1. Involvement of the V2 receptor in vasopressin-stimulated translocation of placental leucine aminopeptidase/oxytocinase in renal cells
Shinako Masuda, Masafumi Tsujimoto, Akira Hattori, Shinobu Miyazawa, Shigehiko Mizutani, Hideko Matsumoto, Yasuhiro Natori Eur J Biochem . 2003 May;270(9):1988-94. doi: 10.1046/j.1432-1033.2003.03570.x.
The placental leucine aminopeptidase (P-LAP)/oxytocinase is a membrane-bound enzyme thought to play an important role during pregnancy. In this study, we identified the presence of P-LAP protein in the renal distal tubules and collecting ducts. In rat NRK52E cells derived from renal tubules, P-LAP was localized mainly in the intracellular compartment. Upon the treatment of cells with 8-arginine-vasopressin (AVP), a significant increase in the surface level of P-LAP was observed. [deamino-Cys1, d-Arg8]-vasopressin (DDAVP), a specific V2 receptor agonist, increased the surface level of P-LAP, while [adamantaneacetyl1, O-Et-d-Tyr2, Val4, aminobutyryl6, Arg8,9]-vasopressin (AEAVP), a potent V2 receptor antagonist, blocked the AVP-stimulated enhancement. Moreover, reagents known to enhance the intracellular level of cAMP have also been shown to increase the surface level of P-LAP. When we examined the colocalization of P-LAP with the cell surface water channel aquaporin-2 (AQP-2) that is regulated by AVP, the P-LAP-containing vesicles had a relatively higher density than the AQP-2-containing vesicles, suggesting that P-LAP and AQP-2 are differently distributed in NRK52E cells. These results suggest that AVP induces the translocation of P-LAP via V2 receptor and P-LAP plays a role in the regulation of excessive AVP level in the renal collecting duct, acting as a negative feedback mechanism for the AVP action of regulating water reabsorption.
2. Oxytocin-induced contractions within rat and rabbit ejaculatory tissues are mediated by vasopressin V1A receptors and not oxytocin receptors
D Hurley, Vm Jackson, Cp Wayman, Rj Russell, J Gupta Br J Pharmacol . 2008 Sep;155(1):118-26. doi: 10.1038/bjp.2008.226.
Background and purpose:Oxytocin is believed to be involved in ejaculation by increasing sperm number and contracting ejaculatory tissues. However, oxytocin may mediate these effects via oxytocin or vasopressin (AVP) receptors. The aim of this study was to determine the effect of oxytocin and AVP on peripheral tissues involved in ejaculation and to identify the receptor subtype(s) involved.Experimental approach:Standard tissue bath techniques were used to measure isometric tension from tissues involved in ejaculation and erection.Key results:Oxytocin and AVP failed to elicit a tonic contractile response in rat and rabbit testes, vas deferens, epididymis, seminal vesicles and prostate. In contrast, oxytocin and AVP elicited large tonic contractions in erectile (corpus spongiosum and corpus cavernosum) and ejaculatory (prostatic urethra, bladder neck and ejaculatory duct) tissues in a concentration-dependent manner. The selective oxytocin agonist, [Thr4,Gly7]-oxytocin and the V2 agonist, [deamino-Cys1,Val4,D-Arg8]-vasopressin (dDAVP), failed to contract tissues. Oxytocin and AVP-induced contractions were weakly antagonized by the selective oxytocin antagonist, L-368899 but potently antagonized by the V1A antagonist, SR49059. The V1B antagonist SSR149415 failed to antagonize AVP contractions except in rabbit bladder neck. Neither L-368899 nor SR49059 antagonized endothelin-1-induced contractions.Conclusions and implications:The contractile effect of oxytocin on rat and rabbit ejaculatory and erectile tissues is mediated via V1A receptors. Endothelin-1-induced contractions are not due to endogenous oxytocin or AVP release. V1A receptor antagonists may have a therapeutic role in both erectile dysfunction and premature ejaculation.
3. Small-molecule vasopressin-2 receptor antagonist identified by a g-protein coupled receptor "pathway" screen
Buranee Yangthara, Aaron Mills, Lukmanee Tradtrantip, A S Verkman, Varanuj Chatsudthipong Mol Pharmacol . 2007 Jul;72(1):86-94. doi: 10.1124/mol.107.034496.
G-protein-coupled receptors (GPCRs) such as the vasopressin-2 receptor (V(2)R) are an important class of drug targets. We developed an efficient screen for GPCR-induced cAMP elevation using as read-out cAMP activation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels. Fischer rat thyroid cells expressing CFTR and a halide-sensing yellow fluorescent protein (H148Q/I152L) were transfected with V(2)R. Increased cell Cl(-) conductance after agonist-induced cAMP elevation was assayed using a plate reader from cell fluorescence after solution I(-) addition. The Z' factor for the assay was approximately 0.7 with the V(2)R agonist [deamino-Cys1, Val4, d-Arg8]-vasopressin (1 nM) as positive control. Primary screening of 50,000 small molecules yielded a novel, 5-aryl-4-benzoyl-3-hydroxy-1-(2-arylethyl)-2H-pyrrol-2-one class of V(2)R antagonists that are unrelated structurally to known V(2)R antagonists. The most potent compound, V(2)R(inh)-02, which was identified by screening 35 structural analogs, competitively inhibited V(2)R-induced cAMP elevation with K(i) value of approximately 70 nM and fully displaced radiolabeled vasopressin in binding experiments. V(2)R(inh)-02 did not inhibit forskolin or beta(2)-adrenergic receptor-induced cAMP production and was more than 50 times more potent for V(2)R than for V(1a)R. The favorable in vitro properties of the pyrrol-2-one antagonists suggests their potential usefulness in aquaretic applications. The CFTR-linked cAMP assay developed here is applicable for efficient, high-throughput identification of modulators of cAMP-coupled GPCRs.
