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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.

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.

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.

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.

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%).

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.

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 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.

A correlative radioimmunological-biochemical-ultrastructural study of the rat pineal gland was undertaken during the summer months when pineal arginine vasotocin (AVT) immunoactivity increases up to 200-fold. RIA confirmed a rapid rise in AVT activity during mid-August regardless of the time of day sampled. Pineal indoles were separated by HPLC and measured using electrochemical detection. Serotonin (5-HT) and 5-hydroxyindoleacetic acid levels were consistently elevated in daytime samples, and there was a significant trend for increased day and nighttime levels of 5-HT from July to September. Mid-dark levels of melatonin also exhibited a significant increase over the sample period. Nighttime levels of N-acetylserotonin mirrored fluctuations in 5-HT in the preceding photoperiod. Ultrastructural components implicated in peptide/protein and/or indole biosynthesis were quantified by stereological morphometry. The greatest amounts of rough endoplasmic reticulum stacks, lipid droplets, and annulate lamellae-like bodies coincided with peak AVT activity. Dense-cored vesicles and synaptic ribbons were consistently more frequent during the dark period. The number of dense-cored vesicles and nucleolar size tended to be greatest before and after the peak in AVT immunoactivity. These observations are consistent with the hypotheses that endoplasmic reticulum and lipid are functionally related to the synthesis and/or storage of peptide/protein factors and that numerical changes in synaptic ribbons and dense-cored vesicles are more closely related to day/night differences in indole metabolism.

A post-proline cleaving enzyme [post-proline endopeptidase: EC 3.4.21.26] was purified from lamb brain by a series of column chromatographies on DEAE-Sephadex, hydroxyapatite and Sephadex G-150. The purified enzyme appeared homogeneous on disc gel and sodium dodecyl sulfate (SDS) gel electrophoreses. The enzyme was most active at pH 7.0 with carbobenzoxy-Gly-Pro-beta-naphthylamide (Z-Gly-Pro-2-NNap) as a substrate and catalyzed the hydrolysis of oxytocin, vasopressin, thyrotropin releasing hormone (TRH), substance P, luteinizing hormone releasing hormone (LH-RH), and angiotensin at the carboxyl side of their proline residues, except for the Pro2-Lys3 bond in substance P. From the results of subsite mapping using synthetic peptides, five subsites, S3 to S2', for substrate interaction with the enzyme were deduced to be present, and high stereospecificity was observed at S2, S1, and S1'. The isoelectric point of the enzyme was at pH 4.9, and the molecular weights estimated by gel filtration and SDS gel electrophoresis were 74,000 and 77,000, respectively. The enzyme was markedly inhibited by diisopropylphosphoro fluoridate (DFP), carbobenzoxy-Gly-Pro-chloromethyl ketone (Z-Gly-Pro-CH2Cl), p-chloromercuribenzoate (PCMB), Hg2+, and Cu2+ ions. These enzymatic and protein chemical properties of post-proline cleaving enzyme from lamb brain closely resemble those of the lamb kidney enzyme, except for the molecular weight. In the present work, however, we decided that the molecular weight of the enzyme from lamb kidney was also 74,000, which is different from that reported previously (J. Biol. Chem. 251, 7593 (1976) but is in accord with the value of post-proline cleaving enzyme from lamb brain.

Most neuropeptides are known to occur both in the central nervous system and in blood. This, as well as the occurrence of central nervous peptide effects after peripheral administration, show the importance of studying the relationships between the peptides in the two compartments. For many peptides, such as the enkephalins, TRH, somatostatin and MIF-1, poor penetration of the blood-brain barrier was shown. In other cases, including beta-endorphin and angiotensin, peptides are rapidly degraded during or just after their entry into brain or cerebrospinal fluid. Some peptides, such as insulin, delta-sleep-inducing peptide, and the lipotropin-derived peptides, enter the cerebrospinal fluid to a slight or moderate extent in the intact form. Many peptide hormones, such as insulin, calcitonin and angiotensin, act directly on receptors in the circumventricular organs, where the blood-brain barrier is absent. Oxytocin, vasopressin, MSH, and an MSH-analog alter the properties of the blood-brain barrier, which may result in altered nutritient supply to the brain. In conclusion, the diffusion of most peptides across the brain vascular endothelium seems to be severely restricted. There are, however, several alternative routes for peripheral peptides to act on the central nervous system. The blood-brain barrier is a major obstacle for the development of pharmaceutically useful peptides, as in the case of synthetic enkephalin-analogs.

Previous studies have indicated that administration of the serotonin releaser p-chloroamphetamine HCl produces a dose-dependent increase in renin secretion through a blood-borne renin-releasing factor. The present studies were designed to partially characterize this renin-releasing factor using an in vitro kidney slice method for the bioassay of renin-releasing activity. Plasma from p-chloroamphetamine-treated, nephrectomized rats was used to obtain the renin-releasing factor, which was fractionated by ultrafiltration into fractions of molecular weight ranges of 1000 to 5000, 5000 to 10,000, and 10,000 to 20,000. The molecular weight ranges of the renin-releasing factor was determined to be between 5000 and 10,000. Since previous studies have shown that lesions in the hypothalamus prevent the effect of p-chloroamphetamine on renin secretion, we tested whether a hypothalamic extract can release renin from kidney slices. Addition of extracts of boiled rat hypothalamic tissue to the kidney slices caused an increase in renin release. Addition of cerebellar extracts produced a smaller increase in renin release, whereas addition of pituitary extracts had no effect. Fractionation by ultrafiltration of bovine hypothalamic extract revealed that the fraction with a molecular weight range of 5000 to 10,000 possessed the highest renin-releasing ability. The 1000 to 5000 (molecular weight) fraction possessed a sizeable renin-releasing activity, but the 10,000 to 20,000 fraction had no renin-releasing activity. Both bovine hypothalamus fractions (molecular weights between 1000-5000 and 5000-10,000) and plasma fraction lost their renin-releasing activity after digestion with pronase, suggesting that the renin-releasing factor or factors are peptides. These results suggest that a renin-releasing factor originate in the hypothalamus.

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