Biotinyl-(Arg8)-Vasopressin

One attomole of [Arg8]-vasopressin (AVP) was detected by a novel noncompetitive enzyme immunoassay (hetero-two-site complex transfer enzyme immunoassay). AVP was indirectly biotinylated using N-hydroxysuccinimidobiotin and trapped onto an anti-AVP IgG-coated polystyrene ball. After washing, biotinylated AVP was eluted from the polystyrene ball with HCl and was reacted with 2,4-dinitrophenyl-fluorescein disulfide-bovine serum albumin-rabbit anti-AVP IgG conjugate. The complex formed was trapped on [anti-2,4-dinitrophenyl group] IgG-coated polystyrene balls and, after washing, reacted with avidin-beta-D-galactosidase conjugate. The polystyrene balls were washed, and the complex of the three components was eluted with 2,4-dinitrophenyl-L-lysine and transferred to anti-fluorescein IgG-coated polystyrene balls. After washing, the complex was released from the polystyrene balls by reduction with 2-mercaptoethylamine and transferred to [anti-rabbit IgG] IgG-coated polystyrene balls. beta-D-Galactosidase activity bound to the last polystyrene balls was assayed by fluorometry. The detection limit of AVP was 1.1 fg (1 amol)/tube. Interference by proteins in biological fluids was eliminated by separation of peptides from proteins using a molecular sieve. The principle of the present method may be applicable to the measurement of haptens, including peptides, that can be derivatized so as to be bound simultaneously by both anti-hapten antibody and avidin molecules.

The neurohypophysial peptide hormone [Arg8]vasopressin (AVP) has well documented pressor effects in the periphery. These are mediated by vasopressin receptors (VPRs) of the V1a subtype, expressed by vascular smooth muscle cells, which induce vascular contraction when activated. AVP also has effects on the vasculature of the brain, where it has been reported to induce both vasodilation and vasoconstriction. The responsiveness of blood vessels of the spinal cord, however, has received little attention. To determine the morphology and distribution of blood vessels within the spinal cord, vessels were vizualised using a mouse anti-rat smooth muscle alpha actin IgG as primary antibody and fluorescein isothiocyanate-conjugated anti-mouse IgG secondary antibodies. A complementary vizualisation strategy which detected the endogenous peroxidase activity of red blood cells within vessels was also utilised. The characteristics of the structures observed using both visualisation strategies were typical of blood vessels. VPRs were localized using recently characterized high affinity biotinylated analogue of AVP (PhAcAL(Btn)VP), which is selective for the V1a subtype of VPR. PhAcAL(Btn)VP:VPR complexes were subsequently visualized by avidin-Texas red. The pharmacological characteristics of these sites were established using selective analogues of vasopressin and oxytocin. This confirmed that V1a receptors were indeed being visualized. The structures observed following visualization of VPRs had the same morphology as the vasculature revealed by the anti smooth muscle alpha-actin antibody. It can therefore be concluded that the blood vessels of the spinal cord express VPRs and are potentially responsive to AVP. Furthermore, VPRs were detected on capillaries of the microvasculature. As these capillaries are devoid of smooth muscle, VPRs must be expressed by endothelial cells as well as by smooth muscle cells. This distribution of VPRs would enable AVP to regulate local blood flow. The source of the AVP could be the general circulation, or perhaps more likely, to be local release from vasopressinergic hypothalamic neurones which are known to innervate specific regions of the spinal cord.

Transient receptor potential channels TRPC3 and TRPC6 are expressed in principal cells of the collecting duct (CD) along with the water channel aquaporin-2 (AQP2) both in vivo and in the cultured mouse CD cell line IMCD-3. The channels are primarily localized to intracellular vesicles, but upon stimulation with the antidiuretic hormone arginine vasopressin (AVP), TRPC3 and AQP2 translocate to the apical membrane. In the present study, the effect of various activators and inhibitors of the adenylyl cyclase (AC)/cAMP/PKA signaling cascade on channel trafficking was examined using immunohistochemical techniques and by biotinylation of surface membrane proteins. Both in vivo in rat kidney and in IMCD-3 cells, translocation of AQP2 and TRPC3 (but not TRPC6) was stimulated by [deamino-Cys(1), d-Arg(8)]-vasopressin (dDAVP), a specific V2-receptor agonist, and blocked by [adamantaneacetyl(1), O-Et-d-Tyr(2), Val(4), aminobutyryl(6), Arg(8,9)]-vasopressin (AEAVP), a specific V2-receptor antagonist. In IMCD-3 cells, translocation of TRPC3 and AQP2 was activated by forskolin, a direct activator of AC, or by dibutyryl-cAMP, a membrane-permeable cAMP analog. AVP-, dDAVP-, and forskolin-induced translocation in IMCD-3 cells was blocked by SQ22536 and H89, specific inhibitors of AC and PKA, respectively. Translocation stimulated by dibutyryl-cAMP was unaffected by AEAVP but could be blocked by H89. AVP- and forskolin-induced translocation of TRPC3 in IMCD-3 cells was also blocked by two additional inhibitors of PKA, specifically Rp-cAMPS and the myristoylated inhibitor of PKA (m-PKI). Quantification of TRPC3 membrane insertion in IMCD-3 cells under each assay condition using a surface membrane biotinylation assay, confirmed the translocation results observed by immunofluorescence. Importantly, AVP-induced translocation of TRPC3 as estimated by biotinylation was blocked on average 95.2 +/- 1.0% by H89, Rp-cAMPS, or m-PKI. Taken together, these results demonstrate that AVP stimulation of V2 receptors in principal cells of the CD causes translocation of TRPC3 to the apical membrane via stimulation of the AC/cAMP/PKA signaling cascade.

Biotinyl analogues of [Arg8]vasopressin were synthesized with the biotinyl moiety at position 4. This involved the substitution of 2, 4-diaminobutyric acid (Dab) for Gln4 in [1-deamino-Arg8]vasopressin to give the parent peptide des-[Dab4,Arg8]vasopressin. Two biotinyl analogues with different spacers between the side chain of Dab4 and the biotinyl residue were then prepared and characterized in detail. The analogues retained high binding affinities for the V2-receptor in both bovine kidney membranes and LLC-PK1 renal epithelial cells and for the V1-receptor in rat liver membranes. Both analogues were as potent as [Arg8] vasopressin in stimulating the cAMP-dependent protein kinase and the production of urokinase-type plasminogen activator in LLC-PK1 cells, with concentration dependence consistent with receptor binding affinities. Avidin or streptavidin did not appear to reduce receptor binding or biological activity of the biotinyl analogues. The use of the biotinylated vasopressin analogue des-[Dab-(biotinylamido)hexanoyl4, Arg8]vasopressin together with fluorescein-labeled streptavidin as a fluorescent probe for the V2-receptor in LLC-PK1 cells demonstrated the following: 1) Specific binding of the biotinyl analogue shown by quantitative single-cell fluorescence measurements using the technique of fluorescence microphotolysis; 2) the V2-receptor visualized by fluorescence microscopy; and 3) the expression of the V2-receptor detected by flow cytometry.