Cyclic AMP and locust diuretic hormone action: Hormone induced changes in cAMP levels offers a novel method for detecting biological activity of uncharacterized peptide

Abstract

The role of cAMP in diuretic hormone (DH) action on Locusta migratoria Malpighian tubules has been investigated. The rate of fluid secretion by semi-isolated tubules is elevated by the phosphodiesterase inhibitors theophylline (10 −3 M) and 3-isobutyl-1-methylxanthine (IBMX, 10 −3 M), although the latter is more effective. Removal of these inhibitors returns the rate of fluid secretion to basal levels. IBMX (10 −4 M) is shown to elevate the intracellular cAMP levels of tubules, but no significant elevation was observed in response to theophylline (10 −4 M). Stimulation of isolated tubules with DH (methanol extracted storage lobes) increases the intracellular cAMP levels of tubules by 7 to 8-fold, after 3 min stimulation at 30°C; the response to DH stimulation is lower after 10 min. Using plasma membrane preparations of Locusta migratoria Malpighian tubules, DH stimulates adenylate cyclase catalysed synthesis of cAMP from ATP, with optimum stimulation using 0.5–2 mg tubule membrane protein/ml. Dose-response data show that a saturated response occurs at doses of DH of greater than 0.2 storage lobe equivalents/75 μl of incubation medium. The proteolytic enzymes trypsin (0.01 mg/ml) and chymotrypsin (0.01 mg/ml) both destroy diuretic activity, tested using the semi-isolated tubule preparation. Trypsin 0.01 mg/ml also reduces the activity of the “DH” activating adenylate cyclase. Chromatography of methanol extracts of storage lobes on a TSK 2000 SW high performance size-exclusion chromatography column eluted in 0.1% TFA, separate the DH, activating adenylate cyclase, in fractions 7 and 8. Comparison of these active fractions with those tested using semi-isolated Malpighian tubule preparations suggests two distinct receptor sites on Locusta tubules.