Patrick E. Ward

Metabolism of vasoactive peptides by vascular endothelium and smooth muscle aminopeptidase M

The cellular localization of vascular plasma membrane aminopeptidase M (AmM; EC3.4.11.2) was examined in cultured porcine aorta endothelium and smooth muscle cells. AmM was 14-fold higher on smooth muscle (117 +/- 16 units/mg) than on endothelium (8.4 +/- 0.2). Proportional to its cellular distribution, AmM hydrolyzed the N-terminus of kallidin to produce bradykinin, and degraded des(Asp1)angiotensin I, angiotensin III, hepta(5-11)substance P and Met5-enkephalin. In contrast, bradykinin, angiotensin II and substance P were resistant to AmM-mediated hydrolysis. Peptide metabolism was optimal at pH 7.0 and was inhibited by o-phenanthroline, bestatin (Ki = 2.2 +/- 0.1 microM) and amastatin (Ki = 25 +/- 5 nM). Des(Asp1)angiotensin I and angiotensin III had the highest affinity (lowest Km) for AmM (Km = 2.2 +/- 0.5 and 2.0 +/- 0.4 microM respectively), followed by hepta(5-11)substance P (53.9 +/- 1.7 microM) and Met5-enkephalin (75.7 +/- 3.5 microM). In contrast, maximal velocities of hydrolysis were higher for Met5-enkephalin (313 +/- 2 nmol/min/mg) than for hepta(5-11)substance P (109 +/- 18 nmol/min/mg) or angiotensin III (26.5 +/- 1.0 nmol/min/mg). As expected for hydrolysis by a common enzyme, AmM-mediated enkephalin degradation was inhibited competitively by angiotensin III (Ki = 0.34 +/- 0.04 microM), hepta(5-11)substance P (43.7 +/- 6.3 microM) and kallidin (62 microM). These data suggest that vascular AmM may modulate vasoactive peptide levels in vivo, particularly within the microenvironment of endothelial and smooth muscle cell surface receptors.

Vascular, plasma membrane aminopeptidase M metabolism of vasoactive peptides

Aminopeptidase M (EC 3.4.11.2), an enzyme present on the cell surface of vascular endothelium and/or smooth muscle, rapidly hydrolyzes leucyl- and arginyl-2-naphthylamides and a number of vasoactive peptides at physiologic pH. Utilizing both thin-layer chromatography and high pressure liquid chromatography, it was found that vascular aminopeptidase M converted kallidin to bradykinin and inactivated des(Asp1)angiotensin I, angiotensin III, hepta(5-11)substance P and hexa(6-11)substance P. Aminopeptidase M did not, however, hydrolyze bradykinin, angiotensin I, angiotensin II, saralasin, vasopressin, oxytocin or any form of substance P containing a component of the Arg-Pro-Lys-Pro sequence. Both the naphthylamidase and peptidase activities were inhibited similarly by known amino-peptidase M inhibitors including o-phenanthroline, amastatin, bestatin and puromycin. However, inhibitors of angiotensin I converting enzyme (captopril), carboxypeptidase N (MERGETPA), neutral endopeptidase (phosphoramidon), post proline cleaving enzyme and dipeptidyl(amino)peptidase IV (diisopropylphosphofluoridate, DFP) were without effect. These results demonstrate that vascular, cell surface aminopeptidase M can selectively metabolize vasoactive peptides and may play a role in modulating their levels in the circulation and/or within the vessel wall.

Conversion of kinins and their antagonists into B1 receptor activators and blockers in isolated vessels

A carboxypeptidase inhibitor (DL-2-mercaptomethyl-3-guanidoethylthiopropranoic acid) (mergetpa) was used to block the conversion of kinins and B2 receptor antagonists into metabolites devoid of the C-terminal Arg. Experiments were carried out on rabbit isolated aortae (a B1 receptor system) or rabbit jugular veins and dog carotid arteries (two B2 receptor systems). The contractile effect of bradykinin in the rabbit aorta was significantly reduced by mergetpa while that of desArg9-BK was not modified. pA2 values of B2 receptor antagonists, [Thi5,8,D-Phe7]bradykinin and [Thi6,9,D-Phe8]kallidin were markedly reduced by mergetpa. The apparent affinity (pA2) of a B1 receptor antagonist, [Leu9]desArg10-kallidin was not affected. Carboxypeptidases inhibition did not modify the activities of bradykinin or the affinities of B2 receptor antagonists in the rabbit jugular vein and the dog carotid artery. An inhibitor of kininase II (D-3-mercapto-2-methylpropranoyl-L-proline (S,S] (captopril) reduced the contractile effects of angiotensin I in the three preparations and potentiated the stimulatory or inhibitory effects of bradykinin: captopril did not have effect on the affinities of B2 receptor antagonists and did not modify the effects of angiotensin II. Comparative experiments performed in tissues with or without endothelium gave the same results with both mergetpa and captopril. The present findings suggest that bradykinin and B2 receptor antagonists are converted by carboxypeptidases into biologically active B1 receptor agonist or antagonists. This is the reason why B2 receptor antagonists are not selective.

Angiotensin metabolism by cerebral microvascular aminopeptidase A

Porcine cerebral microvessels were isolated by differential sieving and centrifugation and were characterized by microscopic examination and marker enzyme enrichment (gamma-glutamyltransferase; EC 2.3.2.2). Purified microvessels contained a membrane-bound enzyme immunologically indistinguishable from renal aminopeptidase A (AmA; EC 3.4.11.7). AmA hydrolyzed both alpha-glutamyl- and alpha-aspartyl-2-naphthylamide, and hydrolysis was competitively inhibited by angiotensin II. Micro-vessel AmA hydrolyzed the N-terminal Asp1-Arg2 bond of both angiotensin I and angiotensin II, whereas the angiotensin II antagonist saralasin [(Sar1, Ala8)angiotensin II] was resistant to N-terminal hydrolysis. Angiotensin metabolism was optimal at pH 8.5 and was inhibited by EDTA, o-phenanthroline and amastatin. Conversely, inhibitors of neutral endopeptidase (phosphoramidon), post-proline cleaving enzyme (Z-Pro-Prolinal), carboxypeptidase N [D-L-mercaptomethyl-3-guanidinoethylthiopropanoic acid (MERGETPA)] and angiotensin I converting enzyme (captopril) had no effect. The Km values of angiotensin I, angiotensin II and (Asn1, Val5)angiotensin II for microvessel AmA were 40.1 +/- 8.2, 35.3 +/- 4.3 and 156 +/- 22 microM respectively. Cerebral microvascular aminopeptidase A may play a role in vivo in modulating angiotensin-mediated local cerebral blood flow, and in preventing circulating angiotensins from crossing the blood-brain barrier.

Relaxation of isolated mesenteric arteries by des-Arg9-bradykinin stimulation of B1 receptors

The present studies were conducted to determine whether des-Arg-kinins can produce relaxation of isolated vessels. Both des-Arg9-bradykinin and bradykinin produced dose-dependent relaxations of isolated rabbit superior mesenteric arteries. Des-Arg9-bradykinin (ED50 = 7.2 X 10(-9) M) was 8.5 times more potent than bradykinin (ED50 = 6.1 X 10(-8) M). Des-Arg9-bradykinin-mediated relaxation was inhibited by the specific B1 receptor antagonist [Leu8]des-Arg9-bradykinin which produced parallel shifts in the dose-response curve. Schild regression analysis of the data established a pA2 value (6.46) similar to that reported for B1 receptor-mediated contraction. Although the relaxant effect of bradykinin was also inhibited by the B1 antagonist, parallel shifts in the dose response curve were not produced. Relaxation of the mesenteric artery by both des-Arg9-bradykinin and bradykinin was inhibited by the cyclooxygenase inhibitor indomethacin. The results of these studies indicate that in addition to vasoconstriction, des-Arg9-bradykinin can produce vasorelaxation which may be mediated through stimulation of B1 kinin receptors and the subsequent release of prostaglandins.

Metabolism of opioid peptides by cerebral microvascular aminopeptidase M

Aminopeptidase M (EC 3.4.11.2), which can degrade low molecular weight opioid peptides, has been reported in both peripheral vasculature and in the CNS. Thus, we have studied the metabolism of opioid peptides by membrane-bound aminopeptidase M derived from cerebral microvessels of hog and rabbit. Both hog and rabbit microvessels were found to contain membrane-bound aminopeptidase M. At neutral pH, microvessels preferentially degraded low molecular weight opioid peptides by hydrolysis of the N-terminal Tyr1-Gly2 bond. Degradation was inhibited by amastatin (I50 = 0.2 microM) and bestatin (10 microM), but not by a number of other peptidase inhibitors including captopril and phosphoramidon. Rates of degradation were highest for the shorter peptides (Met5- and Leu5-enkephalin) whereas beta-endorphin was nearly completely resistant to N-terminal hydrolysis. Km values for the microvascular aminopeptidase also decreased significantly with increasing peptide length (Km = 91.3 +/- 4.9 and 28.9 +/- 3.5 microM for Met5-enkephalin and Met5-enkephalin-Arg6-Phe7, respectively). Peptides known to be present within or in close proximity to cerebral vessels (e.g., neurotensin and substance P) competitively inhibited enkephalin degradation (Ki = 20.4 +/- 2.5 and 7.9 +/- 1.6 microM, respectively). These data suggest that cerebral microvascular aminopeptidase M may play a role in vivo in modulating peptide-mediated local cerebral blood flow, and in preventing circulating enkephalins from crossing the blood-brain barrier.

Degradation of low-molecular-weight opioid peptides by vascular plasma membrane aminopeptidase M

Since both aminopeptidases and angiotensin I-converting enzyme are reported to degrade circulating enkephalins, we have examined the degradation of low-molecular-weight opioid peptides by a vascular plasma membrane-enriched fraction previously shown to contain both angiotensin I-converting enzyme (EC 3.4.15.1) and aminopeptidase M (EC 3.4.11.2). Except for an enkephalin analog resistant to amino-terminal hydrolysis, [D-Ala2]enkephalin, the purified vascular plasma membrane preferentially degraded low-molecular-weight opioids by hydrolysis of the N-terminal Tyr-1--Gly-2 bond. Enkephalin degradation was optimal at pH 7.0 and was inhibited by the aminopeptidase inhibitors amastatin (I50 = 0.08 microM), bestatin (9.0 microM) and puromycin (80 microM). Maximal rates of hydrolysis, calculated per mg plasma membrane protein, were highest for the shorter peptides (18.3, 15.6 and 16.6 nmol/min per mg for Met5-enkephalin, Leu5-enkephalin and Leu5-enkephalin-Arg6, respectively) and decreased with increasing peptide length (0.7 nmol/min per mg for dynorphin (1-13)). No significant hydrolysis of beta- and gamma-endorphin was detected. Km values decreased significantly with increasing peptide length (Km = 72.9 +/- 2.7, 43.6 +/- 4.7 and 21.4 +/- 0.9 microM for Met5-enkephalin, Leu5-enkephalin-Arg6 and Met5-enkephalin-Arg6-Phe7, respectively). However, no further decreases were seen with even larger sequences, i.e., dynorphin(1-13). Other peptides hydrolyzed by the plasma membrane aminopeptidase (angiotensin III, kallidin and hepta(5-11)-substance P) inhibited enkephalin degradation in a competitive manner. Thus, localization, specificity and kinetic data are consistent with identification of aminopeptidase M as a vascular enzyme with the capacity to differentially metabolize low-molecular-weight opioid peptides within the microenvironment of vascular cell surface receptors. Such differential metabolism may play a role in modulating the vascular effects of peripheral opioids.

Immunoelectrophoretic analysis of vascular, membrane-bound angiotensin I converting enzyme, aminopeptidase M, and dipeptidyL(AMINO)peptidase IV

Antisera raised against specific renal brush border peptidases have been used to characterize vascular surface membrane angiotensin I converting enzyme (ACE; EC 3.4.15.1), aminopeptidase M (AmM; EC 3.4.11.2), and dipeptidyl(amino)peptidase IV (DAP IV; EC 3.4.14.5) by techniques of differential solubilization, fused-rocket immunoelectrophoresis and crossed immunoelectrophoresis. The vascular membrane-bound enzymes are immunologically indistinguishable from their brush border counterparts and can be solubilized by treatment with detergent and/or papain. The electrophoretic mobilities of the papain-treated forms of each enzyme were greater than those of the detergent-treated forms. This increased mobility is associated with the removal of small, hydrophobic, non-antigenic components of the enzymes. Regardless of the method of solubilization, the electrophoretic mobilities of the vascular enzymes were greater than those of the brush border enzymes. However, after treatment with neuraminidase to remove sialic acid, their respective mobilities were similar. The mobilities of serum AmM and DAP IV were identical to the respective papain-solubilized vascular enzymes both before and after neuraminidase. Thus, like the brush border enzymes, the data presented are consistent with the model that vascular ACE, AmM and DAP IV are intrinsic membrane peptidases bound to their surface membranes by small, non-antigenic, hydrophobic anchors associated with the lipid bilayer. In addition, these vascular surface membrane peptidases are similar to and may be a source of the circulating enzymes.

Kinin and angiotensin metabolism by purified renal post-proline cleaving enzyme

Post-proline cleaving enzyme (PPCE; EC 3.4.21.26) is a proline specific endopeptidase capable of hydrolyzing biologically active peptides. The present studies examined the hydrolysis of kinin- and angiotensin-related peptides by cytosolic PPCE purified from porcine kidney. PPCE hydrolysis of the synthetic substrate Z-Gly-Pro-MCA (30.7 +/- 0.3 mumol . min-1 . mg-1) was competitively inhibited by saralasin, bradykinin, des(Arg9)bradykinin, [Leu8], des(Arg9)bradykinin and angiotensin II (IC50 = 0.5 to 7.0 microM). Qualitative TLC studies demonstrated that each peptide was degraded by hydrolysis on the carboxyl side of proline residues (positions 7 or 8). Quantitative HPLC studies established that peptide degradation was optimal at pH 8.2 to 8.7 and was inhibited by the specific PPCE inhibitor Z-Pro-prolinal (IC50 = 0.8 +/- 0.1 nM). Conversely, degradation was unaffected by inhibitors of aminopeptidases (amastatin), neutral endopeptidase (phosphoramidon), carboxypeptidase N (MERGETPA) or angiotensin I converting enzyme (captopril). Apparent Km values, obtained from Lineweaver-Burk analysis, were comparable for all kinin and angiotensin peptides (Km = 5.5 to 12.8 microM), whereas Vmax values ranged from 1.7 mumol . min-1 . mg-1 for angiotensin II to 0.44 mumol . min-1 . mg-1 for saralasin. These data are consistent with a role for PPCE in the degradation of kinins and angiotensin in vivo.

Kallikrein and renin in the membrane fractions of the rat kidney.

SUMMARY Plasma membrane (PM) and endoplasmic reticulum (ER) enriched fractions were isolated from the homogenized rat kidney. Transmission electron micrographs of PM showed empty vesicles but no granules present in the fraction. Kallikrein activity was detected in the homogenate, microsomal, and PM and ER fractions; it was most enriched in PM fraction. PM-kallikrein released a klnin, cleaved the peptide substrate, S-2266, and a radiolabeled arginine ester. The ester was also hydrolyzed by renal enzymes other than kallikrein. PM-kallikrein was activated by Triton X-100, phospbolipase A, lysolecithin, and by a peptide, melittin. Melittin (2 fiM) was most potent; it increased the activity to 750%. Solubilized PM and ER kallikrein were inhibited by antibody to rat urinary kallikrein, but membrane-bound kallikrein was more resistant to inhibition. The Km of S-2266 was higher with renal than with urinary kallikrein. The PM and ER fractions also contained renin. Renin activity was enhanced 30-fold or more by activators of kallikrein, e.g., by phospholipase A, lysolecithin, and melittin. Low sodium diet increased the activity of kallikrein in the homogenate and in the membrane fraction. This diet increased the activity of renin in the homogenate but not in the membrane fraction. It is suggested that prekallikrein is on PM and is activated prior to release from the membrane. Membrane-bound renin may be a form of renin retained in the kidney.