Basal and stimulated cyclic adenosine 3',5'-monophosphate production in rat adenopituitary homogenates: direct determination by high-performance liquid chromatography.
A sensitive and reliable procedure to quantify cAMP in biological samples, without involving radioisotopes, is described. After incubation, the assay mixture was boiled for 4 min, centrifuged, filtered and directly injected onto a mu Bondapak C18 column (10 microns particle size; 150 mm x 3.9 mm I.D.). cAMP was resolved within 40 min in isocratic mode using a mobile phase composed of 96% of 0.01 M ammonium acetate, pH 6.6 and 4% of acetonitrile-water &70:30, v/v) at a flow-rate of 0.5 ml/min. Detection was monitored at 254 nm and quantification was achieved by peak surface integration. The lowest detection limit was 1.6 pmol (signal-to-noise ratio = 2.5). The potential of this assay to perform structure-activity of bioactive peptides is illustrated with growth hormone-releasing factor in rat adenopituitary homogenates.
Lefrançois
L
L
Neuroendocrinology Laboratory, Notre-Dame Hospital Research Center, Montreal, Canada.
Gaudreau
P
P
D000262:Adenylyl Cyclases / Q000032:analysis / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000032:analysis / Q000096:biosynthesis*; D013007:Growth Hormone-Releasing Hormone / Q000031:analogs & derivatives / Q000494:pharmacology; D066298:In Vitro Techniques; D008297:Male; D008969:Molecular Sequence Data; D010316:Particle Size; D010903:Pituitary Gland, Anterior / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011986:Receptors, Somatotropin / Q000037:antagonists & inhibitors / Q000187:drug effects; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship
D011986:Receptors, Somatotropin; D005576:Colforsin; D013007:Growth Hormone-Releasing Hormone; D000242:Cyclic AMP; D000262:Adenylyl Cyclases