A simple and sensitive method for determination of human urinary kallikrein activity (kininogenase activity), using human low molecular weight kininogen

Human low molecular weight kininogen was partially purified and applied to the measurement of human glandular kallikrein as a substrate. The prepared human low molecular weight kininogen did not contain any significant amounts of kinin generating or destroying enzymes. When ethanol was added to the assay tube to stop the enzyme reaction, the substrate was almost completely removed from the incubation solution. Moreover, less than 1.25% ethanol had no effect on the kinin radioimmunoassay. These data suggest that the measurement of generated kinin can be done directly after the addition of ethanol. In this assay system, control tubes were unnecessary since the small volume of the urine samples (0.5 to 2.0 nl) contained negligible amounts of endogenous kinin. In a comparison of the availability as a substrate for human urinary kallikrein among human, dog and bovine low molecular weight kininogens, the enzyme activity was 5 or 100 times as high in the human substrate as in the dog and bovine substrates, suggesting that a human substrate is best for the human enzyme. A significant correlation was found between our previous method using bovine substrate and this method for human urinary kallikrein activity. In both methods, urinary kallikrein excretions were significantly lower in patients with essential hypertension and higher in those with primary aldosteronism, respectively. This simple, specific and sensitive kininogenase assay system seems to be very useful for investigating the physiological or pathophysiological role of the renal kallikrein-kinin system in hypertensive and renal diseases.     

Effect of parathyroid hormone and cyclic AMP on protein phosphorylation in rabbit kidney cortex

Suspensions of renal cortical tubules were incubated with ³³P_i and exposed to parathyroid hormone (40 μg/ml) or 1 mM dibutyryl cyclic AMP. In other experiments homogenates of renal cortex were assayed for protein kinase and phosphoprotein phosphatase activity using [γ-³²P]ATP with or without 5 mM cyclic AMP. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and phosphorylation of proteins measured by liquid scintillation counting of gel slices. The pattern of protein phosphorylation was similar in control tissue from both tubule suspensions and homogenates. In intact tubules, parathyroid hormone stimulated the phosphorylation of four proteins with molecular weights of approx. 1500 000, 125 000, 100 000 and 50 000 by 28%, 24%, 13%, and 20%, respectively. Results with dibutyryl cyclic AMP were comparable but more variable. Stimulation of phosphorylation by cyclic AMP in homogenates was more generalized with the major effect on a 50 000 dalton protein (50% stimulation). No effect of cyclic AMP on dephosphorylation of proteins was observed. The results are interpreted as indicating that increased phosphorylation of cell proteins is part of the cyclic AMP-mediated response of the renal cortex to parathyroid hormone.     

Kininogen-derived peptides for investigating the putative vasoactive properties of human cathepsins K and L.

Macrophages at an inflammatory site release massive amounts of proteolytic enzymes, including lysosomal cysteine proteases, which colocalize with their circulating, tight-binding inhibitors (cystatins, kininogens), so modifying the protease/antiprotease equilibrium in favor of enhanced proteolysis. We have explored the ability of human cathepsins B, K and L to participate in the production of kinins, using kininogens and synthetic peptides that mimic the insertion sites of bradykinin on human kininogens. Although both cathepsins processed high-molecular weight kininogen under stoichiometric conditions, only cathepsin L generated significant amounts of immunoreactive kinins. Cathepsin L exhibited higher specificity constants (kcat/Km) than tissue kallikrein (hK1), and similar Michaelis constants towards kininogen-derived synthetic substrates. A 20-mer peptide, whose sequence encompassed kininogen residues Ile376 to Ile393, released bradykinin (BK; 80%) and Lys-bradykinin (20%) when incubated with cathepsin L. By contrast, cathepsin K did not release any kinin, but a truncated kinin metabolite BK(5-9) [FSPFR(385-389)]. Accordingly cathepsin K rapidly produced BK(5-9) from bradykinin and Lys-bradykinin, and BK(5-8) from des-Arg9-bradykinin, by cleaving the Gly384-Phe385 bond. Data suggest that extracellular cysteine proteases may participate in the regulation of kinin levels at inflammatory sites, and clearly support that cathepsin K may act as a potent kininase.     

Estimation of ouabain specifically bound to dog renal (Na+ + K+)-ATPase in vivo.

Suspensions of viable renal cortical cells hydrolyzed a synthetic ester substrate (alpha-N-tosyl-L-arginine methyl ester, Tos-Arg-OMe) and generated kinins from a kininogen substrate. This kallikrein-like esterase activity increased linearly with cell number, or time of exposure to substrate. No radiolabelled substrate or product was found within the cells. Most of the activity appeared to be on cell surfaces as supernatant media had less than 20% of the Tos-Arg-OMe esterase activity on the cell suspensions. Cell surface Tos-Arg-OMe esterase activity was inhibited by aprotinin, benzamidine, pentamidine, and a tris-amidine derivative (alpha,alpha',alpha'-tris(3-amidinophenoxy)mesitylene). Preincubation of cells with phospholipase A2 increased renal cell surface esterase activity up to 76% while only slightly increasing supernatant activity. In contrast, preincubation with deoxycholate caused clearing of suspensions and a marked increase in supernatant esterase activity. Renal cell kininogenase (EC 3.4.21.8) activity was inhibited by preincubation with aprotinin, the tris-amidine derivative, or anti-rat urinary kallikrein antibody. Kallikrein elaborated by renal cells formed a single precipitin line with an antibody to rat urinary kallikrein but the two enzymes were not immunologically identical. We conclude that kallikrein's active sites are facing the external environment of renal cortical cells in suspension with access to substrates, inhibitors, and antibody.     

Effect of parathyroid hormone and cyclic AMP on protein phosphorylation in rabbit kidney cortex.

Suspensions of renal cortical tubules were incubated with 33Pi and exposed to parathyroid hormone (40 mlg/ml) or 1 mM dibutyryl cyclic AMP. In other experiments homogenates of renal cortex were assayed for protein kinase and phosphoprotein phosphatase activity using [gamma-32P]ATP with or without 5 mM cyclic AMP. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and phosphorylation of proteins measured by liquid scintillation counting of gel slices. The pattern of protein phosphorylation was similar in control tissue from both tubule suspensions and homogenates. In intact tubules, parathyroid hormone stimulated the phosphorylation of four proteins with molecular weights of approx. 150 000, 125 000, 100 000 and 50 000 by 28%, 24%, 13%, and 20%, respectively. Results with dibutyryl cyclic AMP were comparable but more variable. Stimulation of phosphorylation by cyclic AMP in homogenates was more generalized with the major effect on a 50 000 dalton protein (50% stimulation). No effect of cyclic AMP on dephosphorylation of proteins was observed. The results are interpreted as indicating that increased phosphorylation of cell proteins is part of the cyclic AMP-mediated response of the renal cortex to parathyroid hormone.     

Localisation of immunoreactive kininogen and tissue kallikrein in the human nephron

Summary The cellular localisation of kininogen and its relationships with tissue kallikrein containing cells was studied in the human kidney by the peroxidase-antiperoxidase method using antisera to human LMW kininogen and to human tissue kallikrein. Immunoreactive kininogen was localised in the principal cells of collecting ducts. Immunoreactive tissue kallikrein was detected in the connecting tubule cells segment of the nephron preceeding the cortical collecting ducts. The co-existence of tissue kallikrein and kininogen in the same transitional tubule, but in different cells, was established by the use of serial sections and double immunostaining. This anatomical relationship is in accordance with known studies that describe intermingling of principal cells and connecting tubule cells where connecting tubules merge into cortical collecting ducts in the human nephron. the close relationship between cells that contain tissue kallikrein and its substrate, kininogen, suggests that kinins could be generated in the lumen of distal cortical segments of the human nephron.     

Localisation of immunoreactive kininogen and tissue kallikrein in the human nephron

The cellular localisation of kininogen and its relationships with tissue kallikrein containing cells was studied in the human kidney by the peroxidase-antiperoxidase method using antisera to human LMW kininogen and to human tissue kallikrein. Immunoreactive kininogen was localised in the principal cells of collecting ducts. Immunoreactive tissue kallikrein was detected in the connecting tubule cells, segment of the nephron preceding the cortical collecting ducts. The co-existence of tissue kallikrein and kininogen in the same transitional tubule, but in different cells, was established by the use of serial sections and double immunostaining. This anatomical relationship is in accordance with known studies that describe intermingling of principal cells and connecting tubule cells where connecting tubules merge into cortical collecting ducts in the human nephron. The close relationship between cells that contain tissue kallikrein and its substrate, kininogen, suggests that kinins could be generated in the lumen of distal cortical segments of the human nephron.     

Kinins mediate the inhibition of atrial natriuretic peptide diuretic effect induced by pepsanurin.

Pepsanurin is a peptidic fraction resulting from pepsin digestion of plasma globulins, that inhibits ANP renal excretory actions. We studied whether kinin-like peptides mediate the anti-ANP effect by testing if pepsanurin: 1) was blocked by the kinin B2 receptor antagonist HOE-140, 2) was produced from kininogen, and 3) was mimicked by bradykinin. Anti-ANP activity was assessed in anesthetized female rats by comparing the excretory response to two ANP boluses (0.5 microgram i.v.) given before and after i.p. injection of test samples. Pepsanurin from human or rat plasma (1-5 mL/kg), and bradykinin (5-20 micrograms/kg), dose-relatedly inhibited ANP-induced water, sodium, potassium and cyclic GMP urinary excretion, without affecting arterial blood pressure. The same effect was exerted by pepsin hydrolysates of purified kininogen, whereas hydrolysates of kininogen-free plasma had no effect. HOE-140 (5 micrograms, i.v.) did not alter baseline, or ANP-induced excretion, but blocked the anti-ANP effects of pepsanurin. Histamine (15 micrograms/kg) plus seroalbumin hydrolysates did not affect ANP response, despite inducing larger peritoneal fluid accumulation as compared with pepsanurin or bradykinin. We concluded that kinins cleaved from kininogen mediate the anti-ANP effects of pepsanurin by activation of kinin B2 receptors, independently of changes in systemic arterial pressure or peritoneal fluid sequestration.     

Canine Plasma Kininogen: Evidence for the Presence of Two Kininogens and Purification of High Molecular Weight Kininogen and Characterization as Multi-Functional Molecule

The ratio of kininogen that is substrate of plasma kallikrein to kininogen, which is not substrate of plasma kallikrein in canine plasma, was about 1:3.6 by differential assay of kininogens. When the plasma was gel-filtered through a column of Sephacryl S-300 superfine, two fractions, which released kinin by trypsin, were obtained. These results indicate that two kininogens with different molecular weights are present in the plasma and they show different susceptibility to plasma kallikrein. One kininogen was purified by ion-exchange and zinc-chelating affinity chromatographies. Purified kininogen showed a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing condition and its molecular weight was 125 kDa. Released kinin from the kininogen by trypsin was bradykinin. The kininogen inhibited papain and ficin but did not inhibit bromelain at the concentration used. The kininogen bound to carboxymethylated-papain and this binding was dissociated by 3M NaSCN. Canine plasma shortened the abnormal clotting time of human high molecular weight kininogen-deficint plasma. The kininogen also shortened the abnormal clotting time of the plasma. From these results, the purified kininogen was high molecular weight kininogen and it was multi-functional protein.     

The localisation of sorbitol pathway activity in the rat renal cortex and its relationship to the pathogenesis of the renal complications of diabetes mellitus.

A series of in vivo and in vitro investigations was performed to examine the localisation of sorbitol pathway activity in the rat renal cortex and to investigate the possible relation that the acculumation of sorbitol pathway intermediates in renal cortical tissue may have to the pathogenesis of renal complications in diabetes mellitus. Neither of the sorbitol pathway intermediates, sorbitol or fructose, were detected either in intact glomeruli which had been isolated from rats rendered chronically diabetic with streptozotocin, or in metabolically active glomeruli which had been incubated in vitro in high glucose media. Such data agreed with previously published observations that the enzyme aldose reductase is not present in renal glomeruli, and suggested that changes in sorbitol pathway activity cannot be directly related to the pathogenesis of diabetic glomerulosclerosis. Sorbitol was detected in low concentrations (3.1 mu-mol/g protein) in cortical tubules which had been isolated from the renal cortex of rats rendered chronically diabetic with streptozotocin. This concentration of sorbitol was higher than that in the intact renal cortex of the diabetic animal (0.3 mu-mol/g protein) or in the cortical tubules of non-diabetic animals (0.5 mu-mol/g protein). It is apparent that the renal cortical tubule is a major site of sorbitol pathway activity in the renal cortex. However, there is presently no obvious causal relationship between the accumulation of such relatively low concentrations of sorbitol in the renal cortical tubule and the pathogenesis of glomerulosclerosis or cortical tubular lesions in diabetes.     

Changes in the kinin system components and structure of the myocardium under the influence of antikinin preparations in allergic myocarditis in rabbits]

Experimental allergic myocarditis was induced in rabbits; a study was made of the effect on the kininogen content and on the activity of kininase in the myocardium, and on its morphological structure produced by anginine, contrical, aspirin, indometacine and epsilon-aminocapronic acid. The former two preparations were much more potent (in comparison with the others) in inhibiting the activation of the myocardial kinin system and promoted restoration of the morphological structure of the myocardium. Development of an allergic inflammation in the myocardium could be arrested or decreased by inhibition of the kinin system of the plasma and myocardium by specific agents.     

The tissue kallikrein-kinin system protects against cardiovascular and renal diseases and ischemic stroke independently of blood pressure reduction

Tissue kallikrein (hK1) cleaves low-molecular-weight kininogen to produce kinin peptide, which binds to kinin receptors and triggers a wide spectrum of biological effects. Tissue kallikrein levels are reduced in humans and in animal models with hypertension, cardiovascular and renal diseases. Transgenic mice or rats over-expressing human tissue kallikrein or kinin B2 receptor are permanently hypotensive, and somatic kallikrein gene delivery reduces blood pressure in several hypertensive rat models. Moreover, kallikrein gene delivery or kallikrein protein infusion can directly improve cardiac, renal and neurological function without blood pressure reduction. Kallikrein has pleiotropic effects in inhibiting apoptosis, inflammation, proliferation, hypertrophy and fibrosis, and promoting angiogenesis and neurogenesis in different experimental animal models. Kallikrein's effects can be blocked by kinin B2 receptor antagonists. Mechanistically, tissue kallikrein/kinin leads to increased nitric oxide levels and Akt activation, and reduced reactive oxygen species formation, TGF-beta1 expression, MAPK and nuclear factor-kappaB activation. Our studies indicate that tissue kallikrein, through the kinin B2 receptor and nitric oxide formation, can protect against oxidative damage in cardiovascular and renal diseases and ischemic stroke. These novel findings suggest that kallikrein/kinin may serve as new drug targets for the prevention and treatment of heart failure, renal disease and stroke in humans.     

Guanylyl cyclase-C receptor mRNA distribution along the rat nephron

Guanylin (GN) and uroguanylin (UGN) are two recently identified peptides that have been shown to affect water and electrolyte transport in both the intestine and the kidney. Mechanistically, the effects of both peptides are thought to be mediated by intracellular cGMP which results from ligand binding to a plasma membrane guanylyl cyclase-C (GC-C) receptor. To date, the specific intrarenal site(s) of GN and UGN action have not been established. To begin to address this issue, the present studies utilized semi-quantitative RT-PCR to assess the distribution of GC-C mRNA in specific microdissected segments of the rat nephron. GC-C mRNA expression was highest in the cortical collecting tubule, followed by the proximal convoluted tubule, medullary thick ascending limb and collecting tubule, and thin limbs of Henle's loop. Expression levels were significantly lower in all other segments tested, including the glomerulus. The renal tubular expression pattern for cGMP-dependent protein kinase II (cGK-II) mRNA, which is activated in response to GN/UGN-dependent cGMP accumulation, was similar to that for GC-C. Notably, both GN and UGN mRNAs were also expressed along the nephron. The highest levels of expression for both peptides were detected in the medullary collecting tubule. Lower, but comparable levels of GN and UGN expression also occurred in the cortical collecting tubule, cortical and medullary thick ascending limb, and thin limbs of Henles loop. In the proximal convoluted tubule, GN mRNA expression was also quite high, while UGN mRNA was almost undetectable. The presence of renal GC-C and cGK-II in the kidney are consistent with a proposed endocrine function for GN and UGN. In addition however, the present data suggest that intrarenally synthesized GN and UGN may also contribute to the regulation of renal tubular transport.     

Kininogen substrates for trypsin and cathepsin D in human,rabbit and rat plasmas

Studies have compared “total”, HMW kininogen and leukokininogen levels in human, rabbit and rat plasmas using trypsin, glass powder and cathepsin D as kininogenases or activators of kininogenases. Rat plasma was found to have about 10 fold more leukokininogen than the other plasmas assayed. When trypsin was used to estimate total kininogen, rat plasma liberated maximal amounts of kinin only in the presence of high concentrations of trypsin (1 mg/ml incubation mixture). In addition, it was found that trypsin in these concentrations liberated from rat plasma both bradykinin and a previously unidentified kinin which we have termed “T-kinin”. The results overall indicate that in the case of rat and rabbit plasma, currently used methods for estimations of total kininogen may not be accurate. T-kinin may represent a leukokininogen or a hitherto undescribed kininogen.     

Cardiac kinin level in experimental diabetes mellitus: role of kininases

Diabetes mellitus impairs the cardiac kallikrein-kinin system by reducing cardiac kallikrein (KLK) and kininogen levels, a mechanism that may contribute to the deleterious outcome of cardiac ischemia in this disease. We studied left ventricular (LV) function and bradykinin (BK) coronary outflow in buffer-perfused, isolated working hearts (n = 7) of controls and streptozotocin (STZ)-induced diabetic rats before and after global ischemia. With the use of selective kininase inhibitors, the activities of angiotensin I-converting enzyme, aminopeptidase P, and neutral endopeptidase were determined by analyzing the degradation kinetics of exogenously administered BK during sequential coronary passages. Basal LV function and coronary flow were impaired in STZ-induced diabetic rats. Neither basal nor postischemic coronary BK outflow differed between control and diabetic hearts. Reperfusion after 15 min of ischemia induced a peak in coronary BK outflow that was of the same extent and duration in both groups. In diabetic hearts, total cardiac kininase activity was reduced by 41.4% with an unchanged relative kininase contribution compared with controls. In conclusion, despite reduced cardiac KLK synthesis, STZ-induced diabetic hearts are able to maintain kinin liberation under basal and ischemic conditions because of a primary impairment or a secondary downregulation of kinin-degrading enzymes.     

Tissue distribution and kininogen gene expression after acute-phase inflammation

A kinin-directed monoclonal antibody to kininogens has been developed by the fusion of murine myeloma cells with mouse splenocytes immunized with bradykinin-conjugated hemocyanin. The hybrid cells were screened by an enzyme-linked immunosorbent assay (ELISA) and a radioimmunoassay (RIA) for the secretion of antibodies to bradykinin. Ascitic fluids were produced and purified by a bradykinin-agarose affinity column. The monoclonal antibody (IgG1) bound to bradykinin, Lys-bradykinin, Met-Lys-bradykinin, and kininogens in ELISA. Further, this target-directed monoclonal antibody recognized purified low and high molecular weight bovine, human, or rat kininogens and T-kininogen in Western blotting. After turpentine-induced acute inflammation, rat kininogen levels increased dramatically in liver and serum as well as in the perfused pituitary, heart, lung, kidney, thymus, and other tissues, as identified by the kinin-directed kininogen antibody in Western blot analyses. The results were confirmed by measuring kinin equivalents of kininogens with a kinin RIA. During an induced inflammatory response, rat kininogens were localized immunohistochemically with the kinin-directed monoclonal antibody in parenchymal cells of liver, in acinar cells and some granular convoluted tubules of submandibular gland, and in the collecting tubules of kidney. Northern and cytoplasmic dot blot analyses using a kinin oligonucleotide probe showed that kininogen mRNA levels in liver but not in other tissues increase after turpentine-induced inflammation. The results indicated that rat kininogens are distributed in various tissues in addition to liver and only liver kininogen is induced by acute inflammation. The target-directed kininogen monoclonal antibody is a useful reagent for studying the structure, localization, and function of kininogens or any protein molecule containing the kinin moiety.     

Solubilization and partial purification of the thiazide diuretic receptor from rabbit renal cortex

This study was designed to solubilize, characterize and begin to purify the thiazide-sensitive Na/Cl transporter from mammalian kidney. Metolazone, a thiazide-like diuretic drug, binds to receptors in rat renal cortex closely related to the thiazide-sensitive Na/Cl transport pathway of the renal distal tubule. In the current study, [3H]metolazone bound to receptors in rabbit renal cortical microsomes. The portion of [3H]metolazone binding that was inhibited by hydrochlorothiazide reflected binding to a high-affinity class of receptor. The affinity (Kd 2.0 +/- 0.1 nM) and number (Bmax = 0.9 +/- 0.4 pmol/mg protein) of high-affinity receptors in rabbit renal cortical membranes were similar to values reported previously for rat. When proximal and distal tubule fragments were separated by Percoll gradient centrifugation, receptors were restricted to the fraction that contained distal tubules. When compared with cortical homogenates, receptor density was enriched 12-fold by magnesium precipitation and differential centrifugation. The zwitterionic detergent CHAPS solubilized 25-35% of the receptors (at 6 mM). Chloride inhibited and Na stimulated binding of [3H]metolazone to solubilized high-affinity receptors. The receptors could be purified significantly by hydroxyapatite chromatography and size exclusion high performance liquid chromatography (HPLC). The combination of magnesium precipitation and differential centrifugation, hydroxyapatite chromatography, and size exclusion HPLC resulted in a 213-fold enrichment of receptors, compared to renal cortical homogenate. The current results indicate that thiazide receptors from rabbit kidney share characteristics with receptors from rat, and that rabbit receptors can be solubilized in CHAPS and purified significantly by hydroxyapatite chromatography and size exclusion HPLC.     

Influence of plasma proteinase inhibitors and aprotinin on trypsin-induced bradykinin release in vitro in man

The consumption of kininogen (measured as kinin-releasable material) was studied in an experimental model in vitro. Analyses were made following the addition of increasing amounts of human cationic trypsin to human serum and plasma. The consumption of kininogen was correlated with the degree of saturation of the plasma proteinase inhibitors alpha 2-macroglobulin (alpha 2-M) and alpha 1-proteinase inhibitor (alpha 1-PI) with trypsin in the presence and absence of aprotinin (Trasylol). The level of kininogen fell dramatically when alpha 2-M was saturated to 70% in spite of 90% free alpha 1-PI. Trypsin-alpha 2-M complexes had no effect on kininogen levels. 60 mumol/l of aprotinin, i.e. approximately 3 X 10(6) KIU/l, blocked only 60% of the trypsin-induced kininogen consumption in serum, while 15 mumol/l of aprotinin blocked 100% of this consumption in plasma. With increasing concentration of aprotinin in serum, a decreasing consumption of alpha 2-M and especially of alpha 1-PI was observed on the addition of trypsin. The high aprotinin concentration needed to block trypsin-induced kininogen cleavage in human serum or plasma may explain the poor clinical effect of aprotinin to date in human acute pancreatitis.     

Further studies of proximal tubular brush border membraned-glucose transport heterogeneity

Summary The properties of two sodium-dependentd-glucose transporters previously identified in renal proximal tubule brush border membrane (BBM) vesicles are studied. The low-affinity system, found in BBM vesicles from the outer cortex (early proximal tubule), is shown to be associated with the high-affinity phlorizin binding site typically found in renal BBM preparations. The high-affinity system, found in BBM vesicles from the outer medulla (late proximal tubule), is almost two orders of magnitude less sensitive to inhibition by phlorizin and is apparently not associated with high-affinity phlorizin binding. The sodium/g;ucose stoichiometry of the outer medullary transporter is found to be 21 by two independent methods. Previous measurements have established that the stoichiometry of the outer cortical system is 11. It is suggested that this arrangement of transporters in series along the proximal tubule enables the kidney to reabsorb glucose from the urine in an energy-efficient fashion. The bulk of the glucose load is reabsorbed early in the proximal tubule at an energetic cost of one Na⁺ per glucose molecule. Then in the late proximal tubule a larger coupling ratio and hence a larger driving force is employed to reabsorb the last traces of glucose from the urine.     

Nitric oxide decreases renal medullary Na+, K+-ATPase activity through cyclic GMP-protein kinase G dependent mechanism.

The aim of this study was to investigate the effect of nitric oxide on renal Na+,K(+)-ATPase and ouabain-sensitive H+,K(+)-ATPase activities. The study was performed in male Wistar rats. The investigated substances were infused under general anaesthesia into abdominal aorta proximally to the renal arteries. The activity of ATPases was assayed in isolated microsomal fraction. NO donor, S-nitroso-N-acetylpenicillamine (SNAP), infused at doses of 10(-7) and 10(-6)mol/kg/min decreased medullary Na+,K(+)-ATPase activity by 29.4% and 45.2%, respectively. Another NO donor, spermine NONOate, administered at the same doses reduced Na+,K(+)-ATPase activity in the renal medulla by 31.7% and 46.5%, respectively. Neither of NO releasers had any effect on Na+,K(+)-ATPase in the renal cortex and on either cortical or medullary ouabain-sensitive H+,K(+)-ATPase. Infusion of NO precursor, L-arginine (100 micromol/kg/min), decreased medullary Na+,K(+)-ATPase activity by 32.2%, whereas inhibitor of nitric oxide synthase, L-NAME (10 nmol/kg/min), increased this activity by 20.7%. The effect of synthetic NO donors was mimicked by 8-bromo-cGMP and blocked by inhibitors of soluble guanylate cyclase, ODQ or methylene blue, as well as by specific inhibitor of protein kinase G, KT5823. In addition, inhibitory effect of either SNAP or 8-bromo-cGMP on medullary Na+,K(+)-ATPase was abolished by 17-octadecynoic acid (17-ODYA), which inhibits cytochrome P450-dependent metabolism of arachidonic acid. These data suggest that NO decreases Na+,K(+)-ATPase activity in the renal medulla through the mechanism involving cGMP, protein kinase G, and cytochrome P450-dependent arachidonate metabolites. In contrast, NO has no effect on Na+,K(+)-ATPase in the renal cortex and on either cortical or medullary ouabain-sensitive H+,K(+)-ATPase.