, exposing the tethered ligand domain, SFLLRN. In the megakaryoblastic CHRF-288 cell line, thrombin receptor activation is followed by the rapid internalization of > 90% of the receptors. In the present studies, antibodies directed at the site of cleavage by thrombin were used to examine changes in receptor structure during activation, internalization, and recovery. As would be expected, the initial rate of receptor cleavage was directly related to the thrombin concentration. However, even after prolonged incubation, receptor cleavage was incomplete until the thrombin concentration exceeded the receptor concentration. Only cleaved receptors were internalized in response to thrombin and only catalytically active thrombin and active variants of SFLLRN-containing peptides caused receptor internalization. Over a 3-h period following receptor activation by thrombin, there was a gradual recovery of approximately one-quarter of the receptors on the cell surface. These receptors were detectable with antibodies directed at retained portions of the receptor N terminus, but not with antibodies directed at the proposed site of cleavage, confirming that they are recycled, rather than new, receptors. At 4 degrees C two-thirds of the receptors cleaved by thrombin were retained on the cell surface. Like recycled receptors these "cold-cleaved" receptors failed to self-activate when warmed to 37 degrees C, but could be activated by SFLLRN. Unlike recycled receptors, however, the cold-cleaved receptors were also internalized and appeared to be activated by a second addition of thrombin. These results 1) provide strong evidence at the protein level that thrombin cleaves its receptors at the predicted site, 2) show that receptor activation is necessary for internalization, 3) suggest that each thrombin molecule may not activate large numbers of receptors, 4) demonstrate that a substantial fraction of internalized thrombin receptors can be recycled, and 5) suggest that the failure of recycled receptors to be reactivated by thrombin may involve a change in the receptor that does not occur at 4 degrees C. Finally, the inability of cold-cleaved receptors to self activate in the absence of thrombin, suggests that in addition to cleaving the receptor, thrombin may also play an important role in guiding the tethered ligand domain to regions on the remainder of the receptor that mediate activation.
Amino Acid Sequence↗
Confocal imaging of N-methyl-D-aspartate receptors in living cortical neurons.
The fluorescence-conjugated N-methyl-D-aspartate receptor-selective antagonist, BODIPY-conantokin-G, was employed to label N-methyl-D-aspartate receptors in living neurons derived from the visual cortex of embryonic rats. The fluorescent labeling was visualized and analysed using confocal microscopy and digital imaging techniques. BODIPY-conantokin-G binding sites were homogeneously distributed across somata four days after neurons (E17-20) were placed in culture. In five-day-old cultures, BODIPY-conantokin-G binding sites became clusters of fluorescently labeled spots which were arranged irregularly on somata and proximal neurites. Distal neurites displayed fluorescent labeling after 10-15 days in culture. Displacement experiments showed that spermine and unlabeled conantokin-G compete with BODIPY-conantokin-G labeling at the N-methyl-D-aspartate receptor-associated polyamine site. The N-methyl-D-aspartate receptor antagonist 2-amino-5-phosphonovaleric acid also depressed the labeling but with a weaker effect, probably due to interactions occurring between the N-methyl-D-aspartate receptor agonist binding site and the polyamine modulatory site. The fluorescent dyes FM 1-43 and FM 4-64 were used in double-labeling studies to compare the distribution of nerve terminals with that of BODIPY-conantokin-G binding sites. BODIPY-conantokin-G binding clusters were associated with presynaptic nerve terminals while isolated BODIPY-conantokin-G binding sites were not always opposed to terminals. The aggregation of receptors to form clusters may lead to the functional formation of excitatory synapses. To investigate whether modulation of membrane potentials affected the formation of N-methyl-D-aspartate receptor clusters, cultured neurons were chronically treated for a week with either tetrodotoxin (to block membrane action potentials) or a high concentration of potassium to depolarize the membrane. While neurons in the tetrodotoxin-treated group showed a similar number of fluorescently labeled clusters compared with the control group, neurons in the high potassium group exhibited a higher number of fluorescently labeled receptor clusters. These results suggest that more active neurons may tend to form more N-methyl-D-aspartate synapses during early development.
Age Factors↗
Highly effective poly(ethylene glycol) architectures for specific inhibition of immune receptor activation.
Architectural features of synthetic ligands were systematically varied to optimize inhibition of mast cell degranulation initiated by multivalent crossing of IgE-receptor complexes. A series of ligands were generated by end-capping poly(ethylene glycol) (PEG) polymers and amine-based dendrimers with the hapten 2,4-dinitrophenyl (DNP). These were used to explore the influence of polymeric backbone length, valency, and hapten presentation on binding to anti-DNP IgE and inhibition of stimulated activation of RBL cells. Monovalent MPEG(5000)-DNP (IC(50) = 50 nM), bivalent DNP-PEG(3350)-DNP (IC(50) = 8 nM), bismonovalent MPEG(5000)-DNP(2) (IC(50) = 20 nM), bisbivalent DNP(2)-PEG(3350)-DNP(2) (IC(50) = 3nM) and DNP(4)-dendrimer ligands (IC(50) = 50 nM) all effectively inhibit cellular activation caused by multivalent antigen, DNP-bovine serum albumin. For different DNP ligands, we provide evidence for more effective inhibition due to (i) preferential formation of intra-IgE cross-links by bivalent ligands of sufficient length, (ii) self-association of monovalent ligands with longer tails, and (iii) higher probability of binding for bisvalent ligands. We also show that larger DNP(16)-dendrimers of higher valency trigger degranulation by cross-linking IgE-receptor complexes, whereas smaller DNP-dendrimers are inhibitory. Thus, features of synthetic ligands can be manipulated to control receptor occupation, aggregation, and inhibition of the cellular response.
Amines↗
Human lymphocyte subpopulations. Effect of corticosteroids.
Normal subjects given 60 mg of prednisone orally at 8:00 a.m. developed a transient lymphopenia at 2:00 p.m. To define the populations of lymphocytes affected the number and type of lymphocytes in the peripheral blood were assayed. "Late" and "early" spontaneous sheep red blood cell rosettes were used as markers for thymus-derived (T) lymphocytes and one of its subpopulations, respectively. Receptors for aggregated gammaglobulin and complement identified bursal-equivalent or bone marrow-derived (B) lymphocytes and one of its subpopulations, respectively. 6 h after administration of 60 mg of prednisone, the blood samples showed a decrease in proportion of T cells from 69.2 +/- 2.1% to 55.9 +/- 2.8% (average +/- SE) and an increase in B-cell proportion from 21.3 +/- 2.0% to 44.8 +/- 4.1%. The changes of "early" rosettes and complement receptor lymphocytes also paralleled these. In all cases the absolute numbers of T cells and of B cells were decreased by prednisone. The density gradient distribution of the lymphocytes did not change after prednisone. These data indicate that both T and B lymphocytes are affected by the prednisone but that the T cell lymphopenia was more pronounced. The lymphopenia might reflect either sequestration in the marrow and/or transient arrest of recirculation.
B-Lymphocytes↗
A molecular mechanism for signaling between seven-transmembrane receptors: evidence for a redistribution of G proteins.
Although activation of one seven-transmembrane receptor can influence the response of a separate seven-transmembrane receptor, e. g., the phenomenon of synergism, the underlying mechanism(s) for this signaling process is unclear. The present study investigated communication between two receptors that exhibit classical synergism, e.g., human platelet thrombin and thromboxane A2 receptors. Activation of thrombin receptors caused an increase in ligand affinity of thromboxane A2 receptors. This effect (i) was shown to be specific, since a similar increase in ligand affinity was not caused by ADP or A23187; (ii) did not require cytosolic components, e.g., kinases, proteases, phosphatases, etc., because it occurred in isolated platelet membranes; (iii) was G protein-mediated because it was blocked by an Galphaq C terminus antibody; and (iv) was associated with a net increase in Galphaq coupling to thromboxane A2 receptors. Collectively, these data provide evidence that seven-transmembrane receptors that share a common Galpha subunit can communicate with each other via a redistribution of their G proteins. Thus, activation of thrombin receptors increases Galphaq association with thromboxane A2 receptors thereby shifting them to a higher affinity state. This signaling phenomenon, which modulates receptor-ligand affinity, may serve as a molecular mechanism for cellular adaptive processes such as synergism.
15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
Disaggregation of aggregated platelets by savignygrin, a alphaIIbeta3 antagonist from Ornithodoros savignyi.
Ticks control their host's hemostatic system by secretion of bioactive components during feeding that inhibit blood coagulation and platelet aggregation. Dissolution of platelets that have already aggregated can enhance control over the hemostatic system. It has been shown that disaggregation of aggregated platelets by the enzyme apyrase was accompanied by a shape change from the aggregated spherical form back to the discoid form associated with un-activated platelets. The present study concerns the disaggregation effect of the alpha IIb/beta3 antagonist, savignygrin. Aggregated platelets that were disaggregated by savignygrin and platelets pre-incubated with savignygrin before activation with ADP, retained a spherical form similar to platelets disaggregated by the fibrinogenolytic enzyme plasmin. The number of pseudopods were however, markedly reduced suggesting a disruption of the focal adhesion points that act as a localization point of alpha IIb/beta3. These results are concurrent with targeting of alpha IIb/beta3 and dissociation of fibrinogen from its receptor, once aggregation has taken place. This is the second mediator of platelet disaggregation found in soft ticks and suggests that disaggregation of aggregated platelets might play an important part in the anti-hemostatic strategy of ticks.
Animals↗
Evaluation of proteinase-activated receptor-1 (PAR1) agonists and antagonists using a cultured cell receptor desensitization assay: activation of PAR2 by PAR1-targeted ligands.
We developed a calcium signaling-based assay, using cultured human embryonic kidney cells (HEK), that evaluates simultaneously, the activation/desensitization or blockade of the proteinase-activated receptors, PAR1 and PAR2. Using this assay, we analyzed the actions of a number of previously described putative PAR1-targeted peptide agonists and antagonists. We found that most of the previously described PAR1-targeted agents can also activate/desensitize PAR2, and most of these peptides can also activate a calcium signaling pathway in a target cell that possesses PAR2 along with PAR1. Furthermore, we used this assay to develop a PAR1 receptor-activating probe [Ala-parafluoroPhe-Arg-Cha-Cit-Tyr-NH2 (Cit-NH2)], which displays a high degree of specificity for PAR1 over PAR2, and we used the assay to quantitate the ability of trypsin to disarm the activation of PAR1 by thrombin. The abilities of the PAR1-targeted agents to desensitize or block PAR1 in the HEK cell assay were compared with their activities in a human platelet aggregation assay. Our data illustrate the usefulness of the HEK cell assay for evaluating the PAR1/PAR2 selectivity of PAR-activating agonists. The PAR1-selective agonist that we developed using the assay should prove useful for studying the effects of selectively activating PAR1 in vivo.
Calcium↗
Intracellular cyclic AMP not calcium, determines the direction of vesicle movement in melanophores: direct measurement by fluorescence ratio imaging.
Intracellular movement of vesiculated pigment granules in angelfish melanophores is regulated by a signalling pathway that triggers kinesin and dyneinlike microtubule motor proteins. We have tested the relative importance of intracellular Ca2+ ([Ca2+]i) vs cAMP ([cAMP]i) in the control of such motility by adrenergic agonists, using fluorescence ratio imaging and many ways to artificially stimulate or suppress signals in these pathways. Fura-2 imaging reported a [Ca2+]i elevation accompanying pigment aggregation, but this increase was not essential since movement was not induced with the calcium ionophore, ionomycin, nor was movement blocked when the increases were suppressed by withdrawal of extracellular Ca2+ or loading of intracellular BAPTA. The phosphatase inhibitor, okadaic acid, blocked aggregation and induced dispersion at concentrations that suggested that the protein phosphatase PP-1 or PP-2A was continuously turning phosphate over during intracellular motility. cAMP was monitored dynamically in single living cells by microinjecting cAMP-dependent kinase in which the catalytic and regulatory subunits were labeled with fluorescein and rhodamine respectively (Adams et al., 1991. Nature (Lond.). 349:694-697). Ratio imaging of F1CRhR showed that the alpha 2-adrenergic receptor-mediated aggregation was accompanied by a dose-dependent decrease in [cAMP]i. The decrease in [cAMP]i was both necessary and sufficient for aggregation, since cAMP analogs or microinjected free catalytic subunit of A kinase-blocked aggregation or caused dispersal, whereas the cAMP antagonist RpcAMPs or the microinjection of the specific kinase inhibitor PKI5-24 amide induced aggregation. Our conclusion that cAMP, not calcium, controls bidirectional microtubule dependent motility in melanophores might be relevant to other instances of non-muscle cell motility.
Adrenergic alpha-Antagonists↗
The opiate receptor: a single 110 kDa recognition molecule appears to be conserved in Tetrahymena, leech, and rat.
We compared the molecular nature of the rat brain opiate receptor with that of the invertebrate leech, Haemopis marmorata, and the protozoan, Tetrahymena, in order to examine the issue of apparent receptor heterogeneity with respect to biochemical structure. A binding study with rat brain membrane verified that [125I]beta-endorphin [( 125I]beta E), a broad specificity ligand, is displaced by the antagonist (-)-naloxone, but not the inactive stereoisomer (+)-naloxone; agonists considered prototypes for mu, delta, and kappa opiate receptors all displayed stereospecific binding displacement. For SDS-PAGE analysis of the opiate receptor [125I]beta-endorphin was covalently affixed to its recognition molecule with the cross-linking reagent DSS. Primary reaction products occur at 110, 58/55, and 29 kDa. Cross-linking products of all 3 molecular weights are effectively reversed by opiate ligands, regardless of their mu, delta, or kappa specificities. Peptide mapping studies in SDS gels, using limited proteolysis, showed that the 110 kDa band can be digested into 58 and 29 kDa fragments and the 58 kDa band into a 29 kDa fragment. Additional smaller molecular weight fragments were generated from the 110, 58/55, and 29 kDa bands which shared their molecular weights. Two possible explanations for the extensive sequence homology between the three major cross-linking products are: (1) the 110 kDa species is the opiate receptor, and the 58 and 29 kDa species are proteolytic fragments; and (2) one of the lower molecular weight species is the opiate receptor, and adjacent receptors are aggregated into the 110 kDa complex through cross-linking.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗