Kinins, receptors, kininases and inhibitors--where did they lead us?

Based on studies presented here and other published experiments performed with surviving tissue preparations, with transfected cells and with cells that constitutively express the human angiotensin I converting enzyme ACE and B2 receptors, we concluded the following: ACE inhibitors and other endogenous peptides that react with the active site of ACE potentiate the effect of bradykinin and its ACE resistant peptide congeners on the B2 receptor. They also resensitize receptors which had been desensitized by the agonist. ACE and bradykinin receptors have to be sterically close, possibly forming a heterodimer, for the ACE inhibitors to induce an allosteric modification on the receptor. When ACE inhibitors augment bradykinin effects, they reduce the phosphorylation of the B2 receptor. The primary actions of bradykinin on the receptor are not affected by protein kinase C or phosphatase inhibitors, but the potentiation of bradykinin or the resensitization of the receptor by ACE inhibitors are abolished by the same inhibitors. The results with protein kinase C and phosphatase inhibitors indicate that another intermediate protein may be involved in the processes of signaling induced by ACE inhibitors, and that ACE inhibitors affect the signal transduction pathway triggered by bradykinin on the B2 receptor.

Potentiation of the effects of bradykinin on its receptor in the isolated guinea pig ileum.

Angiotensin I-converting enzyme (ACE/kininase II) inhibitors potentiated guinea pig ileum's isotonic contractions to bradykinin (BK) and its analogues, shifting the BK dose-response curve to the left. ACE inhibitors added at the peak of the contraction immediately enhanced it further (343 +/- 40%), although the ileum inactivated BK slowly (t(1/2) = 12-16 min). Chymotrypsin and cathepsin G also augmented the activity of BK up to three- or four-fold, but in a manner slower than that of ACE inhibitors. The BK B(2) receptor blocker HOE 140 inhibited all effects. Histamine and angiotensin II were not potentiated. ACE inhibitors potentiate BK independent of blocking its inactivation by inducing crosstalk between ACE and the BK B(2) receptor; proteases activate the receptor by different mechanism.

Human bradykinin B(2) receptor is activated by kallikrein and other serine proteases.

Bradykinin (BK) and kallidin (Lys-BK), liberated from kininogens by kallikreins, are ligands of the BK B(2) receptor. We investigated whether kallikreins, besides releasing peptide agonist, could also activate the receptor directly. We studied the effect of porcine and human recombinant tissue kallikrein and plasma kallikrein on [Ca(2+)](i) mobilization and [(3)H]arachidonic acid release from cultured cells stably transfected to express human BK B(2) receptor (CHO/B(2), MDCK/B(2), HEK/B(2)), and endothelial cells were used as control cells. As with BK, the actions of kallikrein were blocked by the B(2) antagonist, HOE 140. Kallikrein was inactive on cells lacking B(2) receptor. Kallikrein and BK desensitized the receptor homologously but there was no cross-desensitization. Furthermore, 50 nM human cathepsin G and 50 nM trypsin also activated the receptor; this also was blocked by HOE 140. Experiments excluded a putative kinin release by proteases. [(3)H]AA release by BK was reduced by 40% by added kininase I (carboxypeptidase M); however, receptor activation by tissue kallikrein, trypsin, or cathepsin G was not affected. Prokallikrein and inhibited kallikrein were inactive, suggesting cleavage of a peptide bond in the receptor. Kallikreins were active on mutated B(2) receptor missing the 19 N-terminal amino acids, suggesting a type of activation different from that of thrombin receptor. Paradoxically, tissue kallikreins decreased the [(3)H]BK binding to the receptor with a low K(D) (3 nM) and inhibited it 78%. Thus, kallikreins and some other proteases activate human BK B(2) receptor directly, independent of BK release. The BK B(2) receptor may belong to a new group of serine protease-activated receptors.

Protein kinase C and phosphatase inhibitors block the ability of angiotensin I-converting enzyme inhibitors to resensitize the receptor to bradykinin without altering the primary effects of bradykinin.

Angiotensin I-converting enzyme (kininase II) inhibitors (ACEis) are very widely used to treat cardiac conditions and nephropathies, but some of their beneficial activities cannot be attributed to enzyme inhibition alone. We investigated the effects of ACEis on the human bradykinin (BK) B(2) receptor expressed in Chinese hamster ovary cells transfected with the cDNA of human receptor and ACE, and on human pulmonary endothelial cells that constitutively express both proteins. BK and its ACE-resistant peptide analog activated the B(2) receptor to release arachidonic acid and elevate [Ca(2+)](i) and subsequently desensitized it. The release of arachidonic by BK was independent of extracellular Ca(2+). BK enhanced phosphorylation of the immunoprecipitated B(2) receptor but enalaprilat significantly reduced it. ACEi resensitized the receptor by initiating a cross talk between the receptor and ACE. Protein kinase C and phosphatase inhibitors distinguished the signaling by the receptor when activated first by BK from BK acting on the resensitized receptor. Treatment of cells with 1 microM calphostin, 100 nM staurosporine, 100 nM calyculin, or 500 nM okadaic acid did not affect either one of the primary actions of BK on the receptor. Protein kinase C or phosphatase inhibitors, however, blocked the effects of BK on the receptor resensitized by enalaprilat or ramiprilat. The experiments clearly differentiate the primary activation of the receptor by BK from activation of the resensitized receptor after ACEi treatment. The existence of an intermediate component involved in the action of ACEis to enhance release of vasoactive mediators by BK is suggested.

Effects of the N-terminal sequence of ACE on the properties of its C-domain.

Angiotensin I-converting enzyme (ACE, kininase II) has 2 active domains (N and C) in a single peptide chain. Because we found its N-domain more stable than its C-domain, we investigated the effect of the amino-terminus of human ACE on the C-domain with a molecular construct expressed in Chinese hamster ovary cells (CHO) cells and transiently in HEK293 cells. This active N-deleted ACE contained only the first 141 amino acids of the human N-domain but not its active center and was linked to the active C-domain containing the transmembrane and cytosolic portions of ACE. The CHO cells were also transfected with human B(2) bradykinin receptor. ACE inhibitors (5 nmol/L or 1 micromol/L) augmented bradykinin (100 nmol/L) effects, elevated B(2) receptor numbers, and resensitized the receptor desensitized by agonist as measured by arachidonic acid release or [Ca(2+)](i) mobilization. Arachidonic acid release was mediated by pertussis toxin-sensitive G alpha(i), and [Ca(2+)](i) mobilization was mediated by pertussis-insensitive G alpha(q) protein receptor complex. The properties of the construct were compared with wild-type ACE and separate N- and C-domains. The N-deleted ACE differed from wild-type in activation by Cl(-) and [SO(4)](2-) ions, hydrolysis ratios of substrates (both short synthetic and endogenous peptides) and heat stability. Thus, the N-terminal peptide of ACE affected the characteristics of the C-domain active center. ACE inhibitors acting on N-deleted ACE, which had only a single C-domain active center anchored to plasma membrane, induced cross-talk between the enzyme and the B(2) receptor (eg, the inhibitors resensitized the receptor) independent of blocking bradykinin inactivation.

Replacement of the transmembrane anchor in angiotensin I-converting enzyme (ACE) with a glycosylphosphatidylinositol tail affects activation of the B2 bradykinin receptor by ACE inhibitors.

To investigate further the relationship of angiotensin I-converting enzyme (ACE) inhibitors to activation of the B(2) bradykinin (BK) receptor, we transfected Chinese hamster ovary cells to stably express the human receptor and either wild-type ACE (WT-ACE), an ACE construct with most of the cytosolic portion deleted (Cyt-del-ACE), or ACE with a glycosylphosphatidylinositol (GPI) anchor replacing the transmembrane and cytosolic domains (GPI-ACE). BK or its ACE-resistant analogue were the agonists. All activities (arachidonic acid release and calcium mobilization) were blocked by the B(2) antagonist HOE 140. B(2) was desensitized by repeated administration of BK but resensitized to agonist by ACE inhibitors in the cells expressing both B(2) and either WT-ACE or Cyt-del-ACE. In GPI-ACE expressing cells, the B(2) receptor was still activated by the agonists, but ACE inhibitors did not resensitize. Pretreatment with filipin returned the sensitivity to inhibitors. In immunocytochemistry, GPI-ACE showed patchy, uneven distribution on the plasma membrane that was restored by filipin. Thus, ACE inhibitors were inactive as long as GPI-ACE was sequestered in cholesterol-rich membrane domains. WT-ACE and B(2) receptor in Chinese hamster ovary cells co-immunoprecipitated with antibody to receptor, suggesting an interaction on the cell membrane. ACE inhibitors augment BK effects on receptors indirectly only when enzyme and receptor molecules are sterically close, possibly forming a heterodimer.

Enhancement of bradykinin and resensitization of its B2 receptor.

We studied the enhancement of the effects of bradykinin B2 receptor agonists by agents that react with active centers of angiotensin-converting enzyme (ACE) independent of enzymatic inactivation. The potentiation and the desensitization and resensitization of B2 receptor were assessed by measuring [3H]arachidonic acid release and [Ca2+]i mobilization in Chinese hamster ovary cells transfected to express human ACE and B2 receptor, or in endothelial cells with constitutively expressed ACE and receptor. Administration of bradykinin or its ACE-resistant analogue desensitized the receptor, but it was resensitized (arachidonic acid release or [Ca2+]i mobilization) by agents such as enalaprilat (1 micromol/L). Enalaprilat was inactive in the absence of ACE expression. La3+ (100 micromol/L) inhibited the apparent resensitization, probably by blocking the entry of extracellular calcium. Enalaprilat resensitized the receptor via ACE to release arachidonic acid by bradykinin at a lower concentration (5 nmol/L) than required to mobilize [Ca2+]i (1 micromol/L). Monoclonal antibodies inhibiting the ACE N-domain active center and polyclonal antiserum potentiated bradykinin. The snake venom peptide BPP5a and metabolites of angiotensin and bradykinin (angiotensin-[1-9], angiotensin-[1-7], bradykinin-[1-8]; 1 micromol/L) enhanced arachidonic acid release by bradykinin. Angiotensin-(1-9) and -(1-7) also resensitized the receptor. Enalaprilat potentiated the bradykinin effect in cells expressing a mutant ACE with a single N-domain active site. Agents that reacted with a single active site, on the N-domain or on the C-domain, potentiated bradykinin not by blocking its inactivation but by inducing crosstalk between ACE and the receptor. Enalaprilat enhanced signaling via ACE by Galphai in lower concentration than by Galphaq-coupled receptor.

Cellular carboxypeptidases.

This article focuses on four human carboxypeptidases (CPs): two metallo-CPs and two serine CPs. The metallo-CPs are members of the so-called B-type regulatory CP family, as they cleave only the C-terminal basic amino acids Arg or Lys. The plasma membrane-bound CPM and the mainly, but not exclusively, intracellular CPD are surveyed from this group of enzymes. These enzymes can regulate peptide hormone activity at the cell surface and possibly intracellularly after receptor-mediated endocytosis and may also participate in peptide hormone processing. The serine CPs, as their name indicates, contain a serine residue in the active center essential for catalytic activity that reacts with organophosphorus inhibitors. Prolylcarboxypeptidase (PRCP) (angiotensinase C) and deamidase (cathepsin A, lysosomal protective protein) are discussed here. These two enzymes are highly concentrated in lysosomes; however, they may also be active extracellularly after their release from lysosomes in soluble form or in a plasma membrane-bound complex. Whereas deamidase cleaves a variety of peptides with C-terminal or penultimate hydrophobic residues (e.g. substance P, angiotensin I, bradykinin, endothelin, fMet-Leu-Phe). PRCP cleaves only peptides with a penultimate Pro residue (e.g. des-Arg9-bradykinin, angiotensin II). These enzymes may also be involved in terminating signal transduction by inactivating peptide ligands after receptor endocytosis.

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).

Detection of neutral endopeptidase 24.11 (neprilysin) in human hepatocellular carcinomas by immunocytochemistry.

BACKGROUND: We have reported previously that neutral endopeptidase 24.11 (neprilysin; NEP; CALLA, CD10) activity was very high in rat hepatomas and a cultured human hepatocarcinoma cell line (SK-HEP1). MATERIALS AND METHODS: While continuing these studies, we detected the presence of NEP in SK-HEP 1 cells by immunocytochemistry and in paraffin-embedded human hepatocellular carcinomas as well. IgG purified from polyclonal antisera to human NEP was employed as a source of antibody. RESULTS: SK-HEP 1 cells gave a strong positive reaction to the IgG fraction of the antisera. In control studies, where IgG was preabsorbed with recombinant NEP, the results were negative. Of the 18 hepatocellular carcinomas tested, NEP was expressed in 14 (78%) malignant tumors, while adjacent liver tissue did not show the presence of NEP. CONCLUSIONS: It is suggested that, because none of the known hepatocellular carcinoma markers are highly specific, the detection of NEP in these malignant cells can be an additional useful diagnostic tool.

Potentiation of the actions of bradykinin by angiotensin I-converting enzyme inhibitors. The role of expressed human bradykinin B2 receptors and angiotensin I-converting enzyme in CHO cells.

Part of the beneficial effects of angiotensin I-converting enzyme (ACE) inhibitors are due to augmenting the actions of bradykinin (BK). We studied this effect of enalaprilat on the binding of [3H]BK to Chinese hamster ovary (CHO) cells stably transfected to express the human BK B2 receptor alone (CHO-3B) or in combination with ACE (CHO-15AB). In CHO-15AB cells, enalaprilat (1 mumol/L) increased the total number of low-affinity [3H]BK binding sites on the cells at 37 degrees C, but not at 4 degrees C, from 18.4 +/- 4.3 to 40.3 +/- 11.9 fmol/10(6) cells (P < .05; Kd, 2.3 +/- 0.8 and 5.9 +/- 1.3 nmol/L; n = 4). Enalaprilat preserved a portion of the receptors in high-affinity conformation (Kd, 0.17 +/- 0.08 nmol/L; 8.1 +/- 0.9 fmol/10(6) cells). Enalaprilat decreased the IC50 of [Hyp3-Tyr(Me)8]BK, the BK analogue more resistant to ACE, from 3.2 +/- 0.8 to 0.41 +/- 0.16 nmol/L (P < .05, n = 3). The biphasic displacement curve of the binding of [3H]BK also suggested the presence of high-affinity BK binding sites. Enalaprilat (5 nmol to 1 mumol/L) potentiated the release of [3H]arachidonic acid and the liberation of inositol 1,4,5-trisphosphate (IP3) induced by BK and [Hyp3-Tyr(Me)8]BK. Moreover, enalaprilat (1 mumol/L) completely and immediately restored the response of the B2 receptor, desensitized by the agonist (1 mumol/L [Hyp3-Tyr(Me)8]BK); this effect was blocked by the antagonist, HOE 140. Finally, enalaprilat, but not the prodrug enalapril, decreased internalization of the receptor from 70 +/- 9% to 45 +/- 9% (P < .05, n = 7). In CHO-3B cells, enalaprilat was ineffective. ACE inhibitors in the presence of both the B2 receptor and ACE enhance BK binding, protect high-affinity receptors, block receptor desensitization, and decrease internalization, thereby potentiating BK beyond blocking its hydrolysis.

Angiotensin I-converting enzyme inhibitors potentiate bradykinin's inotropic effects independently of blocking its inactivation.

The positive inotropic effects of bradykinin (BK) and 2 analogs resistant to angiotensin I-converting enzyme (ACE) were potentiated on isolated guinea pig atrial preparations by enalaprilat. The stable BK analogs, dextran-BK and [Hyp3-Tyr(Me)8]-BK, were as active as BK. Pretreatment for 5 min with enalaprilat augmented the maximal positive inotropic effect of [Hyp3-Tyr(Me)8]-BK 2.8-fold, from 19% to 53% and that of BK from 28% to 42% over baseline; inotropic responses to dextran-BK (1 microM) were similarly increased. The activity of atrial ACE, a zinc-requiring enzyme, was completely inhibited by 8-hydroxyquinoline-5-sulfonic acid (QSA, 10 mM), which raised the maximal inotropic effect of BK to 39% above baseline. This value rose to 67% when in addition to QSA, 1 microM enalaprilat was added; enalaprilat thus, potentiated the effects of BK independently of enzyme inhibition. The positive inotropic effects to BK and its analogs decline with time in the presence of these agonists. After 10 min of exposure, the response to 1 microM [Hyp3-Tyr(Me)8]-BK decreased to about half, and after 20 min, to 0. Enalaprilat, when present in the tissue bath, prevented the decline in inotropy; even after tachyphylaxis occurred, it reversed this decrease in activity when added. The effects of 1 microM [Hyp3-Tyr(Me)8]-BK, in the absence or presence of enalaprilat, were abolished by the BK B2 receptor antagonist icatibant (0.75 microM). The results indicate that ACE inhibitors, by potentiating the BK effects and blocking BK B2-receptor desensitization, may contribute to the beneficial cardiac effects of BK independently of blocking its inactivation.

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.

Single-domain angiotensin I converting enzyme (kininase II): characterization and properties.

Somatic angiotensin I converting enzyme (ACE; kininase II) has two active sites, in two (N and C) domains. We studied the active centers with separate N-domain ACE (N-ACE), testicular C-domain ACE (germinal ACE) and, as control, renal somatic ACE. Germinal ACE cleaved the nonapeptide bradykinin about two times faster than N-ACE in 20 mM Cl-. Bradykinin1-7 was hydrolyzed further to bradykinin1-5 by N-ACE four times faster in the absence of Cl-, but at 300 mM Cl- the C-domain hydrolyzed it twice as fast. The hematopoietic system regulatory peptide acetyl-Ser-Asp-Lys-Pro was split to two dipeptides by N-ACE, depending on the chloride concentration, 8 to 24 times faster than by germinal ACE; at 100 mM Cl-, the Kcat with N-ACE was eight times higher. One millimolar 1-fluoro-2,4-dinitrobenzene inhibited germinal ACE 96% but it inhibited N-ACE by only 31%. [3H]Ramiprilat was displaced by other unlabeled ACE inhibitors to establish their relative affinities. Captopril had the lowest IC50 (0.5 nM) with N-ACE and the highest IC50 (8.3 nM) with the germinal ACE. The IC50 values of ramiprilat and quinaprilat were about the same with both active sites. The association and dissociation constants of [3H]ramiprilat indicated faster association with and faster dissociation from N-ACE than from germinal ACE. After exposure to alkali or moderate heat, somatic ACE was cleaved by plasmin and kallikrein, releasing N-ACE and apparently inactivating the C-domain. These studies affirm the differences in the activity, stability and inhibition of the two active sites of ACE.

Removal of Arg141 from the alpha chain of human hemoglobin by carboxypeptidases N and M.

Both human plasma carboxypeptidase N (CPN) and membrane-bound carboxypeptidase M (CPM) released the C-terminal arginine (alpha-Arg141) of the alpha chain of human adult hemoglobin. An arginase contamination present in the hemoglobin preparation, which converted the released arginine to ornithine, was removed by gel filtration. CPM was about 20 times more efficient than CPN or its active subunit in hydrolyzing oxyhemoglobin and cleaved oxyhemoglobin twice as fast as deoxyhemoglobin. The hydrolysis of the peptide bond of alpha-Arg141 accelerated the dissociation rate of the tetramer deoxy-des-alpha-Arg141 hemoglobin to dimers 2500-fold over that of deoxyhemoglobin, as measured by haptoglobin binding. Moreover, the dissociation of the deoxy-des-alpha-Arg141 hemoglobin tetramer to dimers was not affected by 2,3-diphosphoglyceric acid. Des-alpha-Arg141 hemoglobin had a higher oxygen affinity (P50, 5.51 mm Hg; control, 19.94 mm Hg [P50 is the partial pressure of oxygen that gives 50% of the saturation of hemoglobin]) and a lower apparent cooperativity (Hill coefficient: n, 1.02; control, 2.24) than unhydrolyzed hemoglobin. After hemoglobin was incubated in human plasma, its oxygen-binding parameters, the P50, and the Hill coefficient decreased drastically due to cleavage by CPN. In the perfused rat heart, des-alpha-Arg141 hemoglobin was a more effective coronary vasoconstrictor than hemoglobin, possibly because it dissociated to dimers in the coronary vascular bed. A covalently cross-linked hemoglobin was less active than native hemoglobin. The coronary vasoconstriction was caused by multiple factors, including interference with vasodilation by nitric oxide and eicosanoids. Thus, the hydrolysis of hemoglobin by CPM and CPN demonstrated the contribution of the alpha-Arg141 residue to sustaining the tetrameric structure of hemoglobin and its normal oxygen affinity and vasoactivity.

Kininase II-type enzymes. Their putative role in muscle energy metabolism.

Because of the importance of bradykinin in improving heart function in some conditions or in enhancing glucose uptake by skeletal muscle, we investigated kininases in these tissues. In P3 fraction of the heart and skeletal muscles, angiotensin I-converting enzyme (ACE) and neutral endopeptidase 24.11 (NEP) are the major kininases, as determined first with specific substrates and second with bradykinin. ACE activity was highest in guinea pig heart (2.7 +/- 0.07 mumol.h-1.mg protein-1) but decreased in other species in this order: dog atrium, rat heart, dog ventricle, and human atrium. The specific activity of NEP was lower: 0.45 mumol.h-1.mg protein-1 in cultured neonatal cardiac myocytes and varying between 0.12 and 0.05 mumol.h-1.mg protein-1 in human, dog, rat, and guinea pig heart. In the skeletal muscle P3, ACE was most active in guinea pig and rat (1.2 and 1.1 mumol.h-1.mg protein-1, respectively) but less so in dog (0.09 mumol.h-1.mg protein-1). NEP activity was higher in dog P3 (0.28 mumol.h-1.mg protein-1) but lower in rat and guinea pig (0.19 and 0.1 mumol.h-1.mg protein-1, respectively). Continuous density gradient centrifugation enriched NEP activity in dog and rat (from 0.3 to 1.0 and 0.49 mumol.h-1.mg protein-1, respectively). Immunoprecipitation with antiserum to purified NEP proved the specificity of the rat enzyme. Bradykinin (0.1 mmol/l) was inactivated in the presence and absence of inhibitors by rat skeletal muscle NEP, as measured by high-performance liquid chromatography. Here, 36% of the activity was caused by NEP and 19% by ACE. In radioimmunoassay (bradykinin 10 nmol/l), 46 and 55% of kininase in rat and dog skeletal muscle P3, respectively, was due to ACE; 36 and 28%, respectively, was due to NEP. Aside from these enzymes, an aminopeptidase in rat P3 also inactivates bradykinin. Thus, in conclusion, heart and skeletal muscle membranes contain kininase II-type enzymes, but their activity depends on the species.

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)

Carboxypeptidase M activity is increased in bronchoalveolar lavage in human lung disease.

Carboxypeptidase M (CPM) cleaves the C-terminal arginine and lysine of peptides; it is expressed in the lung, especially on the plasma membrane of alveolar type I cells. Here, we report on CPM in human bronchoalveolar lavage (BAL) collected from 69 patients and analyzed for activity, cell number and type, and protein level. Seventy-six percent of CPM activity, measured at pH 7.5 with 5-dimethylamino-naphthalene-1-sulfonyl-alanyl-arginine (Dansyl-Ala-Arg) substrate, was immunoprecipitated with polyclonal antibody to purified human enzyme. In patients without active lung disease, CPM activity in BAL was 7.69 (+/- 2.12) nmol/h/mg protein, but in patients with acute pneumonia, it was 29.25 (+/- 4.06) (p < 0.01). In patients with Pneumocystis carinii pneumonia, CPM activity was elevated to 26.00 (+/- 4.85) (p < 0.01) and in patients with lung cancer, to 30.95 (+/- 4.12) (p < 0.01). The activity was not associated with the cellular elements of BAL. The highest specific activity was in the large aggregate fraction of surfactant, which also contained the highest concentration of phosphorus. Transmission electron microscopy of this fraction revealed the presence of typical lamellar bodies and tubular myelin structures. The high CPM activity may stem from its induction and release in acute lung disease. In addition, CPM may be a marker of infection with certain pathogens and an indicator of type I cell injury in parenchymal lung diseases.