Bradykinin metabolism in the isolated perfused rabbit heart.

BACKGROUND: Bradykinin is a potent cardioprotective hormone, the beneficial role of which in vivo appears to be limited by its rapid metabolism. Inhibitors of peptidases that degrade endogenously formed bradykinin are themselves cardioprotective, presumably by increasing local bradykinin concentrations. As bradykinin-degrading peptidases are potential therapeutic targets, it is important to identify these enzymes in different animal models of cardiac function. OBJECTIVE: To determine the mechanism of bradykinin degradation in the coronary circulation of the rabbit, using an isolated perfused heart preparation. DESIGN AND METHODS: [3H]Bradykinin (16 nmol/l) was perfused as a bolus through the isolated rabbit heart in the presence and absence of specific peptidase inhibitors. The effluent was collected and the radiolabeled metabolites of [3H]bradykinin were separated by high performance liquid chromatography, identified, and quantified. RESULTS: [3H]Bradykinin was metabolized to the extent of 62 +/- 3% in a single passage through the rabbit coronary circulation at a physiological flow rate. The metabolites were identified as [3H]bradykinin(1-5) and [3H]bradykinin(1-7),accounting for 50 +/- 4 and 12 +/- 2% of the radioactivity, respectively. Co-perfusion with the angiotensin converting enzyme inhibitor, ramiprilat, completely blocked formation of these metabolites. CONCLUSIONS: Angiotensin-converting enzyme fully accounts for the metabolism of [3H]bradykinin in the rabbit coronary circulation. This result contrasts with data obtained using rat heart, which demonstrated a prominent role for aminopeptidase P in bradykinin metabolism in this species.

Vascular catabolism of bradykinin in the isolated perfused rat kidney.

Kinins in the circulation are rapidly metabolized by several different peptidases. The purpose of this study was to evaluate the contribution of membrane-bound peptidases to kinin metabolism in the renal circulation. Experiments were performed in vitro, in isolated rat kidneys perfused at a constant flow rate (8 ml/min) with Tyrode's solution. The effects of peptidase inhibitors were evaluated on the functional vasodilator response caused by bradykinin (30 nM) or [Tyr(Me)(8)]bradykinin (10 nM) via activation of bradykinin B2 receptors in kidneys precontracted with prostaglandin F2alpha. Angiotensin converting enzyme inhibitors, enalaprilat (3 microM), ramiprilat (1 microM) or lisinopril (1 microM), increased the bradykinin-induced renal vasodilation by 40% or more. Inhibitors of neutral endopeptidase (thiorphan or phosphoramidon, 10 microM), basic carboxypeptidase (DL-2-mercaptomethyl-3-guanidino-ethylthiopropanoic acid or MGTPA, 10 microM) and aminopeptidase P (apstatin, 20 microM) however did not enhance the renal vasodilator response elicited by kinins, whatever tested alone or in the presence of lisinopril. These findings indicate that angiotensin converting enzyme is the major peptidase whose inhibition potentiates the renal bradykinin B2 receptor mediated vasodilator response of kinins. The relative contribution in this potentiation of inhibition of kinin inactivation and of cross-talk of angiotensin converting enzyme with bradykinin B2 receptor remains however to be clarified.

Inhibition of aminopeptidase P potentiates wheal response to bradykinin in angiotensin-converting enzyme inhibitor-treated humans.

Bradykinin is a nonapeptide that contributes to the cardioprotective effects of angiotensin-converting enzyme (ACE) inhibitors. During ACE inhibition, an increased proportion of bradykinin is degraded through non-ACE pathways. Studies in animals suggest that aminopeptidase P (EC 3.4.11.9) may contribute to the metabolism of bradykinin. The purpose of the present study was to determine the contribution of aminopeptidase P to the degradation of bradykinin in humans in the presence and absence of ACE inhibition. To do this, we measured the wheal response to intradermal injection of bradykinin (0, 1, or 10 nicrog) in the presence or absence of intradermal administration of the specific aminopeptidase P inhibitor apstatin (5 or 10 microg) and oral administration of the ACE inhibitor quinapril (10 mg) in six healthy subjects. Both bradykinin (ANOVA; F = 101.18, P <.001) and apstatin alone (F = 7.01, P =.049) caused a wheal of dose-dependent size. There was no significant interaction between apstatin and bradykinin (F = 4.94, P =.175). Pretreatment with 10 mg of quinapril significantly shifted the dose-response curve for bradykinin to the left (effect of quinapril; F = 77.96, P <.001) and there was significant interaction between quinapril and bradykinin (F = 7.82, P =.041). The effect of quinapril was significantly potentiated by coinjection of 10 microg of apstatin (effect of apstatin; F = 21.60, P =.006), such that there was significant interactive effect of quinapril and apstatin (F = 20.83, P =.006) on the wheal response to bradykinin. Collectively, these data suggest that aminopeptidase P plays a minor role in the degradation of bradykinin in human skin in the absence of ACE inhibition but contributes significantly to the degradation of bradykinin in the presence of ACE inhibition.

Cardioprotective effects of the aminopeptidase P inhibitor apstatin: studies on ischemia/reperfusion injury in the isolated rat heart.

Aminopeptidase P and angiotensin-converting enzyme (ACE) are responsible for the metabolism of exogenously administered bradykinin in the coronary circulation of the rat. It has been shown that ACE inhibitors decrease cytosolic enzyme release from the ischemic rat heart and reduce reperfusion-induced ventricular arrhythmias by increasing endogenous levels of bradykinin. It was hypothesized that the aminopeptidase P inhibitor apstatin could do the same. In an isolated perfused rat heart preparation subjected to global ischemia and reperfusion, both apstatin and ramiprilat (an ACE inhibitor) significantly decreased creatine kinase (CK) and lactate dehydrogenase (LDH) release. The difference between the postischemia and preischemia levels of released CK was reduced 68% by apstatin and 68% by ramiprilat compared with control. The corresponding reductions in LDH release were 74% for apstatin and 81% for ramiprilat. A combination of the inhibitors was not significantly better than either one alone. Apstatin and ramiprilat also significantly reduced the duration of reperfusion-induced ventricular fibrillation by 69 and 61%, respectively. The antiarrhythmic effect of apstatin was reversed by HOE140, a bradykinin B2-receptor antagonist, suggesting that apstatin is acting by potentiating endogenously formed bradykinin. The results demonstrate that the aminopeptidase P inhibitor apstatin is cardioprotective in this model of cardiac ischemia/ reperfusion injury.

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.

Apstatin analogue inhibitors of aminopeptidase P, a bradykinin-degrading enzyme.

Membrane-bound aminopeptidase P (AP-P) participates in the degradation of bradykinin in several vascular beds. We have developed an inhibitor of AP-P called apstatin (1) (N-[(2S, 3R)-3-amino-2-hydroxy-4-phenyl-butanoyl]-L-prolyl-L-prolyl-L-al aninam ide); IC50,human = 2.9 microM. In the rat, apstatin can potentiate the vasodilatory effect of bradykinin, reduce blood pressure in an aortic-coarctation model of hypertension, and reduce cardiac damage and arrhythmias induced by ischemia/reperfusion. In this study, we have determined structure-activity relationships for apstatin analogues as well as for other chemical classes of inhibitors using AP-P isozymes from different sources. The most potent inhibitor was one in which the N-terminal residue of apstatin was replaced with a (2S,3R)-3-amino-2-hydroxy-5-methyl-hexanoyl residue (6, IC50,human = 0.23 microM). The (2R,3S)-analogue of 6 was equipotent with 6 while the (2S,3S)- and (2R,3R)-analogues were considerably less potent. Apstatin analogues lacking the L-alanine or having hydroxyproline in place of the proline in the second position had reduced affinity. Certain thiol-, carboxylalkyl-, and hydroxamate-containing compounds were inhibitory in the low micromolar range. Human cytosolic AP-P isozymes and Escherichia coli AP-P exhibited different inhibitor profiles than mammalian membrane-bound AP-P isozymes. The effects of the compounds on X-Pro dipeptidase (prolidase) and leucyl aminopeptidase are also presented.

Effects of aminopeptidase P inhibition on kinin-mediated vasodepressor responses.

We studied in anesthetized rats whether aminopeptidase P (AMP) may be involved in bradykinin (BK) metabolism and responses. For this we inhibited AMP with the specific inhibitor apstatin (Aps). Studies were done with Aps alone or together with the angiotensin-converting enzyme inhibitor lisinopril (Lis). Aps increased the vasodepressor response to an intravenous bolus of BK (400 ng/kg): vehicle, -3.0 +/- 0.7 mmHg; Aps, -7.8 +/- 0.7 mmHg (P < 0.01 vs. vehicle); Lis, -23.8 +/- 1.8 mmHg; Aps + Lis, -37.5 +/- 1.9 mmHg (P < 0.01 vs. Lis). Aps did not affect the vasodepressor response to BK given into the descending aorta. Plasma BK increased only in Aps + Lis-treated rats (in pg/ml): control, 48.0 +/- 1.4; Lis, 57.5 +/- 7.6; Aps + Lis, 121. 8 +/- 30.6 (P < 0.05 vs. control or Lis), whereas in rats infused with BK (400 ng. kg-1. min-1 for 5 min), Aps increased plasma BK (in pg/ml): control, 51.9 +/- 2.5; Aps, 83.5 +/- 20.5; Lis, 725 +/- 225; Aps + Lis, 1,668 +/- 318 (P < 0.05, Aps vs. control and Lis vs. Aps + Lis). In rats with aortic coarctation hypertension, the acute antihypertensive effects of Aps plus Lis were greater than Lis alone (P < 0.01). Hoe-140, a BK B2-receptor antagonist, abolished the difference. We concluded that in the rat AMP contributes to regulation of BK metabolism and responses.

Inhibition of both aminopeptidase P and angiotensin-converting enzyme prevents bradykinin degradation in the rat coronary circulation.

Bradykinin (Bk), which is produced locally in the heart, exhibits potent cardioprotective effects. However, these effects appear to be limited by rapid degradation of the peptide. To determine the mechanism of Bk metabolism in the coronary circulation, [3H]Bk was perfused through the isolated rat heart via the aorta in the presence and absence of specific peptidase inhibitors. The radiolabeled metabolites were collected from the pulmonary artery and then separated, identified, and quantified by reversed-phase high-performance liquid chromatography (HPLC) by using a radioactive flow detector. In the absence of inhibitors, only 45 +/- 2% of the radioactivity eluted from the coronary circulation as intact [3H]Bk. The chromatograms suggested that Bk was being hydrolyzed at the Arg1-Pro2 bond by aminopeptidase P and at the Pro7-Phe8 bond by angiotensin-converting enzyme. When the aminopeptidase P inhibitor, apstatin (200 microM), was coperfused with [3H]Bk, cleavage at the Arg1-Pro2 bond was blocked and the amount of intact [3H]Bk in the perfusate increased to 57 +/- 5% (p < 0.05 vs. control). Coperfusion with the angiotensin-converting enzyme inhibitor, ramiprilat (0.5 microM), alone blocked cleavage at the Pro7-Phe8 bond and increased intact [3H]Bk to 75 +/- 3% (p < 0.001 vs. control). When both apstatin and ramiprilat were present, almost all of the radioactivity (96 +/- 1%) eluted as intact [3H]Bk (p < 0.01 vs. ramiprilat alone). The results indicate that the degradation of Bk in the rat coronary circulation can be fully accounted for by aminopeptidase P (approximately 30%) and angiotensin-converting enzyme (approximately 70%).

Influence of several peptidase inhibitors on the pro-inflammatory effects of substance P, capsaicin and collagenase.

Injection of substance P (SP) in a rat hindpaw induced extravasation of 125I-labelled albumin in both hindpaws and salivation. Intravenous injection of SP dose-dependently increased vascular permeability. This latter effect was increased in rat paws by captopril, an inhibitor of angiotensin-converting enzyme (ACE), administered locally in combination with diprotin A, an inhibitor of an dipeptidyl(amino)peptidase IV (DAP IV) or phosphoramidon, an inhibitor of neutral endopeptidase (NEP). The increase in permeability induced by SP was inhibited by RP 67580, a NK-1-receptor antagonist. Intravenous injection of capsaicin induced labelled albumin extravasation in rat paws. This effect was increased by combination of captopril with diprotin A or phosphoramidon, but not by captopril associated with amastatin, an inhibitor of aminopeptidase M (AmM). It was suppressed by RP 67580. Injection of collagenase in rat paws triggered a swelling and a local plasma exudation. These responses were reduced by RP 67580 but not by RP 68651, its inactive enantiomer. They were increased by combination of captopril with diprotin A or phosphoramidon in normal rats. The potentiating effects of captopril and diprotin A were suppressed by RP 67580 in normal rats but did not develop in kininogen-deficient rats. The oedema induced by collagenase was also increased by lisinopril, another ACE inhibitor, administered locally in combination with apstatin, an inhibitor of aminopeptidase P (AmP). In rats pretreated by methysergide, collagenase-induced oedema was reduced and can be increased by captopril, by lisinopril, administered alone or by lisinopril associated with apstatin. It is concluded that SP is mainly inactivated in rat paws by ACE, DAP IV and NEP. In collagenase-induced oedema, a low amount of SP would be released from afferent nerve terminals by bradykinin formed in low amounts. Bradykinin is inactivated in rat paws by ACE and AmP. In collagenase-oedema, the pro-inflammatory effects of bradykinin are concealed by the effects of the other mediators.

Potentiation of the pro-inflammatory effects of bradykinin by inhibition of angiotensin-converting enzyme and aminopeptidase P in rat paws.

The influence of some peptidase inhibitors on oedema and plasma extravasation induced by bradykinin and carrageenan in rat paw was evaluate. Bradykinin-induced oedema in normal rats was increased by o-phenanthroline (3.10(-2) M), by captopril (10(-6) M to 10(-4) M), by lisinopril (10(-6) M to 10(-4), or by lisinopril (10(-5) M) in combination with apstatin (8.10(-5) M or 1.4 10(-4) M). It was not modified by phosphoramidon (10(-6) M to 10(-5) M) and by diprotin A (10(-3) M). It was increased by mergepta at high concentrations (2.10(-4) M). Mergepta did not increase the potentiating effect of captopril. Carrageenan-oedema in normal rats was increased by captopril (10(-5) M), lisinopril (10(-5) M) and apstatin (1.4 10(-4) M. It was not modified by mergepta (10(-4) M), phosphoramidon (10 (-5) M) and diprotin A (109-3) M). Des-Arg1-bradykinin and Des-Arg9-bradykinin have low oedema-promoting effects. Captopril (10(-5) M) increased the effects of bradykinin but not those of carrageenan in kininogen-deficit Brown Norway rats. Angiotensin-converting enzyme and aminopeptidase P appear to be main kinin-inactivating enzymes in rat paws. Carboxypeptidase N, neutral endopeptidase 24.11 and dipeptidyl(amino)peptidase IV do not play a significant role in this inactivation.

Effect of a new aminopeptidase P inhibitor, apstatin, on bradykinin degradation in the rat lung.

Bradykinin (Bk), a potent vasoactive and cardioprotective peptide hormone, is almost completely inactivated during a single circulation through the rat lung. It has been hypothesized that membrane-bound aminopeptidase P, which can hydrolyze the Arg1-Pro2 bond of Bk, and angiotensin-converting enzyme (ACE) act in concert to degrade Bk in the pulmonary circulation. To test this hypothesis, an inhibitor of aminopeptidase P was designed and synthesized. N-[(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]-L-prolyl-L-prolyl-L - alaninamide (apstatin) inhibited purified rat lung membrane-bound aminopeptidase P with a Ki value of 2.6 microM (linear mixed-type inhibition, alpha = 5.1, beta = 0). Apstatin did not inhibit ACE or other known Bk-degrading enzymes. Apstatin and an ACE inhibitor, ramiprilat, were tested for their ability to inhibit Bk degradation in the isolated perfused rat lung. [2,3-Proly-3,4-3H(N)]-bradykinin ([3H]-Bk) was perfused through the isolated lung in the presence or absence of inhibitors. The perfusate was then subjected to HPLC to identify and quantitate radiolabeled fragments. In the absence of inhibitors, no intact [3H]-Bk was found in the perfusate. In the presence of ramiprilat (0.5 microM), only 22% +/- 6% of the radioactivity in the perfusate was intact [3H]-Bk, and the remaining radioactivity indicated cleavage of the Arg1-Pro2 bond. When apstatin (40 microM) was perfused along with ramiprilat, degradation of [3H]-Bk was almost completely blocked (92% +/- 4% intact [3H]-Bk in the perfusate). The results indicate that the Bk-degrading activity in the rat pulmonary vascular bed can be fully accounted for by aminopeptidase P (30%) and ACE (70%).

Purification and properties of membrane-bound aminopeptidase P from rat lung.

The membrane-bound form of aminopeptidase P (aminoacylprolyl-peptide hydrolase) (EC 3.4.11.9) was purified 670-fold to apparent homogeneity from rat lung microsomes. The enzyme was solubilized from the membranes using a phosphatidylinositol-specific phospholipase C. The purification scheme also resulted in homogeneous preparations of dipeptidylpeptidase IV (EC 3.4.14.5) and membrane dipeptidase (EC 3.4.13.19). Aminopeptidase P had a subunit molecular weight of 90,000, which included at least 17% N-linked carbohydrate. The molecular weight by gel permeation chromatography varied from 220,000 to 340,000, depending on the conditions used. The amino acid composition was determined and the N-terminal sequence was found to be X1-Gly2-Pro3-Glu4-Ser5-Leu6-Gly7-Arg8-Glu9-As p10-Val11-Arg12-Asp13-X14-Ser15- Thr16-Asn17-Pro18-Pro19-Arg20-Leu21- X22-Val23-Thr24-Ala25-. Aminopeptidase P cleaved the Arg1-Pro2 bond of bradykinin with a kcat/Km of 5.7 x 10(5) s-1 M-1. N-Terminal fragments of bradykinin including Arg-Pro-Pro, but not Arg-Pro, were also cleaved. The enzyme was shown to have four binding subsites (S1, S1', S2'. S3'), the first three of which must be occupied for hydrolysis to occur. Neuropeptide Y and allatostatin I were hydrolyzed at the Tyr1-Pro2 bond and Ala1-Pro2 bond, respectively. The pH optimum for Arg-Pro-Pro cleavage was 6.8-7.5 in most buffers. The enzyme was most stable in the range of pH 7.0-10.5 in the presence of poly(ethylene glycol). NaCl inhibited activity completely at 2 M. Mn2+ had variable effects on activity, depending on its concentration and the substrate used. Various peptides having an N-terminal Pro-Pro sequence were inhibitory. The enzyme was also inhibited by EDTA, o-phenanthroline, 2-mercaptoethanol, dithiothreitol, p-(chloromercuri)benzenesulfonic acid, apstatin, and captopril. The carboxyalkyl angiotensin-converting enzyme inhibitors, ramiprilat and enalaprilat, inhibited activity in the micromolar range only in the presence of Mn2+.

Mammalian tissue trypsin-like enzymes: substrate specificity and inhibitory potency of substituted isocoumarin mechanism-based inhibitors, benzamidine derivatives, and arginine fluoroalkyl ketone transition-state inhibitors.

Amino acid and peptide thioesters which contained Arg or Lys in the P1 position were tested as substrates for rat skin tryptase, and the kinetic constants Kcat/KM for the better substrates such as Z-Aba-Arg-SBzl, and Z-Gly-Arg-SBzl were over 5,000,000 M-1 s-1. The inhibitory potency of arginine fluoroalkyl ketones, benzamidine derivatives, and substituted isocoumarins containing basic functional groups was studied with rat skin tryptase, human lung tryptase, human skin tryptase, and bovine trypsin. 1-Naphthoyl-Arg-CF3 was the best arginine fluoroalkyl ketone reversible inhibitor for rat skin tryptase with a KI of 0.9 microM. 1-(4-Amidino-phenyl)-3-(4-phenoxyphenyl) urea showed competitive inhibition against bovine trypsin and rat skin tryptase with KI values of 2 and 4 microM, respectively. Isocoumarin derivatives with isothioureidoalkoxy substituents at the 3 position were potent irreversible inhibitors of these three tryptases with Kobs/[I] values of 10(4)-10(5) M-1 s-1. 4-Chloro-3-(2-isothioureido)ethoxy-7-phenylcarbamoylaminoisocou marin and 7-benzylcarbamoylamino-4-chloro-3-(3-isothioureido)propox yisocoumarin inactivated trypsin and formed stable trypsin-inhibitor complexes which regained less than 8% of activity upon standing in the pH 7.5 buffer and regained 30-75% of activity in the presence of 0.3 M NH2OH after 1 day. In contrast, the complexes with rat skin tryptase regained activity rapidly, indicating differences in the inhibition mechanism and active site structures of these related enzymes.

Potentiation by aminopeptidase P of blood pressure response to bradykinin.

We examined whether a specific aminopeptidase P (APP) inhibitor, apstatin, increases vasodepressor responses to bradykinin in anaesthetized rats, and whether it would augment blood pressure responses further after treatment with the angiotensin-converting enzyme inhibitor (ACEi), lisinopril. Apstatin doubled the maximum blood pressure response to bradykinin. The area under the curve (AUC), which incorporates both peak blood pressure changes and duration of response, was doubled in apstatin-treated rats vs controls and in the apstatin+lisinopril group vs lisinopril alone. These data demonstrate that APP is an important kinase in vivo.

Substrate specificity of aminopeptidase P from Escherichia coli: comparison with membrane-bound forms from rat and bovine lung.

The substrate specificity of recombinant E. coli aminopeptidase P (aminoacylprolylpeptide hydrolase) (EC 3.4.11.9) was studied using about 150 synthetic peptides. E. coli aminopeptidase P released the N-terminal amino acid from most peptides containing a penultimate proline, although the relative rates of hydrolysis varied over two orders of magnitude. Dipeptides (X-Pro) were hydrolyzed relatively slowly. Detailed kinetic analysis using peptides of different lengths suggested that the enzyme has at least four subsites for interaction with substrates, namely S1, S'1, S'2, and S'3. S1 and S'1 have high stereo-specificity Various Pro-X dipeptides where X is a hydrophobic amino acid were competitive inhibitors of the enzyme. The substrate specificity of E. coli aminopeptidase P was compared to that of purified bovine lung and rat lung membrane-bound aminopeptidase P. The mammalian enzymes had much more restricted substrate specificities. The differences appeared to be due primarily to differences in the S'2 subsite. The E. coli enzyme could accommodate bulky amino acid side chains in the S'2 subsite, whereas the mammalian membrane-bound enzymes could not.

Localization of rat tryptase to a subset of the connective tissue type of mast cell.

We examined the cellular distribution of rat tryptase in rat skin, lung, small intestine, and peritoneal lavage cells by immunohistochemical techniques. Tryptase purified to apparent homogeneity from rat skin was used to generate a goat polyclonal anti-rat tryptase antibody. Tryptase-containing cells were detected in lung, skin, and peritoneal lavage cells. Small intestine mucosa, on the other hand, showed few if any tryptase-positive cells. Sequential staining with Alcian blue and anti-tryptase antibody showed that tryptase is located only in mast cells. Sequential staining with safranin to identify the connective tissue type of mast cell and anti-tryptase antibody showed that tryptase resides only in this mast cell type. However, only a subpopulation of the safranin-stained mast cells contained tryptase. In lung, 53% of the mast cells stained with safranin; 94% contained tryptase. In skin, 80% stained with safranin; only 6% contained tryptase. In peritoneal cells, more than 95% of the mast cells were stained with safranin; 20% contained tryptase. In the bowel mucosa, where few cells are stained by safranin, no cells with tryptase were detected. The percentages of cells with chymase I that also contained tryptase were 80% and 84% for lung, 4% and 7% for skin, and 15% and 13% for peritoneal cells by respective simultaneous and sequential double labeling with anti-tryptase and anti-chymase I antibodies. This study suggests that the rat connective tissue type of mast cell is subdivided into two forms on the basis of the presence or absence of tryptase, whereas rat mucosal mast cells lack this enzyme. These results contrast with those in humans, in which tryptase is present in all mast cells, but are similar to mice, in which tryptase mRNA has been detected only in the connective tissue type.

Dipeptidase activities in rat brain synaptosomes can be distinguished on the basis of inhibition by bestatin and amastatin: identification of a kyotorphin (Tyr-Arg)-degrading enzyme.

The neuropeptide kyotorphin (Tyr-Arg) was degraded by rat brain synaptosomes via a synaptic membrane-bound peptidase which was inhibited by bestatin but not by amastatin. The Km for kyotorphin was 8 x 10(-6) M and the Ki for bestatin was 1 x 10(-7) M. The kyotorphin-degrading enzyme was distinguished from at least one other dipeptide-hydrolyzing activity in synaptosomes which was inhibited by both bestatin and amastatin. Gel permeation chromatography of detergent-extracted synaptosomes resulted in the separation of the dipeptide-hydrolyzing activities. A single kyotorphin-degrading enzyme peak was observed which had a M(r) = 52,000. The activity peak could degrade other dipeptides including Phe-Arg, a synaptic membrane-generated metabolic of bradykinin. The kyotorphin-degrading enzyme appears to be novel and can be distinguished from other known dipeptidases on the basis of substrate specificity, subcellular localization, and inhibition profile.

Membrane-bound aminopeptidase P from bovine lung. Its purification, properties, and degradation of bradykinin.

The membrane-bound form of aminopeptidase P (aminoacylprolyl-peptide hydrolase) (EC 3.4.11.9) was purified to apparent homogeneity from bovine lung microsomes. The enzyme was solubilized using phosphatidylinositol-specific phospholipase C (Bacillus thuringiensis), indicating that bovine lung amino-peptidase P is attached to membranes via a glycosylphosphatidylinositol anchor. The enzyme was purified 1900-fold with a yield of 25% by chromatography on decyl-agarose, omega-aminodecyl-agarose, a second decylagarose column, DEAE-Sephacel, and an ultrafiltration step. Native gradient polyacrylamide gel electrophoresis revealed a single stained protein band whose position in the gel corresponded to cleavage of the Arg1-Pro2 bond of bradykinin. The Mr was 360,000 by gel permeation chromatography and 95,000 by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The substrate specificity of aminopeptidase P was determined using approximately 50 peptides with proline in the second position. The enzyme could hydrolyze lower NH2-terminal homologs of bradykinin, including Arg-Pro-Pro, which was used as the routine substrate in a rapid fluorescence assay performed in the absence of added Mn2+. Some peptides having NH2-terminal amino acids other than arginine were also cleaved. Aminopeptidase P appeared to favor peptides that had 2 proline residues or proline analogs in positions 2 and 3 of the substrate. In general, tripeptides having a single proline residue in position 2 were poor substrates. Aminopeptidase P was inhibited by a series of peptides, 3-8 residues long, having an NH2-terminal Pro-Pro sequence. The enzyme was also inhibited by metal-chelating agents, 2-mercaptoethanol (4 mM), p-chloromercuribenzenesulfonic acid, and NaCl at concentrations greater than or equal to 0.25 M. The purified enzyme had a pH optimum of 6.5-7.0 and was most stable in the basic pH range. A role for membrane-bound aminopeptidase P in the pulmonary inactivation of circulating bradykinin is proposed.

Aminopeptidase P: purification of a membrane-bound bradykininase from rat lung.

Aminopeptidase P that hydrolyzes the Arg1-Pro2-bond of bradykinin was solubilized from rat lung microsomes using phosphatidylinositol-specific phospholipase C. The enzyme was purified 420-fold by chromatography on decylagarose (two steps), omega-aminodecyl-agarose and DEAE-Sephacel. A single stained band was observed following native gradient (4-15%) polyacrylamide gel electrophoresis. Dipeptidylaminopeptidase IV-like activity was also present in the final preparation and co-migrated with aminopeptidase P in the above gel system.

Tryptase from rat skin: purification and properties.

Tryptase was purified 13,000-fold to apparent homogeneity from rat skin. The two-step procedure involved ammonium sulfate fractionation of the initial extract followed by combined sequential affinity chromatography on agarose-glycyl-glycyl-p-aminobenzamidine and concanavalin A-agarose. The purified enzyme had a specific activity toward N-benzoylarginine ethyl ester (BzArgOEt) of 170 mumol/min mg-1 and was obtained in a yield of 28% as determined by the specific substrate, H-D-Ile-Pro-Arg-p-nitroanilide. Rat skin tryptase was thermal labile, losing 50% of its activity when preincubated for 30 min at 30 degrees C. The presence of NaCl (1 M) improved thermal stability and was necessary for long-term storage. Heparin did not stabilize the enzyme against thermal denaturation, and heparin-agarose failed to bind the enzyme. Rat skin tryptase was inhibited by diisopropylphosphofluoridate, antipain, leupeptin, and aprotinin but not by alpha 1-antitrypsin, ovomucoid, or soybean or lima bean trypsin inhibitors. Substrate specificity studies using a series of tri- and tetrapeptidyl-p-nitroanilide and peptidyl-7-amino-4-methylcoumarin substrates demonstrated the existence of an extended substrate binding site. Rat skin tryptase hydrolyzed [Arg8]vasopressin, neurotensin, and the oxidized B-chain of insulin at the -Arg8-Gly9-NH2, -Arg8-Arg9-, and -Arg22-Gly23-bonds, respectively. No general proteinase activity was observed toward casein, hemoglobin, or azocoll. Rat skin tryptase had a Mr of 145,000 by gel filtration. The subunit Mr was either 34,000 or 30,000 depending on the electrophoretic technique used. Treatment of the enzyme with peptide N-glycosidase F (N-glycanase) decreased the subunit Mr by 4000. The enzyme exhibited multiple isoelectric forms (pI's of 4.5-4.9). Rat skin tryptase was found to be related statistically to other tryptases on the basis of amino acid composition. The N-terminal amino acid sequence was Ile1-Val2-Gly3-Gly4-Gln5-Glu6-Ala7-+ ++Ser8-Gly9-Asn10-Lys11-Trp12-Pro13- Trp14- Gln15-Val16-Ser17-Leu18-Arg19-Val20- --21-Asp-22Thr23-Tyr24-Typ25-, with a putative glycosylation site at residue 21. This sequence was 72-80% homologous with the N-terminus of other tryptases but only 40% homologous with that of bovine trypsin.