Differential processing of substance P and neurokinin A by plasma dipeptidyl(amino)peptidase IV, aminopeptidase M and angiotensin converting enzyme

Peptides,Vol. 12, pp. 1357-1364. ©PergamonPress plc, 1991.Printedin the U.S.A.

0196-9781/91 $3.00 + .00

Differential Processing of Substance P and Neurokinin A by Plasma Dipeptidyl(amino)peptidase IV, Aminopeptidase M and Angiotensin Converting Enzyme LINHONG W A N G , SALEEM AHMAD, IBRAHIM F. BENTER, ANA CHOW, SHIGEHIKO MIZUTANI* AND PATRICK E. W A R D 1

Department of Physiology, The Ohio State University, Columbus, OH 43210 *Department of Obstetrics and Gynaecology, Nagoya University School of Medicine, Nagoya, Japan Received 2 July 1991 WANG, L., S. AHMAD, I. F. BENTER, A. CHOW, S. MIZUTANIAND P. E. WARD. Differentialprocessing of substance P and neurokinin A by plasma dipeptidyl(amino)peptidaseIV, aminopeptidase M and angiotensin converting enzyme. PEPTIDES 12(6) 1357-1364, 1991.--In addition to plasma metabolism of substance P (SP) by angiotensinconverting enzyme (ACE; EC 3.4.15.1) (< 1.0 umol/min/ml),the majority of SP hydrolysisby rat and human plasma was due to dipeptidyl(amino)peptidaseIV (DAP IV; EC 3.4.14.5) (3.15-5.91 umol/min/ml),which sequentiallyconverted SP to SP(3-11) and SP(5-11). In turn, the SP(511) metabolite was rapidly hydrolyzed by rat and human plasma aminopeptidaseM (AtoM; EC 3.4.11.2) (24.2-25.5 nmol/min/ ml). The K~ values of SP for DAP IV and of SP(5-11) for AmM ranged from 32.7 to 123 p,M. In contrast, neurokininA (NKA) was resistant to both ACE and DAP IV but was subject to N-terminalhydrolysis by AtoM (3.76-10.8 umol/min/ml;K~=90.7 I~M). These data demonstrate differentialprocessingof SP and NKA by specific peptidases in rat and human plasma. Angiotensinconvertingenzyme Substance P SubstanceK

AminopeptidaseM

Dipeptidyl(amino)peptidaseIV

CIRCULATING substance P (SP) is inactivated by plasma angiotensin converting enzyme (ACE; EC 3.4.15.1), and ACE inhibitors reduce SP inactivation and potentiate SP-induced salivary secretion in vivo (6). Since peripherally administered SP is a vasodilator, these data suggest that some of the antihypertensive actions of ACE inhibitors may relate to potentiation of endogenous SP. Plasma also contains other peptidases which could participate in SP metabolism. Purified peptidases such as neutral endopeptidase (NEP-24.11; EC 3.4.24.11) (19), post proline cleaving enzyme (EC 3.4.21.26) (4), and dipeptidyl(amino)peptidase IV (DAP IV; EC 3.4.14.5) (15) have been shown to hydrolyze SP, and all three enzymes are present in plasma (33). In view of structure-activity studies supporting differential actions for SP N-terminal and C-terminal sequences (1, 7, 10, 14), and the colocalization/release of SP and neurokinin A (NKA; substance K) in peripheral sites (21), the present study was conducted to identify, characterize and quantify the enzymes in addition to ACE which metabolize SP and related tachykinins in plasma.

Tachykinins

METHOD

Materials SP, SP fragments, NKA, the aminopeptidase M (AmM; EC 3.4.11.2) inhibitor amastatin, and the NEP-24.11 inhibitor phosphoramidon were obtained from Sigma Chemical Co. (St. Louis, MO). The ACE inhibitor captopril was from Squibb (Princeton, NJ), and the DAP IV inhibitor diprotin A (31) was obtained from Peninsula Labs (Belmont, CA). Purified human AmM and AmM antisera were prepared as previously described (17).

Plasma Substance P Metabolism Blood was obtained from anesthetized (sodium pentobarbital; 65 mg/kg IP) male Sprague-Dawley rats via the carotid artery. Five ml of blood was mixed with 100 I~1 of heparin (100 units/ ml), centrifuged (10 min) in a bench-top clinical centrifuge, and the plasma collected and frozen.

~Requestsfor repfin~ should be addressed to Dr. Patrick E. Ward, Departmentof Physiology, 4196Graves Hall, 333West 10thAve., Columbus, OH43210-1239. 1357

1358

WANG ET AL.

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The standard incubation (600 p,l) consisted of plasma (5-30 p,l), peptide substrate (50 p,M), and 50 mM Tris/HC1 buffer (pH 7.5) containing 30 mM NaC1. In selected cases, captopril (10 IxM), diprotin A (100 p,M) and amastatin (10 ixM) were included to inhibit ACE, DAP IV and AmM, respectively. At sequential time intervals aliquots were immersed in a boiling water bath (5 min) to terminate the reaction, cooled on ice, centrifuged, and the supernate collected for HPLC analysis. Peptide hydrolysis was determined by the decrease in substrate (e.g., SP) and the increase in products [e.g., SP(3-11) and SP(5-11)]. For K m determinations, measurements of initial velocity were made over a range of substrate concentrations (10-100 ~M) and data were plotted as 1/V vs. 1/[S] and fit to the best straight line.

HPLC Analysis Peptide substrates and metabolites were separated and quantitated by a modification of the method of Cascieri et ai. (6) on a reverse-phase column (Vydac, 10 micron, C18 ixBondapak, 1.9 × 30 cm) at a constant flow rate of 2 ml/min using a linear gradient from 95% solvent A/5% solvent B to 45% solvent A/55% solvent B (25 min), followed by column reequilibration (5 min). Solvent A was 0.5 M phosphoric acid, buffered to pH 2.5 with triethylamine, and solvent B was acetonitrile. Integration of sample peak areas (monitored at 210 nm) and quantitation of peptide substrates and metabolites against the last-run standards (run every sixth injection) were automatically calculated by the data module. Amino acid metabolites [N-terminal Gin from SP(5-11) and N-terminal His from NKA] were quantified after precolumn derivitization with o-phthalaldehyde as previously described (2,23).

|

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min FIG. 1. Separation of substance P (SP; retention time = 15.04 min) from SP fragments, including: SP(1-4) (RT= 1.53 min), SP(9-11) (RT= 6.70 min), SP(1-9) (RT= 10.97 rain), SP(8-11) (RT= 12.81 min), SP(3-11) (RT=15.32 min), SP(5-11) (RT=15.90 min), and SP(6--ll) (RT= 16.25 min) by HPLC on a Vydac C18 ~Bondapak column as described in the Method section. Injection volume was 80 ILl containing peptide standards at a concentration of 20 I~M. Other standards now shown include SP free acid (RT= 15.51 min), SP(2-11) (RT= 15.70 min) SP(711) (RT= 16.10 min) and neurokinin A (RT= 13.42 min).

|

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FIG. 2. Rat plasma (30 ~1) hydrolysis of substance P (SP) (50 p.M) under control conditions, in the presence of captopril (10 p.M) or diprotin A (100 p.M), or both captopril/diprotin A. Separation and quantitation of SP was assessed by HPLC as shown in Fig. 1.

Immunoelectrophoresis Immunoelectrophoresis (electroimmunoassay) of human plasma and purified AmM against antisera to human AmM was carded out in 1.0 mm thick 1% (w/v) agarose gel containing 1% (v/v) Triton X-100 as previously described (32,34). Samples were vertically electrophoresed (1.5 V/cm for 15 h) directly into antisera-containing gel. After repeated dehydration/hydration of the gel to remove soluble protein, the AmM immunoprecipitin line was stained using alanyl-4-methoxy-2-naphthylamide. Peak area measurements were determined using Sigma Scan (Jandel Scientific). RESULTS

Substance P Metabolism After establishing effective separation of SP from various potential metabolites by the reverse-phase chromatography system described in the Method section (Fig. 1), this system was utilized to separate SP from metabolites generated during incubation with rat and human plasma. In initial studies, intact SP was found to decrease linearly with time in proportion to the amount of rat plasma added (5-30 ILl). As shown in Fig. 2, the decrease in SP was only slightly inhibited by the ACE inhibitor captopril, whereas the DAP IV inhibitor diprotin A produced considerably more inhibition. Captopril and diprotin A together nearly completely inhibited SP hydrolysis (Fig. 2). Subsequent studies examined ACE- and DAP IV-mediated hydrolysis individually. In the presence of diprotin A to inhibit DAP IV, SP hydrolysis produced SP(9-11), previously shown to be due to ACE (6). ACE hydrolysis of SP was 0.94--.0.25 nmol/min/ml (Table 1) and was completely inhibited by captopril. During ACE inhibition, SP was rapidly hydrolyzed to produce the DAP IV metabolites SP(3-11) and, subsequently, SP(511) (Fig. 3A). Conversion to SP(3-11) and SP(5-11) was inhibited more than 95% by the DAP IV inhibitor diprotin A. As shown in Table 1, DAP IV-mediated SP hydrolysis (3.15---0.32 nmol/

PLASMA TACHYKININ METABOLISM

1359

30

TABLE 1 HYDROLYSISOF SUBSTANCEP BY RAT PLASMAPEPTIDASES Enzyme

25

Rate (nmol/min/ml)

SP(5 - 11 )

% Total 20

ACE DAP IV AmM Other Total

0.94 --- 0.25 (n = 5) 3.15 -+ 0.32 (n = 6) 0 0.57 +-- 0.38 (n = 3) 4.90 ___0.62 (n = 3)

20 68

15 E t-

0 12

10

--

Hydrolysis of substance P (50 I~M) inhibited by captopril (10 p.M) (ACE), diprotin A (100 IxM) (DAP IV), amastatin (10 I~M) (AmM), or none of the above (other). Total hydrolysis was determined in the absence of peptidase inhibitors. Values given are the means -+ S.E.M.

min/ml) was more than three-fold faster than that due t o ACE. In the presence of both ACE and DAP IV inhibitors, SP hydrolysis by rat plasma was inhibited nearly 90%, and the residual (other) unidentified hydrolytic activity (0.57---0.38 nmol/min/ ml) was unaffected by the AmM inhibitor amastatin or the NEP24.11 inhibitor phosphoramidon (Table 1). Although the disappearance of the SP(3-11) metabolite was due to DAP IV conversion to SP(5-11), the progressive disappearance of the SP(5-11) metabolite (Fig. 3A) and appearance of an SP(6-11) metabolite (not shown) indicated N-terminal hydrolysis of the DAP IV-generated SP(5-11) metabolite by plasma AmM-like activity. Consistent with this expectation, addition of the AmM inhibitor amastatin increased the recovery of SP(5-11) generated from SP (Fig. 3B). Confirming this AmM-like hydrolysis, incubation of SP(5-11) resulted in rapid conversion to SP(6-11), which was itself degraded by continued N-terminal

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TIME

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hydrolysis (Fig. 4). SP(5-11) N-terminal hydrolysis was unaffected by diprotin A but was completely inhibited by amastatin [no SP(6-11) production]. Thus, although the AmM-like activity did not hydrolyze either SP or SP(3-11), it rapidly degraded the DAP IV-generated SP(5-11) metabolite (25.5--.2.2 nmol/ min/ml) (Table 2). Residual SP(5-11) hydrolysis was due to ACE (1.13--.0.28 nmol/min/ml) and (other) unidentified hydrolytic activity (4.10 ---0.31 nmol/min/ml) (Table 2). Comparable data were obtained with human plasma. SP hydrolysis was only slightly inhibited by captopril, whereas the

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FIG. 4. Rat plasma (30 ~.1) hydrolysis of SP(5-11) (50 p,M) to produce SP(6-11) in the presence of captopril (10 ~.M). Separation and quantitation of SP(5-11) and SP(6-11) were assessed by HPLC as shown in Fig. 1.

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FIG. 3. Rat plasma (30 I~1)hydrolysis of substance P (SP) (50 I~M) in the presence of captopril (10 I~M) (panel A/control), or captopril/amastatin (10 ~M) (panel B/amastatin). Separation and quantitation of SP, SP(3-I1) and SP(5-11) were assessed by HPLC as shown in Fig. 1.

1360

WANG ET AL.

TABLE 2 HYDROLYSISOF SUBSTANCEP(5-11) BY RAT PLASMAPEPTIDASES Rate (nmol/min/ml)

Enzyme ACE

% Total

1.13 --+ 0.28 (n = 3) 0 25.5 _.+ 2.2 (n = 3) 4.10 --- 0.31 (n = 3) 30.5 __+ 2.3 (n = 3)

DAP IV AmM Other Total

TABLE 3 HYDROLYSISOF SUBSTANCEP AND SUBSTANCEP(5-11) BY HUMANPLASMAPEPTIDASES Substance P

4

Enzyme

0 83

ACE

13

DAP IV AmM

-

% Total

0.98 _ 0.18 (n = 3) 5.91 --- 0.34 (n = 6) 0

13

Other

79 0

addition of diprotin A nearly completely inhibited hydrolysis (Fig. 5A). ACE hydrolysis was 0.98 ---0.18 nmol/min/ml, whereas that due to DAP IV was six-fold more rapid (5.91---0.34 nmol/ min/ml) (Table 3). As with rat plasma, human plasma DAP IV hydrolysis generated SP(3-11) and SP(5-11) metabolites (Fig. 5B), and AmM-like activity hydrolyzed the SP(5-11) metabolite to SP(6-11) (Fig. 5C), which was subsequently degraded with continued incubation (not shown). N-terminal hydrolysis of SP(511) was rapid (24.2---0.2 nmol/min/ml) (Table 3) and completely inhibited by amastatin [no SP(6-11) production]. Residual SP(5-11) hydrolysis was due to ACE (1.04_+0.53 nmol/min/ml) and (other) unidentified hydrolytic activity (3.93 _+0.43 nmol/ min/ml) (Table 3). The K m values of SP for rat plasma DAP IV and of SP(511) for rat and human plasma and purified human AmM ranged from 32 to 123 IxM (Table 4).

Total

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Neurokinin A Metabolism Incubation of rat plasma with NKA resulted in significant hydrolysis (4.74---0.29 nmol/min/ml), the majority of which (3.76---0.32 nmol/min/ml) (Table 5) was inhibited by the AmM inhibitor amastatin (Fig. 6A). However, since neither captopril nor diprotin A produced any additional inhibition, neither ACE nor DAP I V participated in rat plasma NKA metabolism. Residual (other) unidentified hydrolytic activity was 1.00---0.05 nmol/ min/ml (Table 5). Comparable data were obtained with human plasma (Table 5). Nearly all NKA metabolism by human plasma was inhibited by amastatin and was unaffected by the addition of captopril and diprotin A (Fig. 6B). Confirming metabolism as due to N-termi-

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FIG. 5. Panel A: Human plasma (15 i~1) hydrolysis of substance P (SP) (50 ;xM) under control conditions, in the presence of captopril (10 I~M), or both captopfil/diprotin A (10 ~M/100 IJ,M). Panel B: Plasma (15 ~1) hydrolysis of substance P in the presence of captopril/amastatin (10 IJ,M), or panel C: SP(5-11) in the presence of captopril (10 ~M). Separation and quantitation of SP, SP(3-11), SP(5-11) and SP(6-11) were assessed by HPLC as shown in Fig. 1.

PLASMA TACHYKININ METABOLISM

1361

TABLE 5 HYDROLYSISOF NEUROKININA BY RAT ANDHUMANPLASMAPEPTIDASES

TABLE 4 KINETICSOF SUBSTANCEP AND SUBSTANCEP(5-11) HYDROLYSIS Substance P Enzyme

KM p.M

DAP IV Rat plasma 51 • 1.7 AtoM Rat plasma Human plasma -Purified human -AmM

Substance P(5-11)

Vmax nmol/min/ml

KM ~M

Rat Plasma

Vm~ nmol/min/ml Enzyme

8.6 - 0 . 1 m

m

32.7 --- 1.5 76.9 - 1.1 117 - 8 52.9 --. 2.9 123 - 26 3.98 +-- 0.43*

ACE DAP IV AtoM Other Total

Hydrolysis of substance P and substance P(5-11) given as means --+ S.E.M. of three experiments. *Activitygiven as i~mol/mirdmgprotein.

nal hydrolysis, the progressive decrease in NKA (retention time = 13.42 min) was matched by a corresponding increase in a metabolite (RT= 13.94 min) identifiable as NKA(2-10) in that its generation was matched by production of N-terminal His (determined by amino acid analysis). Continued incubation resulted in progressive N-terminal hydrolysis with production of N-terminal Lys and NKA(3-10) (RT = 14.92 min). In order to confLrm that the plasma N-terminal hydrolysis of NKA was due to AmM, comparative studies were conducted with purified human AmM. Incubation of NKA with purified AtoM resulted in the same hydrolytic sequence described above for human plasma. As shown in Fig. 7 (inset), quantitative rocket immunoelectrophoresis (electroimmunoassay) (34) of pudried human AmM and human plasma against antisera to AmM produced rocket-shaped precipitates with peak areas proportional to the amount of sample used (2-8 ixl). When peak areas (immunoreactive AmM; iAmM) were plotted against the units of N-terminal NKA hydrolysis inoculated onto the gels, the relationships of iAmM to hydrolytic activity were comparable for purified AtoM and plasma (Fig. 7). Comparable data were obtained for SP(5-11) hydrolysis (not shown). Thus immunoreactive AmM accounted for essentially all of the N-terminal hydrolysis of NKA and SP(5-11) by human plasma. The Km of NKA for human plasma AmM was 90.7 ___11.9 IxM (n = 3). DISCUSSION Previous studies established that SP is present in the circulation (5), is inactivated by ACE, and that ACE inhibitors reduce SP inactivation in plasma and potentiate SP-induced salivary secretion in vivo (6). The present studies confirm ACE-mediated SP metabolism, supporting the concept that ACE inhibitors may act in part through potentiation of SP (6). If circulating SP mediates antihypertensive and/or other effects, it is important to determine what other plasma enzymes metabolize circulating SP either under normal conditions or during ACE inhibition. Although previous studies have demonstrated plasma peptidase-mediated SP metabolism (3,18), data regarding enzyme identification and quantification have been lacking. Conversely, although purified peptidases, including NEP24.11 (19), post proline cleaving enzyme (4), and DAP IV (15), have been shown to hydrolyze SP, their relative contributions to plasma metabolism have not been previously assessed. The present data do not support a significant role for plasma NEP-24.11 and post proline cleaving enzyme in SP degradation.

Rate (nmol/min/ml) 0 0 3.76 + 0.32 (n = 3) 1.00 _+ 0.05 (n = 4) 4.74 ___0.29 (n = 3)

Human Plasma

% Total 0 0 79 21 --

Rate (nmol/min/ml) 0 0 10.8 -.+ 0.2 (n = 3) 0.44 _ 0.12 (n = 3) 11.3 -+ 0.1 (n = 3)

% Total 0 0 96

Hydrolysis of neurokininA (NKA) (50/zM) inhibitedby captopril (10 p,M) (ACE), diprotin A (100 p,M) (DAP IV), amastatin (10 p,M) (AtoM), or none of the above (other). Total hydrolysis was determined in the absence of peptidase inhibitors. Values given are the means ± S.E.M. Consistent with the relatively low levels of plasma NEP-24.11 and post proline cleaving enzyme relative to ACE and DAP IV (33), plasma SP metabolism was unaffected by the NEP-24.11 inhibitor phosphoramidon, and no post proline cleaving enzyme generated SP(1-4) metabolite (4) was detected. Rather, rat and human plasma metabolized SP by sequential removal of Arg-Pro and Lys-Pro dipeptides to produce SP(3-11) and SP(5-11). Hydrolysis was inhibited by the specific DAP IV inhibitor diprotin A and was unaffected by inhibitors of ACE, AmM, and NEP24.11. Only SP and SP(3-11) (i.e., those with X-Pro N-termini) were subject to diprotin A-sensitive hydrolysis. Collectively, these data identify DAP IV as the responsible enzyme. DAP IV has a unique substrate specificity for N-terminal X-Pro dipeptides (16,24). In contrast, most aminopeptidases cannot hydrolyze N-terminal bonds involving proline (33). Thus, with the exception of aminopeptidase P (EC 3.4.11.9) (35), DAP IV is the only well-characterized aminopeptidase capable of hydrolyzing SP. Further, DAP IV may be considered somewhat specific for SP in that few vasoactive peptides contain the necessary N-terminal sequence. Although bradykinin has an X-Pro N-terminus, the presence of Pro in position three (Arg-Pro-pro) confers resistance to DAP IV (15). In contrast to ACE, DAP IV accounted for the majority of SP hydrolysis by rat and human plasma, and it is likely that this predominance is relevant to metabolism of endogenous plasma SP. DAP IV has a neutral pH optimum and has been localized on the cell surface of vascular endothelium and smooth muscle, microvasculature and blood cells, and numerous tissue sites including kidney, liver and salivary glands (8, 16, 28, 32). Further, in contrast to the poor affinity of SP for purified DAP IV (Km=2000 p.M) originally reported by Kato et al. (15), we found that the K~ of SP for plasma DAP IV (51 I.LM) is comparable to that of angiotensin I for ACE. Although we can only speculate regarding the reason for the difference in affinity found between Kato et al. (15) and our data, it may be related to the former study's use of chemically unstable Arg-Pro and Lys-Pro standards for quantification purposes. Since variable biologic actions have been associated with the N- and C-terminal sequences of SP, the physiologic significance of DAP IV hydrolysis of SP may vary considerably. Regarding receptor binding and actions associated with the N-terminal re-

1362

WANG ET AL.

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FIG. 6. Rat plasma (30 izl) and human plasma (15 p.l) hydrolysis of neurokininA (NKA) (50 p.M) under control conditions, in the presence of amastatin (10 IxM), or a combinationof amastatin/captopril/diprotinA. Separation and quantitationof NKA were assessed by HPLC as shown in Fig. 1.

gion (14), it is likely that DAP IV hydrolysis terminates activity. Some of the physiologic actions thought to be associated with N-terminal sequences include algesia and flare reaction (25), mast cell activation (7,20), macrophage and polymorphonuclear leukocyte phagocytosis (1), and baroreceptor reflex activity (9,10). The N-terminal region may also play a role in the

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FIG. 7. Relationshipof peak area (immunoreactiveAmM) to the N-terminal hydrolysisof NKA (pmol/min)by purified human aminopeptidase M (0) and human plasma (11). Separation and quantitation of NKA were assessed by HPLC as shown in Fig. 1. Inset: Typical rocket immunoelectrophoresis experiment using increasing mounts (2-8 I,d) of purified AtoM and human plasma dilutions migrated into gel containing anti-humanAmM immunoglobulin(1.5 V/cm for 15 h). Immunoprecipitin lines were visualized by staining for alanyl-4-methoxy-2-naphthylamide hydrolysis.

development of SP tachyphylaxis (11,27). DAP IV hydrolysis could be particularly significant in the termination of biologic activities associated with the N-terminal region in that SP hydrolysis by ACE and NEP-24.11 occurs primarily within the C-terminal sequence (i.e., hydrolysis of the C-terminal region may not result in inactivation) (6,19). For instance, the NEP24.11-generated SP(1-7) metabolite is more potent than SP in altering baroreceptor reflex activity (9,10). Since the demonstrated actions of SP(1-7) occur in CNS sites following neural release of SP (9, 11, 14, 27), the present data regarding peripheral metabolism suggest that DAP IV may also be involved in N-terminal degradation within the CNS. Regarding biologic activity associated with C-terminal sequences, the relevance of DAP IV may be more indirect. Since SP(6-11) and larger C-terminal sequences display depressor potencies similar to SP, DAP IV conversion of SP to SP(5-11) does not, in itself, inactivate SP. Rather, removal of the protective Arg-Pro and Lys-Pro sequences exposes SP(5-11) to rapid inactivation by AmM. Further, the affinity of SP(5-11) for AmM is comparable to that reported for physiologically proven peptide/peptidase pathways (33). Thus, despite its inability to hydrolyze SP (i.e., AmM does not hydrolyze N-terminal X-Pro bonds) (34), AmM can act indirectly by degrading DAP IV-generated SP(5-11). Although this multi-step process would seem inefficient compared to one-step depressor inactivation by ACE (i.e., hydrolysis of the C-terminal region), the significantly faster plasma metabolism by DAP IV (>3.0 nmol/min/ml) and AmM (>20 nmol/min/ml) compared to ACE (<1.0 nmol/min/ml) suggests that this DAP IV/AmM pathway may be significant not only in degrading SP depressor activity during treatment with ACE inhibitors, but also under normal conditions. Nevertheless, this hypothesis remains to be tested in vivo, where the relative contributions of cell surface vascular and tissue ACE, DAP IV, and AmM (2, 12, 23, 30, 32) can be comparatively examined. Finally, since SP(5-11) (but not SP) is subject to cellular uptake (22), DAP IV may also act to terminate the actions of SP via conversion to a form which is removed by cellular processes. Sequence differences between SP and NKA affect their rela-

PLASMA TACHYKININ METABOLISM

1363

tive specificities for NK-1, NK-2 and NK-3 receptors (21,26). The present data demonstrate that these sequence differences also substantially alter enzymatic processing by plasma ACE, DAP IV and AmM. Although a previous study reported that NKA is resistant to ACE (13), others reported that some isozymes of ACE do metabolize NKA (29). We found that neither rat nor human plasma ACE metabolized NKA. Thus, as suggested by Hooper et al. (13), replacement of Phe 8 in SP by Val in NKA may represent a critical change for resistance to ACE. Further, these data collectively indicate that the actions of ACE inhibitors, although possibly related to potentiation of SP, are independent of NKA. The N-terminus of NKA also confers complete resistance to DAP IV, consistent with the specificity of DAP IV for N-termi-

nal X-Pro dipeptides. Thus NKA is resistant to the two plasma enzymes primarily involved in SP metabolism. Rather, NKA was subject to N-terminal hydrolysis by AmM. Although the physiologic significance of this A m M hydrolysis is speculative, removal of N-terminal residues could affect N K A ' s receptor specificity and/or possible biologic actions related to the N-terminal region. In addition, N K A ' s resistance to ACE and DAP IV and its nearly complete recovery during A m M inhibition demonstrate circulatory stability beyond that of SP. ACKNOWLEDGEMENT This work was supposed by grants R 0 1 H L 45791 and R01DK 28184.

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