Heparin uncouples alpha 2-adrenoceptors from the Gi-protein in membranes of human platelets.
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ABSTRACT:
SUBMITTER: Willuweit B
PROVIDER: S-EPMC1148785 | biostudies-other | 1988 Feb
REPOSITORIES: biostudies-other
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Project description:Stimulation of G(q)-coupled receptors activates phospholipase C and is supposed to promote both intracellular Ca(2+) mobilization and protein kinase C (PKC) activation. We found that ADP-induced phosphorylation of pleckstrin, the main platelet substrate for PKC, was completely inhibited not only by an antagonist of the G(q)-coupled P2Y1 receptor but also upon blockade of the G(i)-coupled P2Y12 receptor. The role of G(i) on PKC regulation required stimulation of phosphatidylinositol 3-kinase rather than inhibition of adenylyl cyclase. P2Y12 antagonists also inhibited pleckstrin phosphorylation, Rap1b activation, and platelet aggregation induced upon G(q) stimulation by the thromboxane A(2) analogue U46619. Importantly, activation of phospholipase C and intracellular Ca(2+) mobilization occurred normally. Phorbol 12-myristate 13-acetate overcame the inhibitory effect of P2Y12 receptor blockade on PKC activation but not on Rap1b activation and platelet aggregation. By contrast, inhibition of diacylglycerol kinase restored both PKC and Rap1b activity and caused platelet aggregation. Stimulation of P2Y12 receptor or direct inhibition of diacylglycerol kinase potentiated the effect of membrane-permeable sn-1,2-dioctanoylglycerol on platelet aggregation and pleckstrin phosphorylation, in association with inhibition of its phosphorylation to phosphatidic acid. These results reveal a novel and unexpected role of the G(i)-coupled P2Y12 receptor in the regulation of diacylglycerol-mediated events in activated platelets.
| S-EPMC2661997 | biostudies-literature
Project description:Several polyanionic compounds antagonize the interaction between receptors and the G-proteins that regulate adenylate cyclase or K+ channels, possibly by binding to a basic stretch of the receptor that is proposed to mediate its interaction with the G-proteins. We have studied the effects of polyanions on the interaction between the liver alpha 1-adrenoceptor and the G-protein through which it stimulates polyphosphoinositide turnover. Heparin [concn. causing 50% of maximal effect (EC50) = 0.5 microM], Trypan Blue (EC50 7.1 microM) or suramin (EC50 2.1 microM) prevented formation of the high-affinity adrenaline-receptor-G-protein complex without affecting antagonist binding. After alkaline treatment of the membranes, previously reported to cause G-protein removal, binding of agonists was insensitive to both guanine nucleotides and heparin. We conclude that these polyanions uncouple the alpha 1-adrenoceptor from its G-protein, suggesting that similar coupling mechanisms may underlie receptor activation of the G-proteins that activate polyphosphoinositide hydrolysis and those that regulate adenylate cyclase. This action of heparin severely limits its utility as a selective antagonist of the Ins(1,4,5)P3 receptor in intact cells.
| S-EPMC1130523 | biostudies-other
Project description:α2-adrenoceptors, (α2A, α2B and α2C-subtypes), are Gi-coupled receptors. Central activation of brain α2A and α2C-adrenoceptors is the main site for α2-agonist mediated clinical responses in hypertension, ADHD, muscle spasm and ITU management of sedation, reduction in opiate requirements, nausea and delirium. However, despite having the same Gi-potency in functional assays, some α2-agonists also stimulate Gs-responses whilst others do not. This was investigated. Agonist responses to 49 different α-agonists were studied (CRE-gene transcription, cAMP, ERK1/2-phosphorylation and binding affinity) in CHO cells stably expressing the human α2A, α2B or α2C-adrenoceptor, enabling ligand intrinsic efficacy to be determined (binding KD /Gi-IC50 ). Ligands with high intrinsic efficacy (e.g., brimonidine and moxonidine at α2A) stimulated biphasic (Gi-Gs) concentration responses, however for ligands with low intrinsic efficacy (e.g., naphazoline), responses were monophasic (Gi-only). ERK1/2-phosphorylation responses appeared to be Gi-mediated. For Gs-mediated responses to be observed, both a system with high receptor reserve and high agonist intrinsic efficacy were required. From the Gi-mediated efficacy ratio, the degree of Gs-coupling could be predicted. The clinical relevance and precise receptor conformational changes that occur, given the structural diversity of compounds with high intrinsic efficacy, remains to be determined. Comparison with α1 and β1/β2-adrenoceptors demonstrated subclass affinity selectivity for some compounds (e.g., α2:dexmedetomidine, α1:A61603) whilst e.g., oxymetazoline had high affinity for both α2A and α1A-subtypes, compared to all others. Some compounds had subclass selectivity due to selective intrinsic efficacy (e.g., α2:brimonidine, α1:methoxamine/etilefrine). A detailed knowledge of these agonist characteristics is vital for improving computer-based deep-learning and drug design.
| S-EPMC9471048 | biostudies-literature
Project description:Highly selective drugs offer a way to minimize side-effects. For agonist ligands, this could be through highly selective affinity or highly selective efficacy, but this requires careful measurements of intrinsic efficacy. The α1-adrenoceptors are important clinical targets, and α1-agonists are used to manage hypotension, sedation, attention deficit hypersensitivity disorder (ADHD), and nasal decongestion. With 100 years of drug development, there are many structurally different compounds with which to study agonist selectivity. This study examined 62 α-agonists at the three human α1-adrenoceptor (α1A, α1B, and α1D) stably expressed in CHO cells. Affinity was measured using whole-cell 3 H-prazosin binding, while functional responses were measured for calcium mobilization, ERK1/2-phosphorylation, and cAMP accumulation. Efficacy ratios were used to rank compounds in order of intrinsic efficacy. Adrenaline, noradrenaline, and phenylephrine were highly efficacious α1-agonists at all three receptor subtypes. A61603 was the most selective agonist and its very high α1A-selectivity was due to selective α1A-affinity (>660-fold). There was no evidence of Gq-calcium versus ERK-phosphorylation biased signaling at the α1A, α1B, or α1D-adrenoceptors. There was little evidence for α1A calcium versus cAMP biased signaling, although there were suggestions of calcium versus cAMP bias the α1B-adrenoceptor. Comparisons of the rank order of ligand intrinsic efficacy suggest little evidence for selective intrinsic efficacy between the compounds, with perhaps the exception of dobutamine which may have some α1D-selective efficacy. There seems plenty of scope to develop affinity selective and intrinsic efficacy selective drugs for the α1-adrenoceptors in future.
| S-EPMC8343220 | biostudies-literature
Project description:α2-Adrenoceptors, subdivided into α2A, α2B, and α2C subtypes and expressed in heart, blood vessels, kidney, platelets and brain, are important for blood pressure, sedation, analgesia, and platelet aggregation. Brain α2C-adrenoceptor blockade has also been suggested to be beneficial for antipsychotic action. However, comparing α2-adrenoceptor subtype affinity is difficult due to significant species and methodology differences in published studies. Here, 3 H-rauwolscine whole cell binding was used to determine the affinity and selectivity of 99 α-antagonists (including antidepressants and antipsychotics) in CHO cells expressing human α2A, α2B, or α2C-adrenoceptors, using an identical method to β and α1-adrenoceptor measurements, thus allowing direct human receptor comparisons. Yohimbine, RX821002, RS79948, and atipamezole are high affinity non-selective α2-antagonists. BRL44408 was the most α2A-selective antagonist, although its α1A-affinity (81 nM) is only 9-fold greater than its α2C-affinity. MK-912 is the highest-affinity, most α2C-selective antagonist (0.15 nM α2C-affinity) although its α2C-selectivity is only 13-fold greater than at α2A. There are no truely α2B-selective antagonists. A few α-ligands with significant β-affinity were detected, for example, naftopidil where its clinical α1A-affinity is only 3-fold greater than off-target β2-affinity. Antidepressants (except mirtazapine) and first-generation antipsychotics have higher α1A than α2-adrenoceptor affinity but poor β-affinity. Second-generation antipsychotics varied widely in their α2-adrenoceptor affinity. Risperidone (9 nM) and paliperidone (14 nM) have the highest α2C-adrenoceptor affinity however this is only 5-fold selective over α2A, and both have a higher affinity for α1A (2 nM and 4 nM, respectively). So, despite a century of yohimbine use, and decades of α2-subtype studies, there remains plenty of scope to develop α2-subtype selective antagonists.
| S-EPMC8882856 | biostudies-literature
Project description:Mammalian neuroglobin (Ngb) protects neuronal cells under conditions of oxidative stress. We previously showed that human Ngb acts as a guanine nucleotide dissociation inhibitor (GDI) for the α-subunits of heterotrimeric Gi/o proteins and inhibits the decrease in cAMP concentration, leading to protection against cell death. In the present study, we used an eukaryotic expression vector driving high-level expression of human wild-type Ngb or Ngb mutants that either exhibit or lack GDI activities in human cells. We demonstrate that the GDI activity of human Ngb is tightly correlated with its neuroprotective activity. We further demonstrate that Glu53, Glu60, and Glu118 of human Ngb are crucial for both the neuroprotective activity and interaction with Gαi1. Moreover, we show that Lys46, Lys70, Arg208, Lys209, and Lys210 residues of Gαi1 are important for binding to human Ngb. We propose a molecular docking model of the complex between human Ngb and Gαi1.
| S-EPMC4842972 | biostudies-literature
Project description:UnlabelledThis review is based on the JR Vane Medal Lecture presented at the BPS Winter Meeting in December 2011 by J.C. McGrath. A recording of the lecture is included as supporting information. It covers his laboratory's work from 1990 to 2010 on the localization of vascular α1 -adrenoceptors in native tissues, mainly arteries.Main points(i) α1 -adrenoceptors are present on several cell types in arteries, not only on medial smooth muscle, but also on adventitial, endothelial and nerve cells; (ii) all three receptor subtypes (α1 A , α1 B , α1 D ) are capable of binding ligands at the cell surface, strongly indicating that they are capable of function and not merely expressed. (iii) all of these cell types can take up an antagonist ligand into the intracellular compartments to which endocytosing receptors move; (iv) each individual subtype can exist at the cell surface and intracellularly in the absence of the other subtypes. As functional pharmacological experiments show variations in the involvement of the different subtypes in contractions of different arteries, it is concluded that the presence and disposition of α1 -adrenoceptors in arteries is not a simple guide to their involvement in function. Similar locations of the subtypes, even in different cell types, suggest that differences between the distribution of subtypes in model systems do not directly correlate with those in native tissues. This review includes a historical summary of the alternative terms used for adrenoceptors (adrenergic receptors, adrenoreceptors) and the author's views on the use of colours to illustrate different items, given his partial colour-blindness.
| S-EPMC4337695 | biostudies-literature
Project description:Background & aimsColonic musculature contain smooth muscle cells (SMC), interstitial cells of Cajal (ICC), and platelet-derived growth factor receptor α+ cells (PDGFRα+ cells), which are electrically coupled and operate together as the SIP syncytium. PDGFRα+ cells have enriched expression of small conductance Ca2+-activated K+ (SK) channels. Purinergic enteric neural input activates SK channels in PDGFRα+ cells, hyperpolarizes SMC, and inhibits colonic contractions. Recently we discovered that PDGFRα+ cells in mouse colon have enriched expression of α1A adrenoceptors (ARs), which coupled to activation of SK channels and inhibited colonic motility, and α1A ARs were principal targets for sympathetic regulation of colonic motility. Here we investigated whether PDGFRα+ cells in human colon express α1A ARs and share the roles as targets for sympathetic regulation of colonic motility.MethodsIsometric tension recording, intracellular recording, and Ca2+ imaging were performed on muscles of the human colon. Responses to α1 ARs agonists or electric field stimulation with AR antagonists and neuroleptic reagents were studied.ResultsExogenous or endogenous norepinephrine released from nerve fibers inhibited colonic contractions through binding to α1A ARs or enhanced colonic contractions by acting on α1D ARs. Inhibitory responses were blocked by apamin, an antagonist of SK channels. Phenylephrine, α1 AR agonists, or norepinephrine increased intracellular [Ca2+] in PDGFRα+ cells, but not in ICC, and hyperpolarized SMCs by binding to α1 ARs expressed by PDGFRα+ cells.ConclusionsHuman colonic contractions are inhibited by α1A ARs expressed in PDGFRα+ cells and activated by α1D ARs expressed in SMC.
| S-EPMC7474159 | biostudies-literature
Project description:1. Zebrafish has five distinct alpha(2)-adrenoceptors. Two of these, alpha(2Da) and alpha(2Db), represent a duplicated, fourth alpha(2)-adrenoceptor subtype, while the others are orthologue of the human alpha(2A)-, alpha(2B)- and alpha(2C)-adrenoceptors. Here, we have compared the pharmacological properties of these receptors to infer structural determinants of ligand interactions. 2. The zebrafish alpha(2)-adrenoceptors were expressed in Chinese hamster ovary cells and tested in competitive ligand binding assays and in a functional assay (agonist-stimulated [(35)S]GTPgammaS binding). The affinity results were used to cluster the receptors and, separately, the ligands using both principal component analysis and binary trees. 3. The overall ligand binding characteristics, the order of potency and efficacy of the tested agonists and the G-protein coupling of the zebrafish and human alpha(2)-adrenoceptors, separated by approximately 350 million years of evolution, were found to be highly conserved. The binding affinities of the 20 tested ligands towards the zebrafish alpha(2)-adrenoceptors are generally comparable to those of their human counterparts, with a few compounds showing up to 40-fold affinity differences. 4. The alpha(2A) orthologues and the zebrafish alpha(2D) duplicates clustered as close pairs, but the relationships between the orthologues of alpha(2B) and alpha(2C) were not clearly defined. Applied to the ligands, our clustering methods segregated the ligands based on their chemical structures and functional properties. As the ligand binding pockets formed by the transmembrane helices show only minor differences among the alpha(2)-adrenoceptors, we suggest that the second extracellular loop--where significant sequence variability is located --might contribute significantly to the observed affinity differences.
| S-EPMC1575993 | biostudies-literature
Project description:A guanosine 5'-[gamma-[35S]thio]triphosphate-binding activity was detergent-extracted from Trypanosoma cruzi membranes. This binding activity was co-eluted from gel-filtration columns with a factor which, in a heterologous reconstitution system, blocks glucagon stimulation of adenylate cyclase activity in liver membranes. ADP-ribosylation of these membranes by pertussis toxin eliminated this blocking capacity. Incubation of T. cruzi membranes with activated pertussis toxin and [adenylate-32P]NAD+ led to the incorporation of radioactivity into a labelled product with an apparent M(r) of approx. 43,000. Crude membranes were electrophoresed on SDS/polyacrylamide gels and analysed, by Western blotting, with GA/1 anti-alpha common, AS/7 anti-alpha t, anti-alpha i1 and anti-alpha i2 polyclonal antibodies. These procedures led to the identification of a specific polypeptide band of about 43 kDa. Another polypeptide reacting with the SW/1 anti-beta antibody, of about 30 kDa, was also detected in the membrane fraction.
| S-EPMC1133185 | biostudies-other