cyclic-gmp and phosphoramidon

We compared the enzymatic inactivation of major circulating forms of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). Both ANP and BNP induced a significant increase in cyclic GMP (cGMP) formation in cultured epithelial cell line derived from porcine kidney, LLC-PK1. The cGMP formation stimulated by ANP in LLC-PK1 cells was significantly decreased by pre-treatment of the peptide with rat renal brush-border membranes, and the inactivation of ANP was inhibited by neutral endopeptidase inhibitors, phosphoramidon and S-thiorphan. BNP exhibited greater resistance to enzymatic inactivation than did ANP. In addition, phosphoramidon potentiated the natriuresis with a low dose (7.5 pmol min(-1) kg(-1)) of ANP but not of BNP in rats. These results suggest that enzymatic degradation of natriuretic peptides is highly dependent on peptide structure, and that the affinity of BNP to neutral endopeptidase is less than that of ANP.

Topics: Animals; Atrial Natriuretic Factor; Cell Culture Techniques; Cyclic GMP; Glycopeptides; Humans; LLC-PK1 Cells; Male; Natriuresis; Neprilysin; Nitrobenzenes; Protease Inhibitors; Rats; Rats, Wistar; Swine; Thiorphan

Vasodilation of resistance vessels ensues in response to increased perfusion flow to maintain tissue perfusion. The flow-induced vasodilation is mainly dependent on nitric oxide (NO), which also regulates vascular responsiveness to vasoconstrictors. Besides NO, however; high flow increases endothelin-1 (ET-1) production from endothelial cells. It is likely, therefore, that the interaction between NO and ET-1 may play a critical role in the control of arterial vascular tone under high perfusion flow. In this study, the vascular responsiveness (VR) to high flow rate and the role of ET-1 released by vascular smooth muscle cells (VSMC) were evaluated in isolated and in vitro-perfused mesenteric arteries (MA). MA were perfused at constant (3.5 mL/min; CPF) and increased flow rate (4.5, 5.5, 6.5 mL/min; IPF). VR was evaluated by infusing norepinephrine (NE; 5 micromol/L) and potassium chloride (KCl; 80 mmol/L). Mesenteric vascular resistance (MVR), ET-1, and cGMP release were measured under different flow rates. The role of endothelium-derived ET-1 was evaluated by perfusing MA with phosphoramidon (endothelin converting enzyme inhibitor), whereas the role of other endothelium-derived vasoactive substances was excluded by measuring VR in MA without endothelium. Finally, ETA and ETB receptor antagonists were perfused in disendothelized MA. In the IPF group of intact MA, MVR dropped (P<.05) and both ET-1 and cGMP increased in the perfusate (P<.05). VR was enhanced by high flow after NE (101+/-9 v. 56+/-12 mm Hg in CPF, P<.005) and KCl (119+/-12 v. 51+/-10 mm Hg in CPF, P<.005) and it was unaffected by either phosphoramidon or endothelium removal. On the contrary, BQ-610 abolished the flow-dependent increase in VR. No further additive effect was achieved with BQ-788. In conclusion, in MA, high flow reduces MVR and concurrently enhances VR, likely through VSMC-derived ET-1.

Topics: Animals; Antihypertensive Agents; Aspartic Acid Endopeptidases; Blood Flow Velocity; Cyclic GMP; Endothelin Receptor Antagonists; Endothelin-1; Endothelin-Converting Enzymes; Glycopeptides; In Vitro Techniques; Mesenteric Arteries; Metalloendopeptidases; Muscle, Smooth, Vascular; Norepinephrine; Perfusion; Potassium Chloride; Pressure; Protease Inhibitors; Rats; Rats, Sprague-Dawley; Receptor, Endothelin A; Receptor, Endothelin B; Vascular Resistance; Vasoconstrictor Agents; Vasodilation

1. This study examined the activity and mechanisms of action of urodilatin in bovine bronchi. For comparison, the ability of urodilatin to evoke bronchodilatation or protect against subsequent challenge was compared to that of the closely related peptide alpha-human atrial natriuretic peptide (ANP). 2. Urodilatin reversed methacholine-evoked contraction in a concentration-dependent manner in bovine bronchi. In the absence of any attempt to prevent degradation by neutral endopeptidases, urodilatin was more potent than ANP in this tissue. 3. The bronchodilator properties of urodilatin were significantly augmented by the neutral endopeptidase inhibitor, phosphoramidon (3.68 x 10(-5) M). This provides evidence for at least partial degradation of urodilatin by neutral endopeptidases. With phosphoramidon present, urodilatin and ANP were equipotent. 4. In the presence of phosphoramidon (3.68 x 10(-5) M), pre-incubation with urodilatin (10(-6) M) had a protective effect against subsequent methacholine-induced contraction. This action of urodilatin was quantitatively similar to that of ANP in the presence of this endopeptidase inhibitor. 5. The actions of urodilatin appear to involve ATP-sensitive K+ channels since tolbutamide (10(-6) - 10(-5) M) significantly attenuated the relaxations induced by this peptide. 6. Small conductance Ca(2+)-activated K+ channels seem likewise to be implicated in the actions of urodilatin since blockade of these channels with apamin (10(-7) - 10(-6) M) resulted in a marked attenuation of urodilatin-evoked responses. 7. The presence of charybdotoxin (10-9 M-10-M) had no significant effect on subsequent responses tourodilatin suggesting that large conductance Ca2+-activated K+ channels are not involved in the relaxations evoked by this peptide.8. In the presence of phosphoramidon (3.68 x 10-5 M), urodilatin (10-6 M) evoked elevation of cyclic GMP levels within bovine bronchial tissue. Levels of cyclic GMP increased significantly within 5-10 s in response to this peptide and preceded the initiation of relaxant responses. Maximum increases in cyclic GMP levels were reached within 5 min; the time required for maximal relaxation evoked by this peptide.9. In conclusion, urodilatin, like ANP reversed and protected against, subsequent methacholine-induced bronchoconstriction; an action enhanced by the presence of phosphoramidon (3.68 x 1O-5 M).Associated with these actions of urodilatin was a rise in cyclic GMP levels as well as the opening o

Topics: Animals; Apamin; Atrial Natriuretic Factor; Bronchi; Cattle; Cyclic GMP; Dose-Response Relationship, Drug; Glycopeptides; In Vitro Techniques; Methacholine Chloride; Peptide Fragments; Potassium Channels; Tolbutamide

We characterized the endothelin (ET) receptor subtypes responsible for signal transduction in cultured porcine kidney epithelial LLC-PK1 cells. Both ET-1 (IC50, 43 pM) and ET-3 (IC50, 46 pM) inhibited the binding of [125I]ET-1 to LLC-PK1 cells to a similar extent. The binding affinity of LLC-PK1 cells was about 10,000 times higher for the ETB antagonist BQ-788 [N-cis-2,6-dimethyl-piperidinocarbonyl-L-tau-metylleucyl-D-+ ++Nin- methoxycarbonyltryptophanyl-D-norleucine] (IC50, 1.3 nM) than for the ETA antagonist BQ-123 [cyclo-(D-Trp-D-Asp-Pro-D-Val-Leu)] (IC50, 14 microM). ET-1 enhanced cyclic GMP (cGMP) production, but reduced vasopressin- and forskolin-stimulated cyclic AMP (cAMP) production. Both effects of ET-1 were antagonized by BQ-788, but not by BQ-123. The cAMP decrease, but not the cGMP increase, in response to ET-1 was inhibited by pertussis toxin, suggesting that the former response is mediated by pertussis toxin-sensitive Gi, whereas the latter is mediated by a pertussis toxin-insensitive G-protein. Therefore, the ETB receptors in LLC-PK1 cells couple to the two types of signal transduction cascades to reduce cAMP production and stimulate cGMP production via distinct G-proteins. ET-1 and probably also ET-3 may play a role in the regulation of renal epithelial transport by decreasing cAMP and increasing cGMP.

Topics: Amino Acid Sequence; Animals; Arginine Vasopressin; Colforsin; Cyclic AMP; Cyclic GMP; Endothelin Receptor Antagonists; Endothelins; Epithelium; Glycopeptides; Kidney Tubules; LLC-PK1 Cells; Molecular Sequence Data; Oligopeptides; Peptides, Cyclic; Piperidines; Protein Binding; Receptor, Endothelin B; Receptors, Endothelin; Signal Transduction; Swine

The endothelin-converting enzyme (ECE) activity present in endothelial cells and rat and human brains was characterized using a selective and rapid bioassay for endothelin-1 (ET-1) or endothelin-3 (ET-3) together with a sensitive enzyme-linked immunosorbent assay. We found that ECE activity was predominantly in the membrane fraction of endothelial cells from which it could be extracted by treatment with detergent. In rat brain tissue, the ECE activity was in the membrane fraction and was not solubilized by detergent treatment. Further dissection of the brain revealed that there was a strong localization of ECE activity in the hypothalamus, midbrain, and medulla oblongata in agreement with earlier observations of ET-like immunoreactivity and binding sites. Experiments with human brain tissue also showed the presence of ECE activity. In conclusion, our studies confirmed the presence of ECE activity within endothelial cells, and showed ECE to be localized in brain tissue in sites consistent with the selective distribution of the ET-1 synthetic pathway. In all tissues studied, the ECE activity was significantly inhibited by phosphoramidon or ethylenediaminetetra-acetate.

Topics: Animals; Aspartic Acid Endopeptidases; Biological Assay; Brain; Cattle; Cell Line; Cells, Cultured; Cyclic GMP; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Glycopeptides; Hypothalamus; Medulla Oblongata; Mesencephalon; Metalloendopeptidases; Neprilysin; Protein Precursors

Using the endothelin-1 (ET-1)-stimulated elevation in cGMP in LLC-PK1 cells as a biological detector system for the conversion of big ET-1 (bET-1) to ET-1, we detected bET-1-converting activities in subcellular fractions from bovine aortic cultured endothelial cells (BAE) and rat brain. Within the particulate fraction of BAE, we detected two activities, at pH 3.4 and pH 5.4-7.4. The latter but not the former activity was inhibited in a concentration-dependent manner by phosphoramidon (approximate IC50, 1 microM) and converted bET-1 to ET-1 at a rate of 0.6 nmol/hr/mg of protein. It could be solubilized from the particulate fraction by detergent treatment. Phosphoramidon-inhibitable converting activity was also detected in the cytosolic fraction of BAE. Within the rat brain, phosphoramidon-inhibitable conversion of bET-1 to ET-1 was detected principally in the cytoskeletal fraction, i.e., that fraction from the membrane that was not solubilized by detergent treatment. These results show the presence of at least two different endothelin-converting enzyme activities in endothelial cells and a third within the rat brain. They also demonstrate the use of LLC-PK1 cells as a rapid assay that permits the sensitive detection and measurement of the formation of biologically active ET-1 from its precursor bET-1.

Topics: Animals; Aorta; Aspartic Acid Endopeptidases; Brain; Cattle; Cells, Cultured; Cyclic GMP; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Endothelium, Vascular; Epithelial Cells; Epithelium; Glycopeptides; Kidney; Male; Metalloendopeptidases; Protein Precursors; Rats; Rats, Inbred Strains; Subcellular Fractions; Swine

Systemic clearance of atrial natriuretic peptide (ANP) decreases during postnatal development. To determine the relative contribution of ANP clearance (C) receptors and neutral endopeptidase 24.11 (NEP; EC 3.4.24.11) in regulation of plasma ANP concentration ([ANP]) during maturation, 18- to 60-day-old male Sprague-Dawley rats were anesthetized and infused with rat ANP (35 ng.kg-1.min-1). Infusion of the NEP inhibitor phosphoramidon increased [ANP] and urine guanosine 3',5'-cyclic monophosphate (cGMP) excretion in both weanling and adult rats. Infusion of C-ANP, an analogue that binds C receptors selectively, resulted in a greater rise in [ANP] in preweaned than in adult rats, suggesting a maturational decrease in function of C receptors. Despite the increase in [ANP], however, urine flow, cGMP, and sodium excretion failed to increase in preweaned compared with adult rats. Combined infusion of phosphoramidon and C-ANP resulted in a marked increase in [ANP] and cGMP excretion in weanling and adult rats. These results indicate that both C receptors and NEP modulate plasma [ANP] in the physiological range and that each pathway compensates when the other is inhibited. Age-related differences in the renal response to ANP clearance inhibitors may have important physiological implications in the regulation of sodium balance during development.

Topics: Animals; Atrial Natriuretic Factor; Cyclic GMP; Glycopeptides; Infusions, Intravenous; Male; Metabolic Clearance Rate; Neprilysin; Peptide Fragments; Rats; Rats, Inbred Strains; Receptors, Atrial Natriuretic Factor; Receptors, Cell Surface; Thermolysin

Several processes participate in the clearance of atrial natriuretic peptide (ANP) from the circulation, one of which is enzymatic degradation. Endoprotease EC 3.4.24.11 (NEP 24.11), present within the kidney in high concentration, has been shown in vitro to degrade ANP. Phosphoramidon and thiorphan, two potent NEP 24.11 inhibitors, have been shown to prevent the enzymatic degradation of ANP. The purpose of the present study was to determine if phosphoramidon or thiorphan would alter the in vivo time course of the pharmacologic effects of ANP. The magnitude and duration of the ANP-induced increase in urine output and sodium and cyclic GMP excretion were examined with and without either thiorphan or phosphoramidon. Six separate groups of anesthetized rats received either a low, medium, or high infusion rate of thiorphan or phosphoramidon. Renal responses to ANP were potentiated and prolonged during the low phosphoramidon infusion (3 Ki) and the medium thiorphan infusion (150 Ki). At high inhibitor infusion rates in the anesthetized rat, ANP elicited a marked depressor response. In the conscious spontaneously hypertensive rat (SHR), a 15-min intravenous (i.v.) infusion of ANP (1 microgram/kg/min) lowered mean arterial pressure (MAP 23 +/- 6 mm Hg), with an approximately 35-min duration of action. A simultaneous i.v. infusion of phosphoramidon (high dose) produced both a potentiation (33 +/- 3 mm Hg) and a prolongation (greater than 65 min to return to baseline) of the depressor response. These data lend support to the hypothesis that enzymatic breakdown of ANP may play an important role in regulating the actions of atrial natriuretic peptide.

Topics: Animals; Atrial Natriuretic Factor; Blood Pressure; Cyclic GMP; Glycopeptides; Heart Rate; In Vitro Techniques; Kidney; Male; Protease Inhibitors; Rats; Rats, Inbred SHR; Sodium; Thiorphan; Time Factors

To investigate the in vivo effects of inhibition of endopeptidase 24.11, an enkephalinase enzyme shown to be involved in atrial natriuretic peptide (ANP) breakdown in vitro, we infused phosphoramidon, a specific inhibitor of endopeptidase 24.11, into rats with reduced renal mass (and chronic extracellular volume expansion) and into normal rats. Relative to baseline values in rats with remnant kidneys, phosphoramidon led to elevations of plasma ANP levels and concomitant increases in urinary sodium excretion, fractional excretion of sodium, glomerular filtration rate, filtration fraction, and urinary cyclic GMP excretion. Similar changes in renal function and urinary cyclic GMP excretion were obtained with thiorphan, another endopeptidase 24.11 inhibitor. These enhanced ANP levels and renal actions were not observed with phosphoramidon in normal rats. These results show that plasma ANP levels can be modulated in rats with reduced renal mass by inhibition of endopeptidase 24.11.

Topics: Animals; Anti-Bacterial Agents; Atrial Natriuretic Factor; Blood Pressure; Cyclic GMP; Glomerular Filtration Rate; Glycopeptides; Kidney; Male; Nephrectomy; Neprilysin; Rats; Rats, Inbred Strains; Reference Values; Sodium; Thiorphan