Brown Norway Katholiek rats, which have very low levels of plasma kininogens, excreted a much smaller amount of kinin in the urine than normal rats of the same strain. The systolic blood pressure of 7-week-old kininogen-deficient rats fed low (0.3%) NaCI diets (131±4 mm Hg, n=12) was not different from that in normal rats. Two percent NaCI diets given from 7 weeks of age for 4 weeks caused rapid increases in blood pressure (167±4 mm Hg, n=12, 9 weeks old) in deficient rats, although the same diets induced no blood pressure increase in normal rats. Urinary excretion of active kallikrein and prokallikrein remained constant in both rat groups throughout NaCI loading. During this period, the deficient rats secreted less urine (9 weeks old, P<.05) and less urinary sodium (11 weeks old, P<.05). Serum levels of sodium in deficient rats were higher (/ > <.O5) than in normal rats at 9 weeks of age. Intracellular concentrations of sodium in the erythrocytes of deficient rats were higher (P<.05) than in normal rats throughout NaCI loading. Subcutaneous infusion of bovine low molecular weight kininogen with an osmotic pump in NaCI-loaded deficient rats induced a reduction (P<.01) in blood pressure and increases (P<.05) in urine volume and urinary sodium and kinin levels. By contrast, subcutaneous infusion of the bradykinin antagonist Hoe 140 or of aprotinin in NaCI-loaded normal rats induced a hypertensive response. This antagonist treatment reduced urine volume and urinary sodium. These results indicate that the lack of kinin generation observed in the kininogen-deficient rats was related through sodium retention to the hypertensive response to NaCI loading. (Hypertension. 1993;22:705-714.) KEY WORDS • hypertension, sodium-dependent • sodium, dietary • kininogens • bradykinin • aprotininT he blood pressure-lowering effects of urinary kallikrein injected intravenously have been described for more than six decades,' and bradykinin is well known to induce vasodilatation and an increase in renal blood flow and excretion of water and sodium from the kidney.2 Urinary kallikrein therefore has been thought to be involved in hypertension, and its reduction has been reported in human 36 and animal 717 experiments. On the other hand, it is widely accepted that sodium retention may be related to the pathogenesis of hypertension, although the precise mechanisms of its contribution to the elevation of blood pressure are still unclear. The causal relation between these three factors -urinary kallikrein, sodium retention, and hypertension-has not been verified. Recently, we reported that the kallikrein-kinin system may play a suppressive role in deoxycorticosterone acetate (DOCA)-salt hypertension.18 Using kininogendeficient Brown Norway Katholiek (BN-Ka) rats and normal rats of the same strain (Brown Norway Kitasato [BN-Ki]), we were able to demonstrate that the urinary kallikrein-kinin system may contribute to lowering of systemic blood pressure in the initial phase of the development of DOCA-salt hypertension in uninephrectomized ...

Brown Norway Katholiek rats with very low levels of plasma kininogens excreted a much smaller amount of kinin in the urine than normal rats of the same strain. The systolic blood pressure of 7-week-old kininogen-deficient rats (132±2 mm Hg, n=7) was not different from that of normal rats. Angiotensin II (Ang II) (20 jig/d SC) from 7 weeks of age for 2 weeks with a micro-osmotic pump caused significant increases in blood pressure (181 ±5 mm Hg, n=7, 9 weeks old) in the deficient rats, although the same treatment induced no blood pressure increase in the normal rats. Also during this period, the deficient rats had significantly higher heart rates, tended to excrete less urinary sodium, and showed significantly higher sodium levels in serum, erythrocytes, and cerebrospinal fluid compared with the normal rats. Ang II increased urinary excretion of aldosterone in both deficient and normal rats (P<.05). Spironolactone treatment (50 mg/kg per day) for 7 days in deficient rats restored blood pressure and heart rate to T he biologically active peptide bradykinin is well known to induce increases in renal blood flow and water and sodium excretions. Kinin is generated by the action of kallikrein secreted in the distal tubules, and its receptors are distributed on the tubular cells of the distal tubules.12 It has been claimed that urinary kallikrein may be involved in hypertension in humans 3 " 6 and animal models of hypertension. 7 ' 17Using kininogen-deficient Brown Norway Katholiek (BN-Ka) rats that excreted little kinin in the urine 18 and normal rats of the same strain (Brown Norway Kitasato [BN-Ki]), we previously reported that the urinary kallikrein-kinin system may contribute to lowering systemic blood pressure in the initial phase of the development of deoxycorticosterone acetate (DOCA)-salt hypertension in uninephrectomized rats by acceleration of the excretion of sodium and water. This was confirmed in DOCA-salt-treated Sprague-Dawley rats with the use of a potent and selective bradykinin antagonist, Hoe 140.iy Hypertension was also induced as a result of sodium retention 20 in mutant BN-Ka rats by loading a low concentration (2%) of sodium in the diet, whereas the same treatment did not increase systemic blood pressure in normal BN-Ki rats. These results indicated Received September 22, 1993; accepted in revised form March 23, 1994.From the Department of Pharmacology, Kitasato University School of Medicine, Sagamihara, Kanagawa, and the Department of Pharmacology (S.O.), Kitasato University School of Pharmaceutical Sciences, Tokyo, Japan.Correspondence to Dr Masataka Majima, MD, PhD, Department of Pharmacology, Kitasato University School of Medicine, Kitasato 1-15-1, Sagamihara, Kanagawa 228, Japan.© 1994 American Heart Association, Inc.normal levels and significantly reduced sodium levels in erythrocytes and cerebrospinal fluid. Subcutaneous infusion of bovine low-molecular-weight kininogen with an osmotic pump in Ang II-treated deficient rats induced significant reductions in blood pressure, heart ra...

The introduction of new paramagnetic shift reagents in the nuclear magnetic resonance (NMR) method has made it possible to distinguish intra- and extracellular ions in tissues or organs in vitro. We measured the intra- and extracellular 23Na and 1H in vivo in the gerbil brain and skeletal muscle by NMR spectroscopy employing the shift reagent, dysprosium triethylenetetraminehexaacetate (Dy[TTHA]3-). Without Dy(TTHA)3-, the 23Na and 1H signals were seen only as single peaks, but gradual intravenous infusion of Dy(TTHA)3- separated these signals into two peaks, respectively. The unshifted peaks reflected the intracellular 23Na and 1H signals, while the shifted peaks reflected the extracellular signals. In the brain spectra, an additional small peak, which represented intravascular signals, was detected and its intensity increased after injection of papaverine hydrochloride. The present method is advantageous over the microelectrode technique because of its nondestructiveness and its capability for obtaining intra- and extracellular volume information from measurements of the 1H spectra, the peaks of which reflect the intra- and extracellular water amounts. The intracellular Na+ increase associating with increased cellular volume after ouabain in the muscle was clearly visualized by this method. The technique is clearly of use for physiological and pathophysiological studies of organs.

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...

Two types of kininases were separated from rat urine by gel-filtration on a column of Superdex 200 and characterized as a membrane-bound neutral endopeptidase (EC 3.4.24. ll) and a carboxypeptidase Y-like exopeptidase. The neutral endopeptidase which was found in a void volume fraction, liberated first Pheg-Arg9 and then Phe5-Serf-Pro? from bradykinin (BK)*. The kininase activity was inhibited by phosphoramidon and thiorphan but not by captopril. Another kininase with an apparent molecular weight of 93,000 cleaved rapidly BK into BKl-8 (des Argg BK) and BK1-7 (des Phe8~Arg9 BK) into BK1-6 (des Pro7-Phes-Argg BK). The kininase activity was inhibited by inhibitors for serine proteases and cysteine proteases and by metal ions (Co2+, Hg" or Ag+), but not by inhibitors for metal proteases. The kininase activity was also inhibited by a rabbit antipeptide IgG which was raised against a synthetic peptide with the amino acid sequence of human and mouse carboxypeptidase Y-like peptidases. These results indicate that the exopeptidase is not carboxypeptidases N or M but a member of a family of human and mouse carboxypeptidase Y-like exopeptidases which are identified as protective proteins associated with lysosomal ,8-galactosidase and neuraminidase or platelet peptidase with deamidase activity. When rat urine was incubated with BK, the generation of either BK1-8 or BKl-7 was inhibited in the presence of either DFP* or phosphoramidon. In the presence of both of DFP and phosphoramidon, degradation of BK was inhibited, whereas captopril had no effect on the degradation of BK. These results demonstrate that major kininases in rat urine are neutral endopeptidase and carboXypeptidase Y-like exopeptidase.