Expression of rat kallikrein and epithelial polarity in transfected Madin-Darby canine kidney cells.

Many properties of urinary kallikrein are well characterized, but the intracellular processing of prokallikrein and release by kidney cells have yet to be clarified. We report here on the synthesis of prokallikrein in Madin-Darby canine kidney (MDCK) cells transfected with rat submaxillary gland kallikrein cDNA and on its activation by MDCK cells and by an enriched liver Golgi membrane preparation. Transfected MDCK cells secreted only prokallikrein at both the apical and basolateral sides in about a 4:1 ratio, but cells transfected with kallikrein cDNA in reverse orientation or untreated cells released only traces of the enzyme. Prokallikrein, in culture medium or in homogenized MDCK cells, was fully activated by trypsin but activated only to 44% by thermolysin. Prokallikrein was synthesized and released into the medium at a high rate: the enzyme secreted by 5 x 10(6) cells in 24 hours cleaved 46 nmol/min D-Val-Leu-Arg-7-amino-4-methylcoumarin and liberated 63 ng/min bradykinin after activation. Immunocytology indicated the association of prokallikrein with the Golgi apparatus in the transfected cells. Antiserum to rat urinary kallikrein detected a single band in a Western blot of conditioned medium and also immunoprecipitated the enzyme. Aprotinin inhibited activated prokallikrein. Although MDCK cells released prokallikrein, their homogenates activated prokallikrein at both pH 5.5 and 7.5. Prokallikrein was also activated by a highly enriched liver Golgi membrane fraction and by an endoplasmic reticulum preparation, but the Golgi preparation was 38-fold more active. The activation was blocked significantly by inhibitors of serine proteases and less by cysteine protease inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

N-domain-specific substrate and C-domain inhibitors of angiotensin-converting enzyme: angiotensin-(1-7) and keto-ACE.

We used the isolated N- and C-domains of the angiotensin 1-converting enzyme (N-ACE and C-ACE; ACE; kininase II) to investigate the hydrolysis of the active 1-7 derivative of angiotensin (Ang) II and inhibition by 5-S-5-benzamido-4-oxo-6-phenylhexanoyl-L-proline (keto-ACE). Ang-(1-7) is both a substrate and an inhibitor; it is cleaved by N-ACE at approximately one half the rate of bradykinin but negligibly by C-ACE. It inhibits C-ACE, however, at an order of magnitude lower concentration than N-ACE; the IC50 of C-ACE with 100 micromol/L Ang I substrate was 1.2 micromol/L and the Ki was 0.13. While searching for a specific inhibitor of a single active site of ACE, we found that keto-ACE inhibited bradykinin and Ang I hydrolysis by C-ACE in approximately a 38- to 47-times lower concentration than by N-ACE; IC50 values with C-ACE were 0.5 and 0.04 micromol/L. Furthermore, we investigated how Ang-(1-7) acts via bradykinin and the involvement of its B2 receptor. Ang-(1-7) was ineffective directly on the human bradykinin B2 receptor transfected and expressed in Chinese hamster ovary cells. However, Ang-(1-7) potentiated arachidonic acid release by an ACE-resistant bradykinin analogue (1 micromol/L), acting on the B2 receptor when the cells were cotransfected with cDNAs of both B2 receptor and ACE and the proteins were expressed on the plasma membrane of Chinese hamster ovary cells. Thus like other ACE inhibitors, Ang-(1-7) can potentiate the actions of a ligand of the B2 receptor indirectly by binding to the active site of ACE and independent of blocking ligand hydrolysis. This potentiation of kinins at the receptor level can explain some of the well-documented kininlike actions of Ang-(1-7).

Inactivation of endothelin-1 by an enzyme of the vascular endothelial cells.

We previously investigated the inactivation of endothelin-1 by deamidase (lysosomal protective protein), present in many cells, including vascular smooth muscle cells. This enzyme, which we originally purified from human platelets, preferentially hydrolyzes peptides at the C-terminus with hydrophobic amino acids in the P1 or P1' position or both and thereby inactivates endothelin-1, which has a C-terminal sequence of Ile19-Ile20-Trp21-OH. We tested for the presence of deamidase in cultured bovine aortic endothelial cells. The final supernatant of the homogenized cells (S3) cleaved the deamidase substrate dansyl-Phe-Leu-Arg at a rate of 1.3 nmol/min per 10(6) cells at pH 5.5 at 37 degrees C. Endothelin-1 was completely inactivated by the S3 fraction as determined on rat thoracic aorta strips. The major site of inactivation was the Ile20-Trp21 bond, established by high performance liquid chromatography and by amino acid analysis where the main product was des-Trp21-endothelin-1. The hydrolysis of endothelin-1 (5.9 nmol/min per milligram of protein at pH 5.5 at 23 degrees C) by S3 was blocked mainly by inhibitors of deamidase, including diisopropyl fluorophosphate, but not by inhibitors of some other peptidases. This is the first report of a novel pathway of endothelin-1 metabolism in endothelial cells. Thus, endothelial cells, besides being the source of endothelin-1, contain an enzyme that inactivates it.

Hydrolysis of opioid hexapeptides by carboxypeptidase N. Presence of carboxypeptidase in cell membranes.

Carboxypeptidase N, purified to homogeneity from human plasma, rapidly hydrolyzed Lys6- or Arg6-enkephalins when measured by high pressure liquid chromatography. Comparison of the kinetics of hydrolysis of the enkephalin hexapeptides and bradykinin by carboxypeptidase N revealed the following values for the Km and kcat: Arg6-Met5-enkephalin, 49 microM, 1024 min-1; Arg6-Leu5-enkephalin, 57 microM, 375 min-1; Lys6-Met5-enkephalin, 216 microM, 6204 min-1; bradykinin, 19 microM, 58 min-1. Thus, while bradykinin had the lowest Km, the specificity constants (kcat/Km) for all the enkephalin hexapeptides were higher than that of bradykinin due to their high turnover numbers. Preincubation of the enzyme with 0.1 mM CoCl2 increased both the kcat and Km of bradykinin and Arg6-Met5-enkephalin. Similar results were obtained when the above experiments were conducted with the active 48,000 dalton subunit of carboxypeptidase N. Basic carboxypeptidase activity was found in the amniotic fluid, in membrane fractions of various human and bovine tissues, and in cultured cells in the following order of decreasing specific activity: human placental microvilli, human kidney, human amniotic fluid, human lung, bovine lung, bovine pulmonary artery, human foreskin fibroblasts, human pulmonary arterial endothelial cells, and human lung fibroblasts. The membrane-bound carboxypeptidase activity had a neutral pH optimum and behaved similarly to plasma carboxypeptidase N in the presence of various inhibitors and activators. It was different from the carboxypeptidase activity in bovine adrenal chromaffin granules which had an acid pH optimum and was inhibited by sulfhydryl reagents. These studies show that human carboxypeptidase N, an enzyme found in high concentration in blood, readily hydrolyzes Arg6- or Lys6-enkephalins. It could thus control the levels of these peptides if they are released into the circulation from the adrenal gland. In addition, a membrane-bound carboxypeptidase N-like enzyme in various tissues may regulate the local levels of biologically active peptides containing C-terminal basic amino acids such as hexapeptide enkephalins, kinins, anaphylatoxins or fibrinopeptides.

Metabolism of substance P and bradykinin by human neutrophils.

The catabolism of substance P and bradykinin, two peptides involved in inflammation, by human neutrophils was investigated. Substance P was cleaved by unstimulated neutrophils, but the rate of hydrolysis increased greatly (about 4-fold) when the cells were lysed by freezing and thawing or stimulated to release with fMet-Leu-Phe and cytochalasin B. The enzyme responsible for cleaving substance P was cathepsin G, hydrolyzing the Phe7-Phe8 bond. Neutral endopeptidase 24.11 (enkephalinase) became the main inactivating enzyme only when neutrophil cytoplasts (containing plasma membrane but no subcellular particles) or washed plasma membrane enriched high speed sediments were tested. Subcellular fractionation showed the highest substance P degrading activity to be in the granules. Purified cathepsin G readily cleaved substance P with a Km of 1.13 MK, a kcat of 6.35 sec-1 and a kcat/Km of 5639 M-1 sec-1, similar to kinetic constants previously reported for the best peptide substrates of cathepsin G. Despite the high Km, purified cathepsin G did hydrolyze SP at a much lower substrate concentration (down to 1 nM) as determined by radioimmunoassay. Bradykinin was also hydrolyzed by intact neutrophils but, in contrast, was not inactivated by cathepsin G, but by neutral endopeptidase at the Pro7-Phe8 bond. The inactivation of bradykinin by intact neutrophils was decreased by phorbol 12-myristate 13-acetate, probably due to down-regulation by endocytosis of the neutral endopeptidase on the plasma membrane. Thus, both bradykinin and substance P are inactivated by human neutrophils, although by different enzymes. In spite of the less favorable kinetics in vitro than with neutral endopeptidase, cathepsin G is the main inactivator of substance P in neutrophils. This may be due to the estimated 300 to 3600-fold higher concentration of cathepsin G in neutrophils than that of the neutral endopeptidase.

Differences in the hydrolysis of enkephalin congeners by the two domains of angiotensin converting enzyme.

The hydrolysis of enkephalin (Enk) congeners by the isolated N- (N-ACE) and C-domain of angiotensin I converting enzyme (ACE) and by the two-domain somatic ACE was investigated. Both Leu5- and Met5-Enk were cleaved faster by the C-domain than by N-ACE; rates with somatic ACE were 1600 and 2500 nmol/min/nmol enzyme with both active sites being involved. Substitution of Gly2 by D-Ala2 reduced the rate to 1/3rd to 1/7th of that of the Enks. N-ACE cleaved Met5-Enk-Arg6-Phe7 faster than the C-domain, probably with the highest turnover number of any naturally occurring ACE substrate (7600 min(-1)). This heptapeptide is also hydrolyzed in the absence of Cl-, but the activation by Cl- is unique; Cl- enhances the hydrolysis of the heptapeptide by N-ACE but inhibits it by the C-domain, yielding about a 5-fold difference in the turnover number at physiological pH. This difference may result in the predominant role of the N-domain in converting Met5-Enk-Arg6-Phe7 to Enk in vivo.

Renal metabolism of angiotensin I and II.

The enzymatic hydrolysis of angiotensin I and II is reviewed briefly with emphasis on two enzymes, the angiotensin I converting enzyme and neutral endopeptidase 24.11. Angiotensin I is converted to angiotensin II by converting enzyme present in many tissues and highly concentrated in the human kidney and in kidney of some laboratory animals. In addition, there is mounting evidence, collected mostly in experiments in vitro, that other enzymes may be able to activate angiotensin I, for example by the stepwise release of the C-terminal His and Leu residues. Angiotensin I, instead of being activated, could be inactivated by the cleavage of its C-terminal tripeptide either by neutral endopeptidase 24.11 or by prolyl endopeptidase. Angiotensin II is cleaved by several peptidases widely distributed in the kidney. One of the products, des-Phe8-angiotensin II, is not entirely inactive as it has an effect in the CNS.

Hydrolysis of substance p and neurotensin by converting enzyme and neutral endopeptidase.

Angiotensin I converting enzyme (ACE) and neutral endopeptidase ("enkephalinase"; NEP), were purified to homogeneity from human kidney. NEP cleaved substance P (SP) at Gln6-Phe7,-Phe8, and Gly9-Leu10 and neurotensin (NT) at Pro10-Tyr11 and Tyr11-Ile12. NEP hydrolyzed 0.1 mM SP, NT and their C-terminal fragments at the following rates (mumol/min/mg): SP1-11 = 7.8, SP4-11 = 11.7, SP5-11 = 15.4, SP6-11 = 15.6, SP8-11 = 6.7, NT1-13 = 2.9, and NT8-13 = 4.0. Purified ACE rapidly inactivated SP as measured in bioassay. HPLC analysis showed that ACE cleaved SP at Phe8-Gly9 and Gly9-Leu10 to release C-terminal tri- and dipeptide (ratio = 4:1). The hydrolysis was Cl- dependent and inhibited by captopril. ACE released mainly C-terminal tripeptide from SP methyl ester, but only dipeptide from SP free acid. Modification of arginine residues in ACE with cyclohexanedione or butanedione similarly inhibited hydrolysis of SP, bradykinin and Bz-Gly-Phe-Arg (80-93%) indicating an active site arginine is required for hydrolysis of SP. ACE hydrolyzed NT at Tyr11-Ile12 to release Ile12-Leu13. SP, NT and their derivatives (0.1 mM) were cleaved by ACE at the following rates (mumol/min/mg): SP1-11 = 1.2, SP methyl ester = 0.7, SP free acid = 8.5, SP4-11 = 2.4, SP5-11 = 0.9, SP6-11 = 1.4, SP8-11 = 0, NT1-13 = 0.2, and NT8-13 = 1.3. Peptide substrates were used as inhibitors of ACE (substrate = FA-Phe-Gly-Gly) and NEP (substrate = Leu5-enkephalin).(ABSTRACT TRUNCATED AT 250 WORDS)

Enzymes in placental microvilli: angiotensin I converting enzyme, angiotensinase A, carboxypeptidase, and neutral endopeptidase ("enkephalinase").

Microvilli from human placental syncytiotrophoblast are rich in angiotensin I converting enzyme (ACE), aminopeptidase A, a carboxypeptidase N-like enzyme, and a neutral endopeptidase (NEP). The specific activities of these enzymes were enhanced in microvillus-enriched fractions obtained by differential centrifugation: Purified microvilli were isolated in a discontinuous sucrose gradient. The placental microvilli hydrolyzed angiotensin II, vasopressin and oxytocin as shown by high pressure liquid chromatography. The inhibitors, bestatin, phosphoramidon, and o-phenanthroline, established the specificity of the enzymes. Aminopeptidase A (angiotensinase A) cleaved angiotensin II to angiotensin III and Asp1. NEP from placenta and from human kidney hydrolyzed oxytocin at the Pro7-Leu8 bond to yield oxytocin 1-7 and leucyl-glycine amide, but did not hydrolyze vasopressin. Vasopressin was cleaved by aminopeptidases in the placental membranes. On electroblotting placental NEP appeared as a double band with a molecular weight slightly higher than the 90,000 of the purified kidney enzyme. Neuraminidase treatment reduced the molecular weight of the placental enzyme to approximately 90,000, indicating that it contains a large amount of sialic acid. The microvilli of human placenta are thus rich in enzymes that may regulate passage of peptides at the maternal-fetal interface.

Des-Arg9-bradykinin metabolism in patients who presented hypersensitivity reactions during hemodialysis: role of serum ACE and aminopeptidase P.

Bradykinin (BK) has been proposed as the principal mediator of hypersensitivity reactions (HSR) in patients dialyzed using negatively charged membranes and concomitantly treated with angiotensin-converting enzyme (ACE) inhibitors. We investigated the metabolism of exogenous BK added to the sera of 13 patients dialyzed on an AN69 membrane with a history of HSR (HSR+ patients) and 10 others who did not present such a reaction (HSR- patients) while dialyzed under the same conditions. No significant difference in the t1/2 of BK was found between the patient groups. However, the t1/2 of generated des-Arg9-BK was significantly increased (2.2-fold) in HSR+ patients compared to HSR-subjects. Preincubation of the sera with an ACE inhibitor (enalaprilat) significantly increased the t1/2 of both BK and des-Arg9-BK in both groups. There was no significant difference between the groups with respect to the t1/2 of BK, but there was a significantly greater increase (3.8-fold) in the t1/2 of des-Arg9-BK in HSR+ patients compared to HSR-subjects. The level of serum aminopeptidase P (APP) activity showed a significant decrease in the HSR+ sera when compared to HSR-samples. In HSR- and HSR+ patients, a significant inverse relation (r2 = 0.6271; P < 0.00005) could be calculated between APP activity and des-Arg9-BK t1/2. In conclusion, HSR in hemodialyzed patients who are concomitantly treated with a negatively charged membrane and an ACE inhibitor can be considered as a multifactorial disease in that a decreased APP activity resulting in reduced degradation of des-Arg9-BK may lead to the accumulation of this B1 agonist that could be responsible, at least in part, for the signs and symptoms of HSR.

Identification of a membrane-bound carboxypeptidase as the mammalian homolog of duck gp180, a hepatitis B virus-binding protein.

A unique membrane-bound carboxypeptidase was discovered and characterized in membrane fractions of human skin fibroblasts and the mouse monocyte-macrophage cell line J774A.1 and was partially purified from human placenta. Enzymatic characterization identified it as a new member of the regulatory B-type metallocarboxypeptidases, different from carboxypeptidases B, E, M, N and U. It is, however, similar to the newly described bovine carboxypeptidase D, suggested to be a homolog of duck gp180, a 180 kDa hepatitis B virus-binding protein. To prove this, a partial cDNA encoding a 20 kDa fragment of the human homolog of duck gp180 was expressed in bacteria and the recombinant protein was purified. Antibodies raised to the protein immunoprecipitated 94% or 72% of the low pH carboxypeptidase activity in human skin fibroblasts or J774A.1 cells and gave a 175 kDa protein band in Western blots. Thus, carboxypeptidase D is the mammalian homolog of duck gp180 and is distributed in a variety of different cell types.

Kininase one-an'-a-half: the newest member of the kininase family.

A kininase I-like enzyme (carboxypeptidase) was purified to homogeneity from human urine and compared to the 48,000 mol. wt. (48K) active subunit of carboxypeptidase N. The urinary carboxypeptidase had a mol. wt. of 73,000 in gel filtration and 76,000 in SDS-polyacrylamide gel electrophoresis. It had a pH optimum of 7.0 and differed from the 48K subunit in stability, susceptibility to trypsin, and enzymatic activity. The urinary enzyme did not cross-react with antibody to carboxypeptidase N in "Western blotting". Urine from a patient genetically deficient in plasma carboxypeptidase N (21% of normal) contained normal levels of urinary carboxypeptidase with similar properties to that from pooled human urine. Membrane fractions from several tissues contained a similar carboxypeptidase activity. The activity was highest in a microvillous membrane fraction from human placenta (65 nmol/min/mg with Bz-Gly-Lys as substrate). High specific activities were also found in membrane fractions of human kidney (18 nmol/min/mg) and lung (8 nmol/min/mg). The membrane-bound enzyme was distinguished from lysosomal and catheptic carboxypeptidases as well as "enkephalin convertase" by the use of specific inhibitors. These results show that urine contains a carboxypeptidase capable of cleaving arginine or lysine from the C-terminus of peptides. The enzyme does not arise from plasma carboxypeptidase N, but may be released into the urine from the renal brush border.

Functional significance of the subunits of carboxypeptidase N (kininase I).

Carboxypeptidase N (kininase I; 280K) was purified to homogeneity from human plasma. The inactive 83K and active 48K subunits were separated by gel filtration after treatment of homogeneous 280K with guanidine. The two subunits differ in amino acid composition and immunological reactivity. The activities of the 280K and 48K enzymes with naturally occurring substrates were compared to determine whether the 83K subunit affects enzymatic activity of the 48K. At 60 microM concentration, both the 280K and 48K enzymes cleaved (Lys6)-Met5)-enkephalin fastest followed by (Arg6)-(Met5)-enkephalin, anaphylatoxin C3a, (arg6)-(Leu5)-enkephalin, C3a octapeptide and bradykinin. The activity ratios (280K/48K) were: (Arg6)-(Leu5)-enkephalin, 0.9; bradykinin, 1.0; (Lys6)-(Met5)-enkephalin, 1.1; (Arg6)-(Met5)-enkephalin, 1.2; and anaphylatoxin C3a, 1.7. Thus, while most substrates were cleaved at similar rates, assuming 2 active sites per 280K molecule, anaphylatoxin C3a was cleaved significantly faster by 280K than by 48K. The ratio of activity was similar (1.9) when the C-terminal octapeptide of C3a was the substrate. These results indicate that the larger, inactive 83K subunit may increase the efficiency of cleavage of some peptides by 48K.

Differential regulation of inducible and endothelial nitric oxide synthase by kinin B1 and B2 receptors.

Kinins are vasoactive peptides that play important roles in cardiovascular homeostasis, pain and inflammation. After release from their precursor kininogens, kinins or their C-terminal des-Arg metabolites activate two distinct G protein-coupled receptors (GPCR), called B2 (B2R) or B1 (B1R). The B2R is expressed constitutively with a wide tissue distribution. In contrast, the B1R is not expressed under normal conditions but is upregulated by tissue insult or inflammatory mediators. The B2R is considered to mediate many of the acute effects of kinins while the B1R is more responsible for chronic responses in inflammation. Both receptors can couple to Galphai and Galphaq families of G proteins to release mediators such as nitric oxide (NO), arachidonic acid, prostaglandins, leukotrienes and endothelium-derived hyperpolarizing factor and can induce the release of other inflammatory agents. The focus of this review is on the different transduction events that take place upon B2R and B1R activation in human endothelial cells that leads to generation of NO via activation of different NOS isoforms. Importantly, B2R-mediated eNOS activation leads to a transient ( approximately 5min) output of NO in control endothelial cells whereas in cytokine-treated endothelial cells, B1R activation leads to very high and prolonged ( approximately 90min) NO production that is mediated by a novel signal transduction pathway leading to post-translational activation of iNOS.