phosphoramidon and 2-mercaptomethyl-3-guanidinoethylthiopropionic-acid

Angiotensin-converting enzyme (kininase II [ACE]) inhibitors are capable of potentiating bradykinin (BK) effects by enhancing the actions of bradykinin on B(2) receptors independent of blocking its inactivation. To investigate further the importance of ACE kininase activity on BK-induced vasodilation, we investigated the effect of inhibiting ACE, as well as other kininases, on both BK metabolism and vasodilator effect in preparations that exhibit increased ACE activity. Mesenteric arterial beds obtained from 1-kidney, 1-clip hypertensive rats presented augmented ACE and angiotensin I converting activities compared with normotensive rats. The isolated and perfused mesenteric beds were exposed to BK for 15 minutes in the absence or in the presence of kininase inhibitors; then, the perfusate was collected for analysis of the products of BK metabolism by high-performance liquid chromatography. BK was metabolized to the fragments BK(1-8), BK(1-7), and BK(1-5), and the recovery of intact BK was reduced by 47% in the hypertensive group. Recovery of BK was increased in both groups in the presence of a kininase I inhibitor and in the hypertensive group by neutral endopeptidase 24.11 inhibitor; however, ACE inhibition did not affect BK metabolism in both groups. In contrast, only the ACE inhibitor potentiated the vasodilator effect of BK in a mesenteric bed preconstricted with phenylephrine; the increase in BK effect, nevertheless, was not greater in arteries from hypertensive rats that presented an increased ACE activity when compared with those in the normotensive group. These data demonstrated that ACE inhibitor-induced potentiation of BK vasodilator effects is not related to their actions on BK degradation.

Topics: 3-Mercaptopropionic Acid; Angiotensin-Converting Enzyme Inhibitors; Animals; Blood Pressure; Bradykinin; Drug Synergism; Enzyme Inhibitors; Glycopeptides; Hypertension; In Vitro Techniques; Lysine Carboxypeptidase; Male; Mesenteric Arteries; Metalloendopeptidases; Neprilysin; Peptide Fragments; Peptidyl-Dipeptidase A; Protease Inhibitors; Rats; Rats, Wistar; Vasodilation; Vasodilator Agents

The hydrolysis of bradykinin (BK) by human placental subcellular fractions and pregnancy sera was studied in the presence of inhibitors by measuring amino acids liberated from BK by high-performance liquid chromatography. The effects of the inhibitors DL-2-mercaptomethyl-3-guanidinoethylthiopropionic acid (MGTA, for kininase I), phosphoramidon (for endopeptidase 24.11) and captopril and rentiapril (for angiotensin-converting enzyme [ACE, kininase II]) suggested the essential roles of the above three proteases in BK degradation: among the three proteases, kininase I and endopeptidase 24.11 appeared to be the most important in kininase action in the placenta microsomes, whereas kininase I and ACE appeared to be the most important in kininase action in the placental cytosol, lysosome and pregnancy serum. Measurements of BK concentrations in the umbilical arterial blood, umbilical venous blood and maternal plasma revealed higher concentrations in the mother than in the fetus. The present data suggest that degradation of BK in the placenta and pregnancy serum might contribute to the gradient of BK between mother and fetus.

Topics: 3-Mercaptopropionic Acid; Angiotensin-Converting Enzyme Inhibitors; Arginine; Blood; Bradykinin; Captopril; Endopeptidases; Female; Glycopeptides; Humans; Hydrolysis; Lysine Carboxypeptidase; Microsomes; Neprilysin; Peptidyl-Dipeptidase A; Phenylalanine; Placenta; Pregnancy

1. Bradykinin (BK) instilled directly into the airway lumen caused bronchoconstriction in anaesthetized, mechanically ventilated guinea-pigs in the presence of propranolol (1 mg kg-1 i.v.). The geometric mean dose of BK required to produce 100% increase in airway opening pressure (PD100) was 22.9 nmol (95% c.i. 11.7-44.6 nmol). 2. The dose-response curve for the effect of instilled BK was significantly shifted to the left by the angiotensin converting enzyme (ACE) inhibitor, captopril (5 and 50 nmol instillation, PD100 = 3.0, 95% c.i. 0.98-8.9, and 2.0 nmol, 95% c.i. 0.65-6.2 nmol, respectively). 3. The neutral endopeptidase (NEP) inhibitor, phosphoramidon (5 and 50 nmol instillation) also shifted the dose-response curve for the effect of instilled BK; the PD100 values = 2.2 (95% c.i. 0.40-11.7) and 1.8 nmol (95% c.i. 0.87-3.5 nmol), respectively. 4. After pretreatment with captopril (50 nmol) and phosphoramidon (50 nmol) in combination, the dose-response curve for the effect of instilled BK (PD100 = 1.1 nmol, 95% c.i. 0.37-3.2 nmol) was similar to that obtained in the presence of each inhibitor used alone. 5. The kinase I inhibitor, DL-2-mercaptomethyl-3-guanidinoethylthiopropionic acid (50 nmol instillation) failed to alter the dose-response curve to instilled BK (PD100 = 14.6 nmol, 95% c.i. 6.7-32.0 nmol). 6. These data suggest that both ACE and NEP degrade BK in the airway lumen, but that kininase I is not involved.

Topics: 3-Mercaptopropionic Acid; Animals; Asthma; Bradykinin; Bronchoconstriction; Captopril; Glycopeptides; Guinea Pigs; In Vitro Techniques; Lysine Carboxypeptidase; Male; Muscle, Smooth; Propranolol; Protease Inhibitors

The relative contributions of three kininases to total urinary kininase activity were determined by measuring the hydrolysis of kinins in the presence and absence of inhibitors of kininase I (2-mercaptomethyl-3-guanidinoethylthiopropanoic acid; MGTA), kininase II (captopril) and neutral endopeptidase 24.11 (NEP or enkephalinase A; phosphoramidon). Surprisingly, NEP was responsible for 68 +/- 2% (N = 18) of the total kininase in the rat while kininase I and II contributed only 9 +/- 0.4% and 23 +/- 1%, respectively. To study the effects of NEP inhibition on renal function, phosphoramidon (110 or 330 micrograms/hr/kg; N = 6) or saline (0.1 microliter/min; N = 6) was infused into rats. Urinary kinins, kininases, renal blood flow (RBF), glomerular filtration rate (GFR), UNaV, UKV and UV were measured during control, experimental and recovery periods. Phosphoramidon at the higher dose decreased total urinary kininase activity from 284 +/- 49 to 58 +/- 5 ng/min/kg (77%, P less than 0.01), and increased kinin excretion from 74 +/- 9 to 128 +/- 21 pg/min/kg (73%, P less than 0.02), UV from 72 +/- 10 to 82 +/- 10 microliters/min/kg (15%, P less than 0.01) and UNaV from 12 +/- 2 to 17 +/- 3 microEq/min/kg (37%, P less than 0.02), while BP, RBF, GFR and UKV did not change. 125I-Tyr0-bradykinin infused into the aorta did not appear in the urine intact during simultaneous phosphoramidon and captopril administration. This is the first demonstration of NEP having a major role in the catabolism of kinins. The increase in UNaV and UV after phosphoramidon administration may be due to the inhibition of intrarenal kinin destruction.

Topics: 3-Mercaptopropionic Acid; Animals; Bradykinin; Captopril; Glycopeptides; Kidney; Kinins; Lysine Carboxypeptidase; Metalloendopeptidases; Natriuresis; Neprilysin; Peptidyl-Dipeptidase A; Rats; Rats, Inbred Strains

Utilizing both thin-layer chromatography and high pressure liquid chromatography, it was determined that a vascular plasma membrane preparation contains a carboxypeptidase capable of converting kinins (B2 agonists) to des(Arg)kinins (B1 agonists) by hydrolysis of C-terminal Arg. The plasma membrane carboxypeptidase also converted Leu5-enkephalin-Arg6 to Leu5-enkephalin. Carboxypeptidase activity was significantly higher in cultured endothelial (1.47 +/- 0.4 units/mg) than in cultured smooth muscle cells (0.16 +/- 0.4 units/mg). Both the vascular and endothelial activities had neutral pH optima and were activated 4- to 5-fold by 0.1 mM CoCl2. The carboxypeptidase N inhibitor MERGETPA (D-L-mercaptoethanol-3-guanidino-ethylthiopropanoic acid) inhibited the plasma membrane bound carboxypeptidase with an I50 of 0.3 microM. Conversion was also inhibited by o-phenanthroline and EDTA, whereas inhibitors of aminopeptidases (bestatin, puromycin), endopeptidases (phosphoramidon), "enkephalinase" (ZINCOV) or enkephalin convertase (PCMS) were without effect. The affinity of the endothelial plasma membrane carboxypeptidase for bradykinin (Km = 56.8 +/- 4.7 microM) was higher than that for Leu5-enkephalin-Arg6 (Km = 92.7 +/- 10.1 microM), whereas the maximal rates of conversion (calculated per mg of endothelial plasma membrane protein) were similar (17.1 and 21.3 nmoles/min/mg respectively). These results demonstrate that a carboxypeptidase is present on the cell surface of vascular endothelium which can convert kinins and enkephalins in the micro-environments of vascular cell surface receptors.

Topics: 3-Mercaptopropionic Acid; Animals; Carboxypeptidases; Cell Membrane; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Edetic Acid; Endothelium; Enkephalins; Glycopeptides; Hydrogen-Ion Concentration; Kinetics; Kinins; Leucine; Lysine Carboxypeptidase; Microscopy, Phase-Contrast; Muscle, Smooth, Vascular; Phenanthrolines; Puromycin; Swine