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PubMed1992

Structure-activity studies of the thrombin receptor activating peptide.

Cleavage of the human platelet thrombin receptor by thrombin exposes a new N-terminal which acts as a putative tethered ligand. A synthetic peptide--"SFLL" (SFLLRNPNDKYEPF), corresponding to the new N-terminal region, activates and induces platelet aggregation and serotonin secretion. We have found that the pentapeptide--SFLLR is the minimal peptide length which retains full activity in inducing [14C]serotonin secretion. Structure-activity relationship studies were performed on this pentameric peptide. Systematic replacement of all amino acids with L-Ala indicated the importance of F-2, L-3 and R-5 for activity. Further studies demonstrated that the positive charge at the N-terminus, but not at the C-terminus of the pentapeptide, is crucial for activity.

Amino Acid Sequence↗
PubMed1978

Analogous ultrastructure and surface properties during capping and phagocytosis in leukocytes.

Ultrastructural analyses have revealed striking similarities between Concanavalin A capping and phagocytosis in leukocytes. Both processes involve extensive membrane movement to form a protuberance or pseudopods; a dense network of microfilaments is recruited into both the protuberance and the pseudopods; microtubules are disassembled either generally (capping) or in the local region of the pseudopods (phagocytosis); and cells generally depleted of microtubules by colchicine show polarized phagocytosis via the microfilament-rich protuberance rather than uniform peripheral ingestion of particles via individual pseudopods. Cap formation can thus be viewed as occurring as an exaggeration of the same ultrastructural events that mediate phagocytosis. Similar changes in cell surface topography also accompany capping and phagocytosis. Thus, in nonfixed cells, Concanavalin A-receptor complexes aggregate into the region of the protuberance in colchicine-treated leukocytes (conventional capping) or into the region of pseudopod formation in phagocytizing leukocytes. In the latter case, the movement of lectin-receptor complexes occurs from membrane overlying peripheral microtubules into filament-rich pseudopods that exclude microtubules. These data provide evidence against a role for microtubules as "anchors" for lectin receptors. Rather, they indicate a preferential movement of cell surface Concanavalin A-receptor complexes towards areas of extensive (the protuberance) or localized (pseudopods) microfilament concentration. In conventional capping, Concanavalin A must be added to the colchicine-treated cells before fixation in order to demonstrate movement of receptors from a diffuse distribution into the protuberance. However, Convanavalin A receptors are enriched in the membrane associated with phagocytic particles as compared to the remaining membrane. This particle-induced redistribution of receptors is particularly prominent in colchicine-treated cells that phagocytize and are then fixed and Concanavalin A labeled; both lectin receptors and beads are concentrated over the protuberance. Thus, the final analogy between conventionally capped and phagocytic cells is that in both cases the properties of the plasma membrane in regions of microfilament concentration are modified by Concanavalin A itself (capping) or by the phagocytized particle, to limit locally the diffusion of Concanavalin A receptors.

Animals↗
PubMed2000

Presynaptic clustering of mGluR7a requires the PICK1 PDZ domain binding site.

Aggregation of neurotransmitter receptors at pre- and postsynaptic structures is crucial for efficient neuronal communication. In contrast to the wealth of information about postsynaptic specializations, little is known about the molecular organization of presynaptic membrane proteins. We show here that the metabotropic glutamate receptor mGluR7a, which localizes specifically to presynaptic active zones, interacts in vitro and in vivo with PICK1. Coexpression in heterologous systems induces coclustering dependent upon the extreme C terminus of mGluR7a and the PDZ domain of PICK1. mGluR7a and PICK1 localize to excitatory synapses in hippocampal neurons. Furthermore, whereas transfected mGluR7a clusters at presynaptic sites, mGluR7adelta3 lacking the PICK1 binding site targets to axons but does not cluster. These results suggest that PICK1 is a component of the presynaptic machinery involved in mGluR7a aggregation and in modulation of glutamate neurotransmission.

Animals↗
PubMed1995

Radioligand binding affinity and biological activity of the enantiomers of a chiral melatonin analogue.

Melatonin, a hormone secreted by the pineal gland, can act on the central circadian oscillator in the suprachiasmatic nucleus of the hypothalamus. It has been proposed that melatonin or its analogues may be useful in restoring disturbed circadian rhythms in jet-lag, shift-work and some blind subjects, and as sleep-promoting agents. In the present study, the (-)- and (+)-enantiomers of N-acetyl-4-aminomethyl-6-methoxy-9-methyl-1,2,3,4-tetrahydrocarbazole (AMMTC) were separated and tested. The affinity of the enantiomers at the specific 2-[125I]iodomelatonin binding site in chick brain membranes was compared in competition assays, and their biological activity in a specific melatonin receptor bioassay, aggregation of pigment granules in Xenopus laevis melanophores. The (-)-enantiomer of AMMTC was 130-fold and 230-fold more potent than the (+)-enantiomer in competition radioligand binding assays and melanophores, respectively. Both enantiomers are melatonin receptor agonists; (-)-AMMTC is slightly more potent than melatonin itself. As the tetrahydrocarbazole nucleus holds the C-3 amido side-chain of AMMTC in a restricted conformation, the analogues will be useful in modelling the melatonin receptor binding site.

Animals↗
PubMed1987

The single prostacyclin receptor of gel-filtered platelets provides a correlation with antiaggregatory potency of PGI2 mimics.

Gel-filtered human platelets (GFP) display only a single binding site for [3H]-PGI2: KD = 61nM, 234 fmol/10(8) platelets (1410 sites/platelet). Platelet-rich plasma (PRP) displays the same receptor density but the KD value increases to 123 nM due to protein binding of PGI2 which lowers its effective concentration. The [3H]-PGI2/GFP binding assay has been used to evaluate the molecular basis of aggregation inhibition for prostacyclin analogs and mimics, three PGE type structures, and PGD2. Antiaggregatory IC50s and radioligand binding IC50s correlate for PGE2, E1, and six PGI2 analogs. PGD2, and to a lesser extent 6-oxo-PGE1, display greater antiaggregatory potency than expected based on PGI2-binding site affinity data.

Binding Sites↗
PubMed2003

High-affinity thrombin receptor (PAR-1) ligands: a new generation of indole-based peptide mimetic antagonists with a basic amine at the C-terminus.

A new generation of indole-based peptide mimetics, bearing a basic amine at the C-terminus, was developed by the agency of two complementary, multistep, trityl resin-based approaches. Thus, we obtained several high-affinity thrombin receptor (PAR-1) ligands, such as 32 and 34. Compounds 32 and 34 were found to bind to PAR-1 with excellent affinity (IC(50)=25 and 35 nM, respectively) and to effectively block platelet aggregation induced by SFLLRN-NH(2) (TRAP-6) and alpha-thrombin.

Amines↗
PubMed2005

Overview of the P2 receptors.

The release of nucleotides in extracellular fluids can result from cell necrosis, exocytosis of secretory granules (such as platelet dense granules), or efflux through membrane channels. In addition, recent evidence suggests that vesicular trafficking is an important pathway of nucleotide release. Once in the extracellular fluids, they are rapidly degraded by ectonucleotidases, such as CD39, that play a key role in neutralizing the platelet aggregatory action of adenosine diphosphate (ADP), and act on two families of receptors: the ionotropic P2X receptors and the G-protein-coupled P2Y receptors. The family of P2X receptors encompasses seven genes. Currently, there are eight genuine P2Y receptors that can be subdivided into two structurally distinct subfamilies. Whereas P2X receptors are receptors of ATP, the different P2Y receptors are activated by distinct nucleotides, diphosphates or triphosphates, or purines or pyrimidines, some of them being conjugated to sugars. The study of knockout mice has demonstrated that P2X receptors play important roles in the neurogenic control of smooth muscle contraction, in pain and visceral perception, and in macrophage functions. The phenotype of P2Y null mice so far is more restricted: inhibition of platelet aggregation to ADP and increased bleeding time in P2Y (1)(-/-) and P2Y (12)(-/-) mice and lack of epithelial responsiveness to nucleotides in airways (P2Y (2)(-/-)) and intestine (P2Y (4)(0/-)).

Adenosine Diphosphate↗
PubMed2000

Detergent-resistant microdomains offer no refuge for proteins phosphorylated by the IgE receptor.

When the high affinity receptor for IgE and related receptors become aggregated, they emigrate to specialized microdomains of the plasma membrane that are enriched in certain lipids and lipid-anchored proteins. Among the latter are the kinases that initiate signaling cascade(s) by phosphorylating the receptors. In studying the IgE receptor, we explored whether, in addition to their potential role in enhancing the initiation of signaling by the kinase(s), the microdomains might augment the stimulation by excluding phosphatases. In vitro assessment of phosphatase activity, using either a relevant or irrelevant substrate, suggested that the microdomains were deficient in phosphatase activity, but, in vivo, proteins confined to the microdomains were found to be no less vulnerable to dephosphorylation than those outside such domains. In the course of our experiments, we observed that the procedures routinely used to isolate the detergent-resistant domains dissociated the receptor for IgE, thereby artificially accentuating the observed preferential distribution of phosphorylated subunits in the microdomains.

Adaptor Proteins, Signal Transducing↗
PubMed1989

Inhibitory effect of a selective thromboxane A2 receptor antagonist, EP 092, on platelet aggregation in whole blood ex vivo and in vivo.

1. The inhibitory effect of a selective prostaglandin H2 (PGH2)/thromboxane A2 receptor antagonist, EP 092, on platelet aggregatory responses in whole blood ex vivo (guinea-pig: Rhesus monkey) and intravascular aggregation in vivo (rabbit) has been investigated. 2. Collagen (0.1-10.0 micrograms ml-1) caused a concentration-dependent decrease in single platelet count in samples of both guinea-pig and Rhesus monkey citrated whole blood incubated ex vivo. EP 092 administered to guinea-pigs by intravenous (0.1-3.0 mg kg-1) or oral (1.0-10.0 mg kg-1) routes significantly inhibited the platelet responses to collagen (ED50 values 1.3 +/- 0.2 and 1.4 +/- 0.2 mg kg-1 respectively). Similar potency against collagen-induced whole blood aggregation was observed in Rhesus monkey blood samples following EP 092 given orally (ED50 0.9 +/- 0.3 mg kg-1). 3. The duration of action of EP 092 against collagen aggregatory responses ex vivo in both guinea-pigs and Rhesus monkeys was between 3 and 6 h following oral administration at 3.0 mg kg-1. 4. The inhibitory activity demonstrated by EP 092 against collagen-induced aggregation of Rhesus monkey whole blood ex vivo was not accompanied by any significant reduction in thromboxane A2 formation except at the highest dose tested (10 mg kg-1). 5. The intravascular aggregatory response induced by collagen or thrombin in the anaesthetized rabbit was significantly inhibited by an intravenous infusion of EP 092 (10 mg kg-1). EP 092 appeared less potent and its effect was of shorter duration in this preparation compared with its inhibitory effect on ex vivo aggregation, being evident immediately after infusion of drug but not after a further 30 min. 6. It is concluded that collagen-induced platelet aggregatory response in guinea-pig and Rhesus monkey whole blood ex vivo and rabbit in vivo exhibit a thromboxane-dependent component which can be inhibited in a dose-related fashion by pretreatment with the thromboxane antagonist EP 092. In the rabbit, moreover, the data support the possibility of a role for thromboxane in the intravascular aggregatory response to thrombin.

Administration, Oral↗
PubMed1998

Aggregation of the human high affinity immunoglobulin G receptor (FcgammaRI) activates both tyrosine kinase and G protein-coupled phosphoinositide 3-kinase isoforms.

Phosphoinositide 3-kinases (PI3-kinases) play an important role in the generation of lipid second messengers and the transduction of a myriad of biological responses. Distinct isoforms have been shown to be exclusively activated either by tyrosine kinase-coupled or G protein-coupled receptors. We show here, however, that certain nonclassical receptors can couple to both tyrosine kinase- and G protein-dependent isoforms of PI3-kinase: thus, aggregation of FcgammaRI, the human high affinity IgG receptor, on monocytes unusually leads to activation of both of these types of PI3-kinase. After aggregation of FcgammaRI, phosphatidylinositol 3,4, 5-triphosphate (PIP3) levels rise rapidly in interferon gamma-primed cells, reaching a peak within 30 sec. Moreover, and in contrast to the situation observed after stimulation of these cells with either insulin or ATP, which exclusively activate the tyrosine kinase- and G protein-coupled forms of PI3-kinase, respectively, PIP3 levels remain elevated up to 15 min after receptor aggregation. We show here that although the initial peak results from transient activation of the p85-dependent p110 isoform of PI-3kinase, presumably through recruitment of tyrosine kinases by the gamma chain, the later sustained rise of PIP3 results from activation of the G protein betagamma subunit-sensitive isoform, p110gamma. This finding indicates that receptors lacking an intrinsic signaling motif, such as FcgammaRI, can recruit both tyrosine kinase and G protein-coupled intracellular signaling molecules and thereby initiate cellular responses.

Adenosine Triphosphate↗
PubMed1983

Thrombin-induced protein phosphorylation in resting platelets and fibroblasts: evidence for common post-receptor molecular events.

We have investigated the thrombin-stimulated protein phosphorylation associated with the activation of two cellular processes: 1) reinitiation of DNA synthesis in G0-arrested hamster fibroblasts and 2) stimulation of serotonin release in platelets. We found a rapid 4- to 6-fold increased phosphorylation of a peptide with apparent Mr = 27,000 in SDS-PAGE. In both cell systems, the 27,000 dalton phosphopeptide is cytosolic, is resolved by isoelectric focusing as multiple variants (pHi 5 to 6), and thrombin stimulation generates the most acidic phosphorylated forms. This result and the fact that the 27,000 dalton peptide is also stimulated by a variety of growth factors in fibroblasts, strongly suggests that thrombin action in platelets and growth factor-induced mitogenesis, share common post-receptor molecular events.

Animals↗
PubMed1999

Internalization of the neurokinin 1 receptor in rat myenteric neurons.

Immunoreactivity for the neurokinin 1 receptor is contained in nerve cell bodies that have been deduced to be intrinsic primary afferent neurons in the myenteric plexus of the rat ileum. This study shows that neurokinin 1 receptor immunoreactivity on these neurons represents receptors that can bind agonist and undergo endocytosis, and explores the properties of that endocytosis. Segments of rat ileum were incubated in Hanks' balanced salt solution for 1 h at 4 degrees C, followed by 1 h at 37 degrees C in physiological saline solution with nicardipine and tetrodotoxin, in the presence or absence of substance P. Tissue was then fixed and whole-mount preparations were processed for fluorescence immunohistochemistry, using antibodies raised against the C-terminus of the neurokinin 1 receptor. The intracellular and surface distributions of receptor immunoreactivity were analysed using confocal microscopy and quantified by computer analysis. In tissue not exposed to substance P, most neurokinin 1 receptor immunoreactivity was confined to the surfaces of nerve cells, and 29% was intracellular. Exogenous substance P (10(-6) M) caused an increase in the amount of intracellular receptor to 72%. This internalization was concentration dependent, and maximum receptor internalization was achieved between 10(-6) M and 10(-5) M substance P (EC50 = 4.9 +/-1.6 x 10(-7) M). The specific neurokinin 1 receptor antagonist, SR104333 (10(-6) M), inhibited substance P-induced endocytosis. In tissue that was incubated in 5 x 10(-5) M monensin (to trap endocytosed receptor in the cell), without the addition of substance P, a high level of intracellular neurokinin 1 receptor immunoreactivity (81%) was also present. We deduce that endocytosis in the presence of monensin was stimulated by the release of tachykinins from intrinsic nerve endings, based on the following evidence: when endogenous release of tachykinin was blocked using a high magnesium/low calcium solution, or binding of tachykinins to the receptor was prevented using 10(-6) M SR140333, the intracellular receptor immunoreactivity remained at approximately 40%. Incubation with hypertonic sucrose also trapped receptors on the cell surface. Use of these protocols that modify receptor trafficking showed that agonist induced the neurokinin 1 receptors to aggregate, accumulate in endocytotic vesicles, move to perinuclear organelles and recycle to the surface in less than 1 h. This study indicates that there is sufficient release of endogenous tachykinins in the rat ileum to cause receptor internalization and implies that these intrinsic primary afferent neurons are likely to be under continuous influence from tachykinins in the normal intestine.

Animals↗
PubMed1983

Platelet alpha-adrenergic receptors in obesity: alteration with weight loss.

We studied platelet alpha-adrenergic receptor concentration and function in 19 subjects with simple obesity participating in a double-blind, controlled clinical trial of diet and anorexiants (phentermine, fenfluramine, or a combination of the two) or placebo. From wk 1 to wk 8, weight loss for the group as a whole was 4.9 +/- 0.7 kg (mean +/- SE). Concomitant with this weight loss, the platelet alpha-adrenergic receptor concentration rose from 85.7 +/- 5.8 to 113 +/- 5.8 fmol/mg protein. This increase moved the values for the obese subjects toward or beyond values in lean controls (100 +/- 10.5 fmol/mg protein). The response in the different treatment groups was similar. The receptor concentration increase was accompanied by a corresponding increase in alpha-adrenergic receptor-mediated platelet aggregation. For individual subjects the extent of weight loss over time generally correlated with percent receptor change. Altered adrenergic sensitivity occurring in obese subjects who are losing weight may have important implications in relation to external (therapeutic or inadvertent) administration of catecholamines.

Adult↗
PubMed1986

Influence of dihydrolysergic acid amide on serotonergic and alpha-adrenergic receptors in human blood platelets and femoral veins in vitro.

The effect of dihydrolysergic acid amide (DLSA) on responses of human blood platelets and isolated postmortem femoral veins to serotonin and catecholamines was studied in vitro in comparison to dihydroergotamine (DHE). DLSA inhibited the 5-HT-potentiated, ADP-induced platelet aggregation in approximately the same concentration range as DHE. It was three orders of magnitude less potent in inhibiting the adrenaline-induced aggregation and specific binding of 3H-yohimbine to intact platelets than DHE. In femoral vein strips, DLSA caused an increase in tone in the same concentration range (0.01-1.0 mumol/l) as DHE; however, its efficacy was somewhat lower. At concentrations of 0.1 to 1.0 mumol/l it antagonized the 5-HT-induced contractile response in a noncompetitive manner, the antagonist potency being one order of magnitude lower than that of DHE. DLSA (1 mumol/l) did not inhibit the noradrenaline-induced venoconstriction. The results show that DLSA possesses agonist activity in femoral veins and 5-HT-antagonist activity in platelets and veins.

Binding, Competitive↗
PubMed1988

The effect of inhibition of both diacylglycerol metabolism and phospholipase A2 activity on superoxide generation by human neutrophils.

A 'cocktail' consisting of an inhibitor of diacylglycerol kinase (R59022, 10 microM), an inhibitor of diacylglycerol lipase (RHC80267, 10 microM), and an inhibitor of phospholipase A2 (either 100 microM indomethacin, or 100 microM sodium meclofenamate) markedly enhanced superoxide production by human neutrophils stimulated with post-receptor stimuli, fluoride and gamma-hexachlorocyclohexane. On the other hand, the response to the C3b/Fc receptor stimulus, opsonized zymosan, was marginally decreased whilst that to the Fc receptor stimulus, aggregated IgG, was virtually unaffected. Since the inhibitors used are deemed to inhibit the main routes of arachidonate production, these results call into question the role of arachidonate in the transduction of O2- generation by post-receptor stimuli, but support a role for arachidonate in receptor-mediated transduction.

Cyclohexanones↗