Solubilization of human platelet vasopressin receptors

The human platelet membrane receptor for vasopressin (AVP) has been solubilized with the cholic acid derivative detergent 3-( [3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate. Rapid and simple separation of free tritiated AVP ( [3H]AVP) from the solubilized receptor-hormone complex was done by filtration through polyethylenimine-treated filters. [3H]AVP binds to this soluble receptor with an equilibrium dissociation constant of 11.03 +/- 1.86 nM and a maximal number of binding sites = 288 +/- 66 fmol/mg protein while the corresponding values of the membrane-bound receptor are 1.62 +/- 0.21 nM and 237 +/- 38 fmol/mg of protein, respectively. The Ki value for native AVP derived from competition experiments is 11.02 +/- 2.05 nM for the soluble receptor. Competition experiments with specific vascular and renal antagonists confirm that the solubilized receptor belongs to the V1-vascular subtype.     

Human platelet vasopressin receptors

Specific saturable binding of 125I-arginine-vasopressin to human platelets is described. For ten normal volunteers the mean (+/- S.D.) KD is 5.6 nM (+/- 2.1) and the mean (+/- S.D.) Bmax is 115 fmoles/mg protein (+/- 30). Association studies indicate that equilibrium is reached after 90 minutes on ice. Pharmacological inhibition studies with analogues indicate that the platelet receptor is very similar to the kidney medulla receptor. The function of the receptor may involve serotonin release and platelet aggregation. Vasopressin binding to platelets should provide a readily means of assessing vasopressin receptor function in man.     

Interaction of heterologous fibrinogens with human platelet receptors.

The interaction of ADP-stimulated human platelets with human 125I-fibrinogen as well as with pig and bovine fibrinogens was analysed. It was found that the fibrinogens studied were bound to the same platelet receptors but the affinity of animal preparations was about half the value observed for human fibrinogen (in a homologous system).     

Immunocytochemical evidence for vasopressin receptors.

An electron microscopic study was made of mouse pituitaries immunocytochemically stained with anti-lysine vasopressin (LVP) as the primary antiserum in the unlabeled antibody peroxidase-anti-peroxidase procedure. Vasopressin (VP) was identified in the neurosecretory granules of the neural lobe which stained with peroxidase anti-peroxidase molecules. Electron density was induced in secretory granules of the pars intermedia (PI), both in the melanocyte stimulated hormone and ACTH cell types, probably indicating VP molecules attached to binding (receptor) sites. Omission of anti-LVP abolished staining both in the neural lobe and the PL Anti-LVP absorbed with antigen, by admixing with LVP, abolished staining in the neural lobe but not in the PI; according to optical density measurements the PI showed a +/- 22% staining increase over controls. Staining intensity in the PI probably reflects occupancy of binding (receptor) sites for VP. Exposure of PI granules to LVP before the usual staining sequence resulted in +/- 48% increased staining. In water-deprived mice with high endogenous VP titers, staining was +/- 33% and +/- 40% more intense than in normal mice. Solid phase absorbed and eluted antibodies to LVP provided additional proof that staining in both neural lobe and PI could be attributed to anti-LVP. Results indicate that binding or receptor sites for VP are located on secretory granules in the PL Possible physiological significance is discussed.     

Probes for vasopressin receptors. Attachment of affinity and fluorescent groups in vasopressin

Fluorescent, photoreactive, and biotinylated analogs of vasopressin have been prepared in which one of these three groups has been attached to a reactive amino group in either position 4 or position 7. Using solid phase methodology, we have synthesized two active parent compounds, [1-desamino,4-lysine,7-hydroxyproline]arginine vasopressin and [1-desamino,7-aminoproline]arginine vasopressin, and acylated them to obtain biotinyl, azidobenzoyl, and fluoresceinyl derivatives. We have also prepared analogs in which a "spacer arm" was inserted between lysine in position 4 and the marker group. Some of these derivatives have good antidiuretic activity and could be valuable probes in studying hormone-receptor interaction and in receptor visualization and purification.     

The human platelet vasopressin receptor identification by direct ultraviolet photoaffinity labeling

Tritiated vasopressin ([3H]AVP) was directly crosslinked to its human platelet receptor by using an ultraviolet irradiation procedure. After preincubation with [3H]AVP, the hydrodynamic parameters of the hormone-receptor complexes solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate were derived from Sephacryl S-300 superfine gel filtration and from sucrose density gradient ultracentrifugation experiments. The following values were obtained: Stoke's radius = 5.48 +/- 0.1 nm, apparent sedimentation coefficient = 5.55 +/- 0.1 S, and calculated molecular weight = 132,000. On sodium dodecyl sulfate-8% polyacrylamide slab gel electrophoresis under reducing conditions, [3H]AVP preferentially and specifically labeled a 125,000-dalton protein. The labeling of this protein was suppressed by addition of excess cold vasopressin, whereas angiotensin II did not inhibit incorporation of tritiated vasopressin in this protein. These results suggest that direct UV-photoaffinity labelling with [3H]AVP is a suitable tool for the purification of the human platelet vasopressin receptor.     

Signal transduction of V1-vascular vasopressin receptors.

This review covers the recent developments gained in the exploration of V1-vascular vasopressin (AVP) receptors. We examine the different radioligands available for the pharmacological characterization of these receptors. The immediate transmembrane signaling of V1-vascular AVP receptors involves ligand-receptor complex formation, receptor lateral mobility and internalization, coupling to a Gq protein, activation of phospholipases A2, C and D, translocation and activation of protein kinase C, production of inositol 1,4,5-triphosphate and 1,2-diacylglycerol, mobilization of intracellular calcium, alteration of intracellular pH with activation of the Na+/H+ exchanger, calmodulin activation and myosin light chain phosphorylation. The secondary nuclear signal mechanisms triggered by activation of V1-vascular AVP receptors includes tyrosine phosphorylation, induction of gene expression and protein synthesis.     

Lack of V1 vasopressin receptors in rabbit hepatocytes.

Vasopressin does not induce glycogenolysis in rabbit hepatocytes; glucagon, angiotensin, phenylephrine and ATP are as potent as with rat hepatocytes, whereas isoprenaline is nearly 10000 times more potent in the rabbit. Binding studies of [3H]vasopressin reveal the complete absence of specific vasopressin receptors on rabbit liver plasma membranes. We verified that vasopressin acts as an antidiuretic and vasopressor agent in the rabbit. We conclude that there is a selective lack of V1 vasopressin receptors in rabbit liver.     

Hypothesis for the receptors of human blood platelet aggregation and its inhibition by structure-activity relationship.

We tried to clarify the size and the common charge distribution of the inhibition or stimulation of human platelet aggregation by structure-activity relationship. Numerous inhibiting and stimulating agents were able to enter the receptors. Inhibitory receptor had recess of 14 x 12.5 A in diameter. Stimulatory receptor had recess of 11 x 12 A in diameter. In the recess, there were three charges, two negative and one positive in the inhibitory receptor, and one negative and two positive in the stimulatory receptor, respectively. Charge distributions and conformation of inhibiting or stimulating agents were similar for the inhibitory agents, prostaglandin I2 (PGI2), PGD2, PGE1 adenosine and isoproterenol and conformation of the stimulating agents, thromboxane A2 (TXA2), platelet activating factor (PAF), adenosine diphosphate (ADP) and adrenaline. Each molecule had 3-10 inhibiting and stimulating conformations. The ratio of the number of conformations for inhibition and stimulating of platelet aggregation was highest for PGI2 which showed the strongest inhibitory activity. TXA2 was opposite in both respects.     

Ligand binding properties of human galanin receptors

Abstract

The galanin receptor family comprises of three members, GalR1, GalR2 and GalR3, all belonging to the G-protein-couple receptor superfamily. All three receptors bind the peptide hormone galanin, but show distinctly different binding properties to other molecules and effects on intracellular signaling. To gain insight on the molecular basis of receptor subtype specificity, we have generated a three-dimensional model for each of the galanin receptors based on its homologs in the same family. We found significant differences in the organization of the binding pockets among the three types of receptors, which might be the key for specific molecular recognition of ligands. Through docking of fragments of the galanin peptide and a number of ligands, we investigated the involvement of transmembrane and loop residues in ligand interaction.     

Mediators of the mitogenic action of human V(1) vascular vasopressin receptors

Arginine vasopressin (AVP) activation of V(1) vascular receptors (V(1)Rs) stimulates cell growth and proliferation in different tissues via cellular signaling pathways that remain to be identified. To explore the intracellular mediators of the mitogenic action of V(1)R, Chinese hamster ovary (CHO) cells were stably transfected with the human V(1)R cDNA clone we isolated previously. We assessed AVP effects on kinase activation (immunoblotting with phosphospecific antibodies), DNA synthesis (tritiated thymidine uptake), cell cycle progression (flow cytometry analysis after nuclear labeling with propidium iodide), and cell proliferation (conversion of the colorimetric reagent MTS) in the presence or absence of various pathway inhibitors. AVP stimulation of V(1)Rs leads to the phosphorylation of several kinases, an increase in DNA synthesis, a progression through the S and G(2)-M phases of the cell cycle, and an increase in cell proliferation. The mediators of the mitogenic action of V(1)R activation included calcium mobilization, coupling to a G(q) protein, and the simultaneous and parallel activation of several kinases, mainly calcium/calmodulin-dependent kinase II, phosphatidylinositol 3 kinase, protein kinase C, and p42/p44 mitogen-activated protein kinase.