ABSTRACT-Ebelactone B (EB) (10-'-10-' M) inhibited dose-dependently carboxypeptidase (CP) Y-like exopeptidase, one of the major kininases separated from rat urine, whereas it inhibited neither CPA, CPB or neutral endopeptidase (NEP). Degradation of bradykinin (BK) to BK-(1-8) in rat urine was completely inhibited by EB (10-5 M) with the increased generation of BK-(1-7). Intraduodenal administration of EB (3 mg/kg) to anesthetized rats caused marked diuresis (by 110%) and natriuresis (130%), in parallel with the increase in urinary kinin levels (110%). Intravenous infusion of a BK antagonist, Hoe140 (3 mg/kg/hr), strongly blocked both EB-induced diuresis and natriuresis. EB may be a novel type of diuretic and natriuretic agent that acts by increasing urinary kinin levels. Bradykinin (BK) is very active in renal function because it participates in increasing the renal blood flow and in di uresis and natriuresis (1). We have reported that the degra dation pathway of BK in rat urine is quite different from that in plasma because of the difference in kininases present (2). The major kininases in rat urine were neutral endopeptidase (NEP) and carboxypeptidase (CP) Y-like exopeptidase (3), whereas angiotensin converting enzyme (ACE) and CPN mainly degrade BK in rat plasma (2).Ebelactones, isolated from actinomycetes, were report ed to inhibit some enzymes such as esterase, lipase and N formylmethionine aminopeptidase (4). In the present paper, we report that Ebelactone B (EB) selectively in hibited not only CPY from yeast but also CPY-like exopep tidase in rat urine without inhibition of other kininases in plasma and urine. Furthermore, administration of EB to anesthetized rats caused diuresis and natriuresis via in creased urinary kinin excretion. This suggests that EB is a candidate for a new type of diuretic and natriuretic agent. NEP and CPY-like exopeptidase in rat urine were sepa rated by using a Superdex 200 column as described in the previous paper (3). Plasma samples were prepared from blood collected from the carotid artery under ether anesthesia (2). Enzyme activities of isolated CPA, CPB and CPY were determined with peptide substrates: Z-Gly Phe for CPA (5), Bz-Gly-Arg for CPB (6) and Z-Phe-Leu for CPY (7). Assays of kininase activity and detection of BK fragments were carried out by HPLC (2, 3). The in vivo effects of EB were studied in male Sprague-Dawley strain rats (SPF, 8 to 10-week-old, anesthetized with sodium pentobarbital, 40 mg/kg, i.p.). Urine was collected through a polyethylene cannula inserted into the bladder to estimate the volume of urine and urinary so dium and potassium levels. Physiological saline was in fused (6 ml/kg/hr) via the jugular vein throughout the experimental period. Urine volume was estimated by its weight, and urinary sodium and potassium levels were as sayed by flame photometry (8). For the assay of kinin in the urine, urine was collected directly into plastic tubes containing absolute ethanol through a polyethylene can nula inserted into both ureters under pentobarbital...