The synthesis and biological activity of four novel fluorescent vasopressin analogs.
We synthesized and tested the biological properties of four fluorescent vasopressin analogs: [1-(2-mercapto)propionic acid]-8-lysine-N6-5-dimethylamino-naphthalene-1-sulfonyl vasopressin (D-MLVP), [1-(2-mercapto)propionic acid]-8-lysine-N6-carboxyfluorescein vasopressin (F-MLVP), [1-(2-mercapto)propionic acid]-8-lysine-N6-2-N-methylanthranilamide vasopressin (MA-MLVP), and [1-(2-mercapto)propionic acid]-8-lysine-N6-carboxytetramethylrhodamine vasopressin (R-MLVP). All fluorescent analogs were prepared by coupling the appropriate fluorochrome to the 6-amino group of the lysine residue in [1-(2-mercapto)propionic acid]-8-lysine vasopressin (MLVP) which was synthesized by the Merrifield solid-phase method. The structures of high performance liquid chromatography-purified MLVP and the fluorescent analogs were confirmed by fast atom bombardment mass spectrometry. F-MLVP, MA-MLVP, and R-MLVP effectively competed for 8-arginine vasopressin (AVP)-binding sites in canine renal plasma membranes and on the surface of porcine kidney cells (LLC-PK1, ATCC CL101). Dissociation constants for F-MLVP, MA-MLVP, and R-MLVP of 32, 8.8, and 26 nM, respectively, were calculated from the results of competition binding assays conducted with membranes. D-MLVP did not bind to plasma membranes. Dissociation constants for F-MLVP, MA-MLVP, and R-MLVP of 390, 38, and 160 nM, respectively, were calculated from the results of competition binding assays conducted with cells. F-MLVP, MA-MLVP, and R-MLVP at a concentration of 10(-6) M increased adenylate cyclase activity in canine renal plasma membranes to values 2.4, 2.9, and 2.6 times that of basal activity, respectively. A maximally active concentration of AVP (1 microM) increased adenylate cyclase activity in canine renal plasma membranes to a value 2.7 times that of basal activity. D-MLVP did not stimulate adenylate cyclase activity. F-MLVP, MA-MLVP, and R-MLVP at a concentration of 10(-6) M increased the cAMP content of porcine kidney cells from a basal level of 43 to 267, 160, and 469 pmol/mg of cell protein, respectively. Specific binding of these fluorescent analogs to receptors on the surface of LLC-PK1 cells was observed by fluorescence microscopy. These observations indicate that F-MLVP, MA-MLVP, and R-MLVP are biologically active fluorescent vasopressin analogs which are well-suited to the study of renal vasopressin receptors by fluorescence microscopy.
Lutz
W H
WH
Department of Medicine, Mayo Clinic and Foundation, Rochester, Minnesota 55905.
Londowski
J M
JM
Kumar
R
R
D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000096:biosynthesis; D004285:Dogs; D005456:Fluorescent Dyes; D007668:Kidney / Q000187:drug effects / Q000378:metabolism; D008236:Lypressin / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011945:Receptors, Angiotensin / Q000378:metabolism; D017483:Receptors, Vasopressin; D013552:Swine
D005456:Fluorescent Dyes; D011945:Receptors, Angiotensin; D017483:Receptors, Vasopressin; C063307:vasopressin, (1-(2-mercapto)propionic acid)-N(6)-5-dimethylaminonaphthalene-1-sulfonyl-8-Lys-; C063310:vasopressin, (1-2-mercapto)propionic acid-N(6)-carboxytetramethylrhodamine-8-Lys-; C063308:vasopressin,-(1-(2-mercapto)propionic acid)-N(6)-carboxyfluorescein-8-Lys-; D008236:Lypressin; D000242:Cyclic AMP; D000262:Adenylyl Cyclases