Polarized expression of the vasopressin V2 receptor in Madin-Darby canine kidney cells.

Background: The vasopressin V2 receptor is expressed in the polarized principal cell of the renal collecting duct. Inactivating mutations of the vasopressin V2 receptor gene cause X-linked nephrogenic diabetes insipidus (NDI). Most of the mutant V2 receptors show transport defects, as analyzed in non-polarized cells, but data pertaining to polarized cells have not previously been presented.

A heterotrimeric G protein of the Gi family is required for cAMP-triggered trafficking of aquaporin 2 in kidney epithelial cells.

Vasopressin is the key regulator of water homeostasis in vertebrates. Central to its antidiuretic action in mammals is the redistribution of the water channel aquaporin 2 (AQP2) from intracellular vesicles to the apical membrane of kidney epithelial cells, an event initiated by an increase in cAMP and activation of protein kinase A. The subsequent steps of the signaling cascade are not known.

Vasopressin V2 receptor mutants that cause X-linked nephrogenic diabetes insipidus: analysis of expression, processing, and function.

We investigated the biochemical and functional properties of five vasopressin V2 receptor mutants (L44F, L44P, W164S, S167L, and S167T) that were recently described in families with a history of X-linked nephrogenic diabetes insipidus. COS.M6 cells transfected with cDNA encoding these mutants acquired < 4% specific [3H]arginine vasopressin (AVP) binding sites on the cell surface in comparison with cells transfected with cDNA coding for the wild-type receptor.

Properties of the human arginine vasopressin V2 receptor after site-directed mutagenesis of its putative palmitoylation site.

Most G-protein-coupled receptors have conserved cysteine residues in their C-terminal cytoplasmic domain that appear to be generally palmitoylated. An example is the human arginine vasopressin V2 receptor with cysteine residues at positions 341 and 342. Site-directed mutagenesis of the putative palmitoylation site was used to study the significance of palmitoylation for the V2 receptor.

Identification of Rab3-, Rab5a- and synaptobrevin II-like proteins in a preparation of rat kidney vesicles containing the vasopressin-regulated water channel.

According to the 'shuttle hypothesis', vasopressin increases the water permeability of renal epithelial cells by exocytotic fusion of vesicles containing the water channel AQP-CD with the apical plasma membrane, whereas withdrawal of vasopressin results in endocytotic uptake of AQP-CD. The proteins involved in the redistribution of AQP-CD have not been identified. With a panel of monoclonal antibodies, we detected Rab3-, Rab5a- and synaptobrevin II-like proteins in a kidney preparation enriched in AQP-CD-containing vesicles.

The protein tyrosine kinase pp60c-src is activated upon platelet stimulation.

Stimulation of human platelets causes a dramatic increase in phosphorylation of various proteins at tyrosine residues. The abundance of protein tyrosine kinases of the src-family in platelets, particularly pp60c-src, suggests an important role of these kinases in response to stimulation events. We have shown that pp60c-src is activated on agonist-induced platelet stimulation with respect to its substrate affinity.

Heparin-associated thrombocytopenia: the effects of various intravenous IgG preparations on antibody mediated platelet activation--a possible new indication for high dose i.v. IgG.

The immunologic type of heparin-associated thrombocytopenia (HAT) is caused by antibodies which activate platelets via the Fc-receptor in the presence of polysulfated oligosaccharides. The antigen is formed by a releasable platelet protein (in many cases PF4) complexed to heparin. Since the role of GP IIb/IIIa in platelet activation by HAT antibodies is controversial, we investigated platelet activation by antibodies related to HAT.

[Fc receptor-dependent platelet activation results independently of glycoprotein complex IIb/IIIa].

Platelets of a patient with Glanzmann's thrombasthenia revealed the same activation pattern when stimulated with antibodies of patients with heparin-associated thrombocytopenia (HAT) or immune complexes. This was investigated by the 14C-serotonin release test and by changes in phosphorylation of p20 and p47. Platelet activation by HAT antibodies was completely inhibited by a moab against the platelet FcRII (IV.

Substrate affinity of the protein tyrosine kinase pp60c-src is increased on thrombin stimulation of human platelets.

Human blood platelets contain high levels of non-receptor protein tyrosine kinases of the Src family, particularly pp60c-src, suggesting an important role for these enzymes in platelet physiology. Indeed, in response to various agonists of platelet function, a number of proteins become phosphorylated at tyrosine residues. However, no enzymic activation of an Src-related tyrosine kinase has yet been shown in platelets.

Heparin-associated thrombocytopenia: immune complexes are attached to the platelet membrane by the negative charge of highly sulphated oligosaccharides.

The interaction of sulphated oligosaccharides (SO) with platelets and the antibody of heparin-associated thrombocytopenia (HAT type II) was investigated. 3H-heparin binding to platelets was inhibited by different SO, depending on their grade of sulphation. Dextran sulphate, pentosan polysulphate, and heparin were more effective than were LMW heparins.