Identification of substance P metabolites using a combination of reversed-phase high-performance liquid chromatography and capillary electrophoresis

Gradient elution reversed-phase high-performance liquid chromatographic and capillary electrophoretic separations were optimised to separate substance P (SP) and twelve of its fragments. The methods were applied to a study of the in vivo metabolism of substance P in the rat after intrastriatal injection of the peptide (10 nmol). SP and significant amounts of its N-terminal fragments, SP(1-7) and SP(1-4), were detected but no major C-terminal fragments could be identified. At the concentration studied, the metabolism of SP was shown to follow zero order elimination kinetics with a rate of decay of 0.2 nmol/min. As we have shown that SP(1–4) and SP(1–7) can be produced in vivo in the striatum in relatively large amounts, it is conceivable that these fragments contribute to the overall pharmacological pattern of activity of the parent peptide.     

Modulation of isolation-induced fighting by N- and C-terminal analogs of substance P: evidence for multiple recognition sites

Substance P (SP) significantly reduced fighting in mice made aggressive by prolonged isolation. The N-terminal heptapeptide fragment SP (1-7) also reduced fighting. The C-terminal fragment SP(4-11) was without activity, while the shorter C-terminal fragment analog less than E-SP(7-11) significantly increased isolation-induced fighting. The aggression-enhancing effect of less than E-SP(7-11) was antagonized by naloxone, which by itself had no significant effect. The aggression-reducing effect of SP(1-11) was significantly enhanced by naloxone, while the effect of SP(1-7) was unchanged. These results demonstrate that a behavioral effect of SP may be duplicated by an N-terminal fragment of the SP molecule, and that peptide fragments or analogs of the N- and C-terminal portions of the SP molecule can exert opposing effects on a specific behavior. These findings represent a structure/activity relationship that is strikingly different from any previously described for SP. The differing effects of naloxone on N- and C-terminal fragment analogs suggest that these two effects may be mediated by different mechanisms.     

Active Uptake of Substance P Carboxy-Terminal Heptapeptide (5–11) into Rat Brain and Rabbit Spinal Cord Slices

We previously reported that nerve terminals and glial cells lack an active uptake system capable of terminating transmitter action of substance P (SP). In the present study, we demonstrated the existence of an active uptake system for SP carboxy-terminal heptapeptide, (5-11)SP. When the slices from either rat brain or rabbit spinal cord were incubated with [3H](5-11)SP, the uptake of (5-11)SP into slices was observed. The uptake system has the properties of an active transport mechanism: it is dependent on temperature and sensitive to hypoosmotic treatment and is inhibited by ouabain and dinitrophenol (DNP). In the brain, (5-11)SP was accumulated by means of a high-affinity and a low-affinity uptake system. The Km and the Vmax values for the high-affinity system were 4.20 x 10(-8) M and 7.59 fmol/10 mg wet weight/min, respectively, whereas these values for the low-affinity system were 1.00 x 10(-6) M and 100 fmol/10 mg wet weight/min, respectively. In the spinal cord, there was only one uptake system, with a Km value of 2.16 x 10(-7) M and Vmax value of 26.2 fmol/10 mg wet weight/min. These results suggest that when SP is released from nerve terminals, it is hydrolysed into (5-11)SP before or after acting as a neurotransmitter, which is in turn accumulated into nerve terminals. Therefore, the uptake system may represent a possible mechanism for the inactivation of SP.     

Detection of N-Terminally Extended Substance P but Not of Substance P in Human Cerebrospinal Fluid: Quantitation with HPLC-Radioimmunoassay

A reversed-phase HPLC system was used to concentrate and separate components of substance P-like immunoreactivity (SP-LI) from human CSF. When CSF was injected and fractions collected, no SP-LI could be detected by radioimmunoassay (RIA) at the retention time of SP or SP-sulfoxide. Instead, SP-LI was detected in later eluting fractions. This SP-LI reacted with two different antisera raised against the C-terminal part of SP, but not with an antiserum against the N-terminal part. A compound with similar properties was also found to be present in neutral extracts of rat dorsal spinal cord. When the late-eluting compound from human CSF was treated with trypsin and rechromatographed on HPLC, an immunoreactive component eluting at the position of SP could be detected with both the C- and N-terminally directed SP antisera. These results suggest that an N-terminally extended form of SP is present in human CSF. Trypsinization also gave two other compounds with affinity for the N- but not the C-terminally directed antisera. This may indicate that N-terminal fragments of SP extended at the N-terminus or SP molecules extended at both the N- and the C-terminus (i.e., preprotachykinins) also are present in human CSF. In 32 CSF samples from depressed patients, SP-LI was determined with a C-terminally directed antiserum with and without prior HPLC separation. SP itself could not be detected, but the late-eluting form of SP-LI could be quantitated in all samples by combined HPLC-RIA. In most samples, there was a relatively good agreement between the SP-LI levels measured with and without HPLC.     

Proteolysis Controls Endogenous Substance P Levels

Substance P (SP) is a prototypical neuropeptide with roles in pain and inflammation. Numerous mechanisms regulate endogenous SP levels, including the differential expression of SP mRNA and the controlled secretion of SP from neurons. Proteolysis has long been suspected to regulate extracellular SP concentrations but data in support of this hypothesis is scarce. Here, we provide evidence that proteolysis controls SP levels in the spinal cord. Using peptidomics to detect and quantify endogenous SP fragments, we identify the primary SP cleavage site as the C-terminal side of the ninth residue of SP. If blocking this pathway increases SP levels, then proteolysis controls SP concentration. We performed a targeted chemical screen using spinal cord lysates as a proxy for the endogenous metabolic environment and identified GM6001 (galardin, ilomastat) as a potent inhibitor of the SP _1–9-producing activity present in the tissue. Administration of GM6001 to mice results in a greater-than-three-fold increase in the spinal cord levels of SP, which validates the hypothesis that proteolysis controls physiological SP levels.     

Use of substance P fragments to differentiate substance P receptors of different tissues

The C- and N-terminal fragments of substance P were compared to the parent molecule with respect to their ability to: (a) contract the isolated guinea pig ileum, (b) induce salivation in the rat, (c) excite single cat dorsal horn neurones, and (d) induce scratching by intracranial injections in mice. C-terminal fragments as small as the heptapeptide were potent SP agonists on all assay systems. C-terminal fragments containing five amino acids or less were, at most, only weakly active. The C-terminal hexapeptide was a potent SP receptor stimulant on the isolated guinea pig ileum and, when directly applied by microiontophoresis, on cat dorsal horn neurons. However, the same compound was only 2-5% as potent as substance P in eliciting salivation and scratching in vivo, an indication that this fragment may be especially labile to enzymatic degradation. N-terminal fragments were totally inactive on the isolated guinea pig ileum. On the rat salivation and central nervous system assays, however, N-terminal fragments were capable of weak SP-like activity. It is concluded that SP receptors exist in multiple forms which we have labelled SP1 and SP2 receptors for those insensitive or sensitive to N-terminal fragments, respectively.     

Substance P and behavior: opposite effects of N-terminal and C-terminal fragments

Most of the biological actions of substance P (SP) have been thought to be mediated by the carboxy-terminal portion of the peptide. Some of the behavioral effects produced by exogenous SP exhibit a strikingly different structure-activity relationship. The N-terminal heptapeptide fragment of SP, SP(1-7), inhibits nociceptive, aggressive and grooming behaviors and stimulates investigative motor behavior, but the C-terminal hexapeptide fragment analog pyroglutamyl-SP(7-11) exerts opposite effects. While the C-terminal fragment mimics the effects of administered intact SP on motor behaviors, the N-terminal fragment mimics the effects of intact SP on aggressive and nociceptive behaviors. The significant behavioral effects of SP(1-7) and the consistently opposite behavioral effects of N- and C-terminal fragments are important new findings.     

Tachykinin Systems in the Spinal Cord and Basal Ganglia: Influence of Neonatal Capsaicin Treatment or Dopaminergic Intervention on Levels of Peptides, Substance P–Encoding mRNAs, and Substance P Receptor mRNA

The aim of the study was to test whether the synthesis of substance P (SP) and that of its receptor (also known as NK1 receptor) are coordinately regulated after chronic pharmacologic intervention in two neural systems, the spinal cord and basal ganglia. In one set of experiments, capsaicin was administered subcutaneously during the early postnatal period (day 3 after birth) to induce degeneration of afferent sensory neurons in the spinal cord. In the other set of experiments, interruption of dopaminergic transmission was achieved by two methods: (a) The neurotoxin 6-hydroxydopamine was used to denervate dopaminergic neurons during the early postnatal period, and (b) haloperidol was used in adult animals to block dopaminergic transmission by receptor blockade. The spinal cord, striatum, or both were used for the quantification of tachykinin [SP and neurokinin A (NKA)] and opioid peptides [[Met5]-enkephalin (ME) and dynorphin A (1-8) (DYN)] by radioimmunoassays. The abundance of total SP-encoding preprotachykinin (PPT) mRNA and SP receptor (SPR) mRNA in spinal cord (C5 to T1 segments), striatum, or microdissected substantia nigra was determined by northern blot or solution hybridization analysis. Amines and their acid metabolites were quantified by HPLC. Capsaicin administration (subcutaneously) during the early postnatal period increased latency in a hot-plate test, decreased SP and NKA levels, increased levels of PPT mRNAs, and did not affect SPR mRNA levels in the spinal cord. Intraspinal SP systems may attempt to compensate for the loss of afferent SP input, whereas spinal cord receptor mRNA levels do not appear to be altered.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tachykinin Receptors of the NK1 Type (Substance P) Coupled Positively to Phospholipase C on Cortical Astrocytes from the Newborn Mouse in Primary Culture

Specific 125I-Bolton-Hunter substance P (125I-BHSP) binding sites are present on intact cortical astrocytes of the newborn mouse in primary culture. Therefore, these cells were used to ascertain the existence of functional substance P (SP) receptors coupled positively to phospholipase C. SP stimulated phosphoinositide breakdown with an EC50 value (4.5 x 10(-10) M) similar to its IC50 value (3.8 x 10(-10) M) for inhibiting 125I-BHSP binding. The maximal response to (10(-6) M SP for 60 min) obtained was approximately 500% of control values. The rank order of potency of tachykinins was SP greater than neurokinin (NK) A greater than NKB. Long SP C-terminal fragments were more potent than shorter ones in stimulating the accumulation of 3H-inositol phosphates. SP free acid and SP N-terminal fragments were without effect. [L-Pro9]SP and SP methyl ester, two selective agonists of NK1 receptors, were almost as potent as SP. An excellent correlation was found when the abilities of tachykinins and their analogs for stimulating phosphoinositide breakdown and for inhibiting 125I-BHSP binding were compared. Finally, when used at a concentration of 3 x 10(-6) M, spantide [( D-Arg1, D-Trp7,9, Leu11]SP), an SP antagonist, competitively reduced the stimulatory effect of SP on accumulation of 3H-inositol phosphates. These results demonstrate the presence of functional SP receptors (NK1) on cortical astrocytes from the newborn mouse in primary culture.     

Substance P antagonist-induced spinal cord vasoconstriction: Effects of thyrotropin-releasing hormone and substance P agonists

Local spinal cord vasomotor effects of 3 substance P (SP) antagonists were studied in the rat following intrathecal (IT) administration. Each SP antagonist (3.3 nmol) increased spinal cord vascular resistance and reduced blood flow. A LH-RH antagonist analog (10 nmol) of similar molecular weight and which also contained multiple D-Trp residues did not cause spinal cord vasoconstriction. The vasoconstrictor action of the SP antagonist, [D-Arg¹, D-Pro², D-Trp^7,9, Leu¹¹]-SP ([D-Arg]-SP) was unaffected by pretreatment with a stable SP receptor agonist (5 nmol IT). Given evidence for a cerebral vasodilator action of TRH agonists, the effects of TRH (IV) and a stable TRH analog (MK-771, IT) on [D-Arg]-SP-induced vasoconstriction were also assessed. Neither TRH nor MK-771 prevented the [D-Arg]-SP-induced vasoconstriction. However, TRH (IV) but not MK-771 (IT) partially opposed [D-Arg]-SP-induced reduction in thoracic spinal cord blood flow. Thus, SP antagonists cause spinal cord vasoconstriction by a non-SP receptor mediated phenomenon. In addition, the attenuation of SP-antagonist-induced neuropathological changes previously reported with IV. TRH administration is likely due to less severe consequences of vasoconstriction in the presence of a higher initial baseline blood flow rather than direct prevention of the vasoconstriction.     

Distribution and origin of bombesin,substance P and somatostatin in cat spinal cord

Bombesin (BN), substance P-(SP) and somatostatin (SRIF) were measured in individual laminae of the cervical, thoracic and lumbar (L) spinal cord of control cats, and in the L6 segment of cats receiving a spinal hemisection (L2) or deafferentation via dorsal rhizotomy at L6, 7, S1. The interlaminar distribution of BN, SP, and SRIF was remarkably similar. Highest concentrations were found in the superficial dorsal horn, and progressively less was found proceeding ventrally. Some intersegmental variations in peptide concentration within a single lamina were found. Dorsal rhizotomy caused a significant decline in BN, SP and SRIF in lamina I-III, therefore all three peptides appear to be contained in dorsal root ganglion cells. Evidence is presented for the existence of ascending BN and SP projections originating in lamina I-III and VII, for a descending SRIF pathway terminating in lamina VIII, and for an ascending BN path in lamina VIII. Dorsal root afferents to lamina VIII influence levels of BN, SP and SRIF.     

Regional Distribution of Immunoreactive-Thyrotrophin-Releasing Hormone and Substance P, and Indoleamines in Human Spinal Cord

The regional distributions of thyrotrophin-releasing hormone (TRH) and substance P in postmortem human spinal cord were determined by radioimmunoassay in fresh tissue taken from 22 patients who died without known neurological disease. Dorsal, ventral, and intermediolateral spinal cord regions were obtained from different segmental levels (lumbar L1, 2, 3, and 4; thoracic groups T1-3, T4-6, T7-9, and T10-12) together with selective regions of grey matter of lumbar spinal cord. The effects on peptide levels of the age of the patient, the postmortem time interval, and freezing the tissue samples prior to assay were assessed. Levels of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were determined in regional lumbar and thoracic tissue using HPLC with electrochemical detection. Substance P was found in the highest concentration in the dorsal spinal cord, with no significant segmental differences. In contrast, TRH was present in higher levels in the ventral rather than the dorsal spinal cord, with segmental differences. There was a significant difference in the 5-HT/5-HIAA ratio between dorsal and ventral spinal cord, with the highest ratio in the ventral spinal cord. There were no significant differences in substance P, TRH, or 5-HT levels in spinal cords between 5 and 20 h postmortem or from patients aged between 65 and 90 years. Freezing the tissue (-80 degrees C for 24 h) prior to assay significantly reduced TRH and substance P levels compared to samples assayed immediately without prior freezing.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endomorphins interact with the substance P (SP) aminoterminal SP(1-7) binding in the ventral tegmental area of the rat brain

We have recently identified a specific binding site for the tachykinin peptide substance P (SP) fragment SP(1-7) in the rat spinal cord. This site appeared very specific for SP(1-7) as the binding affinity of this compound highly exceeded those of other SP fragments. We also observed that endomorphin-2 (EM-2) exhibited high potency in displacing SP(1-7) from this site. In the present work using a [(3)H]-labeled derivative of the heptapeptide we have identified and characterized [(3)H]-SP(1-7) binding in the rat ventral tegmental area (VTA). Similarly to the [(3)H]-SP(1-7) binding in the spinal cord the affinity of unlabeled SP(1-7) to the specific site in VTA was significantly higher than those of other SP fragments. Further, the tachykinin receptor NK-1, NK-2 and NK-3 ligands showed no or negligible binding to the identified site. However, the mu-opioid peptide (MOP) receptor agonists DAMGO, EM-1 and EM-2 did, and significant difference was observed in the binding affinity between the two endomorphins. As recorded from displacement curves the affinity of EM-2 for the SP(1-7) site was 4-5 times weaker than that for SP(1-7) but about 5 times higher than that of EM-1. The opioid receptor antagonists naloxone and naloxonazine showed weak or negligible binding. It was concluded that the specific site identified for SP(1-7) binding in the rat VTA is distinct from the MOP receptor although it exhibits high affinity for EM-2.     

Monoclonal Antibodies to Substance P: Production, Characterization of Their Fine Specificities, and Use in Immunocytochemistry

Five hybrid clones secreting antibodies to the neuropeptide substance P have been obtained by somatic cell fusion of mouse myeloma cells with splenocytes from immunized mice of the Biozzi strain. To perform rapid and sensitive screening tests as well as to study the fine specificities of each monoclonal antibody, we developed a new enzyme immunoassay of substance P using acetylcholinesterase as label. All five monoclonal antibodies were directed to the C-terminal pentapeptide of substance P, especially to the Phe7 residue. They cross-reacted with neurokinin A and to some extent with neurokinin B but not with other nontachykinin mammalian peptides. One monoclonal antibody (SP 14) was used for immunocytochemical experiments in the rat spinal cord and spinal ganglion, both at the light and electron microscopic levels. A strong specific neurokinin-like immunoreactivity was observed in cell bodies, nerve fibers, and terminals, with a very low background staining. Finally, the affinities of several analogues of substance P for SP 14 monoclonal antibody were shown to be correlated with their biological activities, as measured by their hypotensive effects in vivo. These findings suggested a strong structural resemblance between the combining site of the antibody and that of the physiological substance P receptor.     

Nitric Oxide Regulates Substance P Release from Rat Spinal Cord Synaptosomes

Abstract: In order to determine whether nitric oxide (NO) acts directly upon nerve terminals to regulate the synaptic transmission at the level of spinal cord, effects of NO-donors on release of substance P (SP) and glutamic acid (Glu) were investigated by superfusion of synaptosomes prepared from the rat spinal cord. Basal levels of endogenous SP and Glu release were 5.99 ± 2.50 fmol/min/mg of protein and 26.2 ± 4.8 pmol/min/mg of protein, respectively. Exposure to a depolarizing concentration of KCI evoked 2.7- and 3.8-fold increases in SP and Glu release in a calcium-dependent manner, respectively. Sodium nitroprusside (NP) caused a reduction in the depolarization-evoked overflow of SP in a concentration-dependent manner without affecting its basal release, although it failed to affect either basal or evoked release of Glu. The reduction in SP overflow was also observed by the perfusion with S -nitroso- N -acetyl-penicillamine or membrane-permeable cyclic GMP, but not with cyclic AMP. NP caused the concentration-dependent increases in cyclic GMP levels in synaptosomes. Together with reports that excitatory amino acids stimulate NO synthase and release NO in the spinal cord, these data suggest that there may be an interaction between nerve terminals containing Glu and SP, and that NO may directly participate in the regulation of synaptic transmission in SP-containing nerve terminals, which may be mediated through the activation of guanylate cyclase and the increase in cyclic GMP levels.     

A fragment of substance P with specific central activity: SP(1-7)

Amino-terminal fragments of substance P (SP), SP(1-7) and SP(1-8), were found to produce naloxone-reversible antinociception in the mouse similar to that produced by SP. Similar to SP, these peptides produce antinociception only within a narrow dose range. They have no activity on smooth muscle or blood pressure. These results suggest that contrary to peripheral effects of SP, which are mediated by receptors which recognize the carboxy-terminal part of the SP molecule, certain central actions of SP are mediated by receptors which recognize the amino-terminal part of the SP molecule. SP may be metabolized to this active fragment prior to its action at these receptors.     

Functional Studies with Substance P Analogues: Effects of N-Terminal, C-Terminal, and C-Terminus-Extended Analogues of Substance P on Nicotine-Induced Secretion and Desensitization in Cultured Bovine Adrenal Chromaffin Cells

Abstract: Substance P (SP) and SP analogues, including C-terminal, N-terminal, and C-terminus-extended analogues, have been investigated for their ability to modulate nicotine-induced secretion from bovine adrenal chromaffin cells in culture. Secretion was monitored by measuring the release of endogenous catecholamines by electrochemical detection following separation on HPLC and the release of endogenous ATP with an on-line luciferin-luciferase bioluminescence technique. SP is known to have the following two effects on nicotine-induced secretion of catecholamines (see Livett and Zhou, 1991): inhibition of the nicotinic response and protection against nicotinic desensitization. Secretion induced by 10^-5M nicotine was inhibited 70-80% by SP, SP-methyl ester, and the C-terminus-extended analogue SP-Tyr¹²-NH₂, 65% by (Ala³)SP-NH₂, 45% by the C-terminal analogue SP(4-11), and 20 and 5% by the N-terminal analogues SP(1-7) and SP(1-5), respectively, when these peptides were present at 3 ×; 10^-5M concentrations. The order of potency was SP = SP-methyl ester = SP-Tyr¹²-NH₂ > (Ala³)SP-NH₂ > SP(4-11) > SP(1-7) > SP(1-5). SP, SP-methyl ester, and (Ala³)SP-NH₂ protected against nicotinic desensitization by 40-55%, and SP(4-11) protected by 20% (all at 3 ×; 10^-5M). In contrast, the N-terminal analogues SP(1-7) and SP(1-5) and the C-terminus-extended analogue SP-Tyr¹²-NH₂ at 3 × 10^-5M did not protect against nicotinic desensitization. Cyclo-SP(3-9), Ac-SP(3-9)-NH₂, SP(3-9), and SP(3-6) had neither inhibitory nor facilitatory effects on secretion. Of the 20 SP analogues extended at the C terminus by one amino acid, there were only three that protected against nicotinic desensitization, whereas the majority inhibited nicotine-evoked catecholamine secretion. The present work indicates that for inhibition of nicotine-evoked secretion, both the C terminus and N terminus of SP are necessary. For the protection against nicotine-induced desensitization, the C terminus of SP is important. This suggests that the two mechanisms, inhibition of nicotine-evoked secretion and protection against nicotinic desensitization, are regulated independently.     

Widespread distribution of substance P-and somatostatin-immunoreactive elements in the spinal cord of the neonatal rat

The distribution of substance P (SP)- and somatostatin (SOM)-immunoreactive elements in the spinal cord of the neonatal rat was examined. With few exceptions, the distribution of SP-immunoreactive elements is similar to that described for the adult. A major difference is the obvious presence of SP-immunoreactive fibers in all funiculi of neonatal cords. In addition, an obvious small bundle of longitudinal SP immunoreactive fibers is seen in the base of the dorsal horn at rostral cervical levels. Unlike that of the adult, the neonatal spinal cord shows a widespread distribution of SOM-immunoreactivity. SOM-immunoreactive fibers are present in all funiculi. SOM-immunoreactive perikarya of various shapes and sizes are widely dispersed throughout the gray matter. The cell density is increased in the superficial laminae of the dorsal horn, in a region ventral-lateral to the central canal and in the ventral horn. SOM-immunoreactive varicosities are present in moderate amounts in the superficial laminae of the dorsal horn but are extremely sparse in other regions of the gray matter. A few SOM-immunoreactive fibers course longitudinally at the base of the dorsal horn at rostral levels of the cord. These fibers are found in the same region occupied by the longitudinal SP-immunoreactive fibers referred to above.     

Calcitonin gene-related peptide is metabolized by an endopeptidase hydrolyzing substance P

Calcitonin gene-related peptide (CGRP) is cleaved by an endopeptidase, also known to hydrolyze substance P (SP). The enzyme which was isolated from human cerebrospinal fluid, converted rCGRP into two products, clearly separable on HPLC. Amino acid analysis showed cleavage to occur at Leu16-Ser17. The carboxy-terminal fragment, rCGRP-(17-37), was weakly active in inhibiting 125I-rCGRP binding to a rat medulla oblongata membrane preparation, but it showed no binding to spinal cord membranes. The N-terminal fragment, rCGRP-(1-16), had very low or no affinity. Autoradiography with 125I-rCGRP showed distinct labelling of rat dorsal spinal cord, while there was no consistent pattern with 125I-rCGRP-(1-16). In the isolated guinea pig ileum preparation, the two fragments showed no CGRP-like activity. The ability of CGRP to interfere with SP degradation is offered as the explanation why CGRP has been reported to potentiate several biologic actions of SP.     

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)