Polarized expression of the GFP-tagged rat V(1a) vasopressin receptor.

We investigated the targeting of the V(1a) receptor fused with the green fluorescence protein (V(1a)R-GFP) in polarized MDCK cells. Cells expressing V(1a)R-GFP displayed binding to vasopressin (AVP) and AVP-induced calcium responses, similar to cells expressing the wild-type V1a receptor. Interestingly, as with the wild-type V(1a)R, V(1a)R-GFP is preferentially distributed in the basolateral side of MDCK cells as monitored by confocal microscopy. Furthermore, AVP induced internalization of GFP-tagged receptors. Therefore, the GFP-tagged V(1a) receptor retains all the sorting signals of the wild-type receptor and offers an excellent system to elucidate the mechanisms of cell trafficking of V(1a) receptors.

Vasopressin stimulates tyrosine phosphorylation by activation of PKC in the rat smooth muscle cell line, A-10.

Arginine vasopressin (AVP)-induced tyrosine phosphorylation was studied in a rat smooth muscle cell line, A-10, by western blotting, using a monoclonal antibody against phosphotyrosine. AVP stimulated the phosphorylation of several cellular proteins of molecular mass 60-130 kDa in a time- and dose-dependent manner. Phosphorylation was mediated largely by V(1)receptor subtype since it was inhibited by selective V(1)antagonist and was only partially elicited by the V(2)agonist, desmopressin. Heterotrimeric G-proteins seemed to be involved in the phosphorylation mechanism because fluoraluminates, an activator of heterotrimeric G-proteins (and thus an uncoupler of the receptor-G-protein interaction) inhibited the AVP-induced phosphorylation. The protein kinase C (PKC) inhibitors: staurosporine, H7 and GF109203X are able to block the AVP-stimulated phosphorylation. The last of these has been shown to be one of the most selective inhibitors of PKC. These results indicate that PKC is upstream of the phosphorylation, a motion which is supported by the fact that the AVP-stimulated phosphorylation was downregulated by phorbol esters.

Trial of a community-based intervention to decrease infestation of Aedes aegypti mosquitoes in cement washbasins in El Progreso, Honduras.

Washbasins and metal drums are important sources of Aedes aegypti mosquitoes in much of Latin America. When manual cleaning was found to be ineffective in eliminating mosquito larvae in a community-based control programme in El Progreso, Honduras, it was decided to develop and evaluate an improved method of removing mosquito eggs based on commonly-available materials. The method, named La Untadita ('The Little Dab', in English), consists of five steps: mixing chlorine bleach and detergent to make a paste, applying the mixture to the walls of the container, waiting 10 min, scrubbing with a brush, and finally rinsing with water. A field trial of the Untadita was conducted in 13 peri-urban neighbourhoods. At the first post-intervention survey, in spite of high levels of exposure to the community-based intervention, high levels of knowledge regarding the Untadita and high levels of its reported use, little or no impact was discernable on mosquito larvae and pupae. The method was then modified by increasing the recommended quantities of bleach and detergent and simplifying the instructions. In the second post-intervention survey, knowledge of the steps and their order increased further; the intervention neighbourhoods had significantly fewer algae on washbasin walls, an indicator of more effective cleaning; and numbers of pupae and 3rd and 4th instar larvae were significantly lower than in untreated neighbourhoods. Effective promotion of the Untadita should be able to control mosquito infestation in many washbasins, especially those in frequent use, thus reducing the need for chemical and biological larvicides that may be either more costly or less acceptable to householders.

Immunolocalization of V1 vasopressin receptors in the rat kidney using anti-receptor antibodies.

By using immunocytochemical techniques we have been able to localize the V1 vasopressin receptor in the rat kidney. Immunoblotting using an antiserum raised against an affinity-purified vasopressin receptor showed a 55,000 daltons protein band that has a molecular mass similar to that of the liver V1 vasopressin receptor, as demonstrated by cross-linking studies. Immunoblotting of the antibody showed a band of 55,000 daltons in A-10 cells, which contains the V1 subtype, whereas it did not stain LLC-PK1 cells, which possess the V2 subtype, showing that the antibody recognizes the V1 vasopressin receptor. The immunostaining of kidney sections with this antiserum showed a strong reaction of the connecting tubules and cortical and medullary collecting ducts. The immunostaining pattern of connecting tubule and collecting duct cells was different, that is, the former showed a staining of both the apical and basal plasma membrane but also in the cytoplasm, whereas the latter showed a strong reaction mainly in the basolateral membrane. Immunostaining of consecutive serial sections with an antiserum raised against tissue kallikrein, an enzyme present exclusively in connecting tubules, and with the anti-receptor serum allowed us to show, for the first time, the presence of the vasopressin receptor in the connecting tubule cells and their absence in intercalated cells, the other cell type present in connecting tubules. These findings support experiments carried in the eighties on the release of renal tissue kallikrein by AVP.

Modulation of the affinity of the vasopressin receptor by magnesium ions.

The binding of [3H]vasopressin (AVP) and the 125I-labelled vasopressin antagonist (VP-AT) d(CH2)5[Tyr2(Me),Tyr9(NH2)]AVP to rat liver membranes was examined with or without the addition of milimolar concentrations of divalent cations. The binding of vasopressin was enhanced by Mg2+ and Co2+ and markedly decreased by EGTA. The addition of EGTA and Mg2+ together restored the binding to a value similar to that of Mg2+ alone. On the contrary, the addition of Mg2+, Co2+, EGTA, and the combination of EGTA and Mg2+ decreased the binding of VP-AT to rat liver membranes. Kinetic analyses showed that Mg2+ increased the Kd twofold for VP-AT; that is from 0.13 nM to 0.28 nM. Moreover, it showed that the receptor with or without the addition of Mg2+ consists of a single population of binding sites, indicating that the receptor is switched from a high affinity to a low affinity state for VP-AT in the presence of 10 mM Mg2+. GTP gamma S was unable to block the effect of Mg2+ on the binding of VP-AT. These results suggest that this divalent cation interacts with receptor itself producing a conformational changes which thus modulates the affinity of the receptor.