Thiorphan, an inhibitor of endopeptidase 24.11, potentiates the natriuretic activity of atrial natriuretic peptide

To evaluate the role of endopeptidase 24.11 in metabolism of atrial natriuretic peptide (ANP) in vivo, we examined the effect of thiorphan, an inhibitor of this enzyme, on plasma ANP concentrations and the cardiovascular and renal actions of ANP(99-126). Thiorphan alone produced a modest increase in urinary sodium excretion in anesthetized rats; however, urine flow, arterial pressure, and basal plasma ANP concentrations were unchanged. When administered during an infusion of ANP(99-126) (330 ng/kg/min i.v.), thiorphan increased the plasma concentration of ANP and enhanced the diuretic and natriuretic activity of this hormone. The effects on urine flow and urinary sodium excretion were most pronounced immediately after the inhibitor was administered and later diminished in magnitude. Thiorphan did not alter the depressor activity of exogenous ANP(99-126). These data suggest that endopeptidase 24.11 participates in metabolism of ANP(99-126) and that thiorphan potentiates the renal actions of this hormone by inhibiting its degradation.     

Effect of neutral endopeptidase inhibitor on endogenous atrial natriuretic peptide as a paracrine factor in cultured cardiac fibroblasts

1. Cardiac remodelling is a fundamental response to hypertension, myocardial infarction and chronic heart failure, and involves cardiac fibroblast proliferation and production of extracellular matrix components such as collagen. The present study was performed to examine the role of endogenous atrial natriuretic peptide (ANP) as a possible paracrine factor for cardiac fibroblasts, and to examine the effects of three neutral endopeptidase (NEP) inhibitors, thiorphan, phosphoramidon and ONO-BB-039-02 (ONO-BB) on endogenous ANP-induced changes in collagen synthesis by cultured neonatal rat cardiac fibroblasts. 2. Each NEP inhibitor singly had no significant effect on collagen synthesis by cardiac fibroblasts, except for maximum concentration (10(-3) M) of thiorphan. 3. Exogenous ANP inhibited collagen synthesis in a concentration-dependent manner (10(-8) - 10(-6) M). Thiorphan (10(-4) and 10(-3) M) and phosphoramidon (10(-5) and 10(-4) M) enhanced the ANP (10(-7) M)-induced decrease in collagen synthesis. ONO-BB (10(-5) and 10(-4) M) slightly enhanced the ANP-induced decrease in collagen synthesis. 4. Myocyte-conditioned medium (MC-CM), as well as exogenous ANP, inhibited collagen synthesis dose-dependently. The decrease in collagen synthesis at 100% MC-CM was augmented by thiorphan (10(-3) M), phosphoramidon (10(-4) M) and ONO-BB (10(-4) M). 5. HS-142-1, a natriuretic peptide receptor antagonist, significantly reduced the MC-CM plus thiorphan- and MC-CM plus ONO-BB-induced decrease in collagen synthesis, by 92 and 62%, respectively and showed a tendency to attenuate the MC-CM plus phosphoramidon-induced decrease in collagen synthesis by 40%. 6. Our observations suggested that endogenous ANP released from cardiomyocytes inhibited collagen synthesis as a paracrine factor and that NEP inhibitors enhanced the activity of this peptide in cardiac fibroblasts.     

Phosphodiesterase-5A and neutral endopeptidase activities in human adipocytes do not control atrial natriuretic peptide-mediated lipolysis

BACKGROUND AND PURPOSE: Atrial natriuretic peptide (ANP) stimulates lipolysis in human adipocyte through a cGMP signalling pathway, the regulation of which is poorly known. Since phosphodiesterases (PDE) and neutral endopeptidase (NEP) play a major role in the regulation of the biological effects of natriuretic peptides in the cardiovascular and renal systems, we investigated whether these mechanisms could regulate cGMP signalling and ANP-mediated lipolysis in human adipocytes. EXPERIMENTAL APPROACH: The presence of cGMP-specific PDE and NEP in differentiated pre-adipocytes and in mature adipocytes was evaluated by real-time qPCR and Western blot. The effect of non-selective and selective inhibition of these enzymes on ANP-mediated cGMP signalling and lipolysis was determined in isolated mature adipocytes. KEY RESULTS: PDE-5A was expressed in both pre-adipocytes and adipocytes. PDE-5A mRNA and protein levels decreased as pre-adipocytes differentiated (10 days). PDE-5A is rapidly activated in response to ANP stimulation and lowers intracellular cGMP levels. Its selective inhibition by sildenafil partly prevented the decline in cGMP levels. However, no changes in baseline- and ANP-mediated lipolysis were observed under PDE-5 blockade using various inhibitors. In addition, NEP mRNA and protein levels gradually increased during the time-course of pre-adipocyte differentiation. Thiorphan, a selective NEP inhibitor, completely abolished NEP activity in human adipocyte membranes but did not modify ANP-mediated lipolysis. CONCLUSIONS AND IMPLICATIONS: Functional PDE-5A and NEP activities were present in human adipocytes, however these enzymes did not play a major role in the regulation of ANP-mediated lipolysis.     

Altered Regulation of Renal Nitric Oxide and Atrial Natriuretic Peptide Systems in Lipopolysaccharide-induced Kidney Injury

Nitric oxide (NO) and atrial natriuretic peptide (ANP) may induce vascular relaxation by increasing the production of cyclic guanosine monophosphate (cGMP), an important mediator of vascular tone during sepsis. This study aimed to determine whether regulation of NO and the ANP system is altered in lipopolysaccharide (LPS)-induced kidney injury. LPS (10 mg.kg(-1)) was injected in the tail veins of male Sprague-Dawley rats; 12 hours later, the kidneys were removed. Protein expression of NO synthase (NOS) and neutral endopeptidase (NEP) was determined by semiquantitative immunoblotting. As an index of synthesis of NO, its stable metabolites (nitrite/nitrate, NOx) were measured using colorimetric assays. mRNA expression of the ANP system was determined by real-time polymerase chain reaction. To determine the activity of guanylyl cyclase (GC), the amount of cGMP generated in response to sodium nitroprusside (SNP) and ANP was calculated. Creatinine clearance decreased and fractional excretion of sodium increased in LPS-treated rats compared with the controls. Inducible NOS protein expression increased in LPS-treated rats, while that of endothelial NOS, neuronal NOS, and NEP remained unchanged. Additionally, urinary and plasma NOx levels increased in LPS-treated rats. SNP-stimulated GC activity remained unchanged in the glomerulus and papilla in the LPS-treated rats. mRNA expression of natriuretic peptide receptor (NPR)-C decreased in LPS-treated rats, while that of ANP and NPR-A did not change. ANP-stimulated GC activity reduced in the glomerulus and papilla. In conclusion, enhancement of the NO/cGMP pathway and decrease in ANP clearance were found play a role in the pathogenesis of LPS-induced kidney injury.     

Enzymatic pathways involved in the generation of endothelin-1(1-31) from exogenous big endothelin-1 in the rabbit aorta

We investigated whether blood vessels contribute to the production of ET-1(1-31) from exogenous big endothelin-1 (BigET-1) in the rabbit and assessed which enzymes are involved in this process. Vascular reactivity experiments, using standard muscle bath procedures, showed that BigET-1 induces contraction in endothelium-intact rabbit aortic rings. Preincubation of the rings with phosphoramidon, CGS35066 or thiorphan reduced BigET-1-induced contraction. Conversely, chymostatin did not affect BigET-1-induced contraction. Thiorphan and phosphoramidon, but not CGS35066 or chymostatin, reduced ET-1(1-31)-induced contraction. None of the enzymatic inhibitors affected the contraction afforded by ET-1.BQ123-, but not BQ788-, selective antagonists for ET(A) and ET(B) receptors, respectively, produced concentration-dependent rightward displacements of the ET-1(1-31) and ET-1 concentration-response curves. By the use of enzymatic assays, we found that the aorta, as well as the heart, lung, kidney and liver, possess a chymase-like activity. Enzyme immunoassays detected significant levels of Ir-ET-1(1-31) in bathing medium of aortas after the addition of BigET-1 (30 nM). Neither thiorphan nor chymostatin altered the levels of Ir-ET-1(1-31). Conversely, the levels of Ir-ET-1(1-31) were increased in the presence of phosphoramidon. This marked increase of the 31-amino-acid peptide was abolished when phosphoramidon and chymostatin were added simultaneously. The major new finding of the present work is that the rabbit aorta generates ET-1(1-31) from exogenously administered BigET-1. Additionally, by measuring the production of ET-1(1-31), we showed that a chymase-like enzyme is involved in this process when ECE and NEP are inhibited by phosphoramidon. Our results also suggest that ET-1(1-31) is an alternate intermediate in the production of ET-1 following BigET-1 administration. Finally, we showed that NEP is the predominant enzymatic pathway involved in the cleavage of ET-1(1-31) to a bioactive metabolite that will act on ET(A) receptors to induce contraction in the rabbit aorta.     

Prolonged neutral endopeptidase inhibition in heart failure.

We studied the hormonal, renal and hemodynamic effects of prolonged treatment with SCH 39370, a new neutral endopeptidase (NEP) inhibitor, in experimental congestive heart failure (CHF). Coronary-ligated CHF rats and sham-operated controls received vehicle or SCH 39370 30 mg/kg s.c. twice daily for six days. In rats with heart failure, SCH 39370 elevated the high plasma atrial natriuretic peptide (ANP) and cyclic guanosine monophosphate (cGMP) levels 2-fold both initially and at the end of the experiment. Initially, water balance was more negative in SCH 39370-treated CHF rats than in those treated with vehicle. In all SCH 39370-treated rats, ANP, cGMP and electrolyte excretion and diuresis were pronounced for 6 h after injection but attenuated thereafter. Blood pressure and pulse remained unchanged. On reverse phase high performance liquid chromatography (HPLC), ANP-(99-126) appeared to be the only circulating form of ANP in rats with heart failure. Three forms have been discovered in patients with heart failure. HPLC revealed only intact ANP in plasma of rats with heart failure during SCH 39370 treatment. NEP inhibitors may provide a new tool for treating chronic heart failure.     

Heart failure augments the cardiovascular and renal effects of neutral endopeptidase inhibition in rats.

We compared the cardiovascular and renal actions of the neutral endopeptidase (NEP) inhibitor, SQ 28,603, in normal rats and in rats with healed myocardial infarcts. The infarcted rats were studied in the conscious state 8 weeks after ligation of the left main coronary artery and 4 h after placement of cardiovascular and renal catheters. Infarct size was 39 +/- 1.2% of left ventricle circumference; right ventricle and lung weight to body weight ratios were twice those of normal rats. These postmortem values were shown to be associated with elevated left ventricular end diastolic pressure and high plasma atrial natriuretic peptide (ANP) concentration in separate groups of rats. SQ 28,603 at 100 mumol/kg intravenously (i.v.) caused urine volume and sodium excretion to increase by 79 +/- 11 microliters/min and 8.2 +/- 1.4 microEq/min, respectively, 20 min after injection in infarcted rats; these changes were significantly greater than those in normal rats (12 +/- 5 microliters/min and 1.6 microEq/min, respectively). Thoracic venous pressure decreased by 1.9 +/- 0.4 mm Hg 80 min after SQ 28,603 in infarcted rats and by only 0.1 +/- 0.1 mm Hg in normal rats (p less than 0.05 vs. infarcted rats). SQ 28,603 had no effects on mean arterial pressure (MAP), cardiac output (CO), or glomerular filtration rate (GFR). The observation that NEP inhibition has more pronounced effects in animals with high ambient ANP level than in those with normal ANP is consistent with previous studies in a variety of animal models and supports the concept that NEP inhibition potentiates endogenous ANP.     

Role of the endopeptidase 24.11 in the disposition and metabolism of endogenous atrial natriuretic factor in the rabbit.

The neutral endopeptidase 24.11 (NEP) has been shown to inactivate the atrial natriuretic factor (ANF) by opening the ring structure. To document the role of NEP in the metabolic fate of ANF in vivo, the effects of an infusion of thiorphan (25 micrograms/min/kg), a specific NEP inhibitor, on the kinetics and metabolism of endogenous ANF were studied in conscious rabbits. A bolus of [125I]ANF(99-126) was injected 50 min after the beginning of the infusion of thiorphan. Plasma samples containing the radioactive peptides were separated by reverse-phase HPLC. The parent compound could be separated from at least two other minor metabolites, corresponding to the elution position of [125I]ANF(99-105/106-126), the inactive ring-opened metabolite, and of [125I]ANF(103-126), an N-truncated analog. The generation of the N-truncated metabolite was increased by thiorphan. Thiorphan also induced an increase in plasma ANF (29%) that was closely associated with a 32% reduction in the systemic clearance of [125I]ANF(99-126), whereas no modification in the estimated secretion rate was detected. These results support a role for NEP in the regulation of endogenous ANF plasma levels. These results also suggest that specific inhibition of NEP may result in an increase in the apparent activity of alternative metabolic pathways.     

Indomethacin decreases particulate guanylyl cyclase activity in rat kidney

1. Effects of non-steroidal anti-inflammatory drugs on the local atrial natriuretic peptide (ANP) and nitric oxide (NO) systems in the kidney were investigated. 2. Male Sprague-Dawley rats were treated with indomethacin (5 mg/kg, every 12 h, i.p.) for 2 days. The expression of ANP and natriuretic peptide receptor-A (NPR-A) mRNA was determined in the kidney, as was that of endothelial NO synthase (NOS) proteins. Particulate and soluble guanylyl cyclase activities were determined separately. 3. Following treatment with indomethacin, urinary sodium excretion decreased significantly. Although the renal expression of ANP was not changed significantly, that of NPR-A decreased significantly. The expression of NOS increased significantly. Particulate guanylyl cyclase activity was decreased, whereas the activity of soluble guanylyl cyclase was increased. The catalytic activity of Na(+)/K(+)-ATPase was increased, with no significant changes in its expression. The expression of the type 3 Na/H exchanger and Na-K-2CL cotransporters increased significantly. 4. The indomethacin-induced decrease in urinary sodium excretion may be attributed, at least in part, to decreased activity of the local ANP/cGMP system. The increased activity of the NO/cGMP system may be a compensatory response to the diminished activity of the prostaglandin system.     

Endothelin generating pathway through endothelin1-31 in human cultured bronchial smooth muscle cells.

The effects of endothelin (ET)-1(1-31) and ET-2(1-31), human chymase products of the corresponding big ETs, on the intracellular free Ca2+ concentration ([Ca2+]i) and [125I]-ET-1 binding were investigated using human cultured bronchial smooth muscle cells (BSMC). ET-1(1-31) (10(-8)M - 3 x 10(-7)M) and ET-2(1-31) (3 x 10(-8)M - 3 x 10(-6) M) caused an increase in [Ca2+]i in a concentration-dependent manner. Big ET-1 (3 x 10(-8)M - 10(-6)M) also caused this increase, but not big ET-2 at concentrations up to 10(-6)M. The [Ca2+]i increase induced by ET-1 was inhibited by both BQ123, an ET(A)-receptor antagonist, and BQ788, an ET(B)-receptor antagonist, whereas that induced by ET-3 was inhibited by BQ788 but not by BQ123. Increases in [Ca2+]i caused by ET-1(1-31), big ET-1 and ET-2(1-31) were completely inhibited by 10(-4)M phosphoramidon, a dual neutral endopeptidase (NEP)/endothelin-converting enzyme (ECE) inhibitor, and 10(-5)M thiorphan, a NEP inhibitor. Scatchard plot analyses of the saturation curves of [125I]-ET-1 and [125I]-ET-3 showed that both ET(A)- and ET(B)- receptors at the ratio of 4:1 were expressed on BSMC. ET-1(1-31), big ET-1 and ET-2(1-31) inhibited [125I]-ET-1 binding in a concentration-dependent manner, and these effects were attenuated by treatment with thiorphan. On the other hand, big ET-2 slightly inhibited the binding at a high concentration and this was not affected by thiorphan. These results suggest that ET-1(1-31), big ET-1 and ET-2(1-31) cause an increase in [Ca2+]i by being converted into the corresponding ET-1 and ET-2 by NEP, but this did not occur with big ET-2 in human BSMC. ET-2(1-31), produced by human chymase from big ET-2 might be important for the generation of ET-2 in human bronchial tissue.     

The role of membrane composition in ATPase activities of cirrhotic rat liver: effect of silymarin

The activities of Ca2(+)- and Na+, K(+)-ATPases were studied in liver plasma membranes from CCl4-cirrhotic rats and from livers of rats treated with silymarin in addition to CCl4. CCl4 chronic treatment produced significant decreases in Na+, K(+)- and Ca2(+)-ATPase activities; however, the animals treated with silymarin along with CCl4 showed no differences in ATPase activities as compared to controls. The lipid analysis performed in plasma membranes revealed increases in the cholesterol/phospholipid (CH/PL) and sphingomyelin/phosphatidylcholine (SM/PC) ratios in the cirrhotic group. Again, the membranes isolated from rats receiving CCl4 + silymarin showed normal CH/PL and SM/PC values. Considering that CH/PL and SM/PC ratios are related to membrane microviscosity, this study suggests that a lower fluidity of the membrane may be responsible for the observed decreases in ATPase activities in the cirrhotic group. Additionally, the role of silymarin to improve liver function in CCl4-cirrhosis can be attributed partially to its action at membrane level by preventing the increases in CH/PL and SM/PC ratios.     

Renal and depressor effects of SQ 29,072, a neutral endopeptidase inhibitor, in conscious hypertensive rats.

The depressor and renal responses to the neutral endopeptidase (NEP) inhibitor, SQ 29,072, were characterized in both the conscious spontaneously hypertensive rat (SHR) and the conscious deoxycorticosterone acetate (DOCA)/salt hypertensive rat. Inhibition of tissue NEP activity by pharmacologically active doses was also ascertained in both hypertensive models. Intravenous administration of 300 mumol/kg of SQ 29,072 significantly reduced mean arterial pressure (MAP), produced modest natriuretic and diuretic responses, and inhibited renal NEP activity by approximately 40% in conscious SHR. Doses of 100 and 300 mumol/kg of SQ 29,072 elicited greater depressor responses (-36 +/- 7 and -41 +/- 8 mm Hg, respectively) in DOCA/salt hypertensive rats than in SHR (-11 +/- 24 and -31 +/- 5 mm Hg, respectively). SQ 29,072 (300 mumol/kg, i.v.) also inhibited renal NEP activity to a greater extent (70%) in DOCA/salt hypertensive rats. Similarly, the depressor responses to exogenous ANP 99-126 (1, 3, and 10 nmol/kg, i.v.) were greater in DOCA/salt hypertensive rats (-16 +/- 4, -38 +/- 6, and -73 +/- 6 mm Hg, respectively) than in the SHR (0 +/- 6, -17 +/- 3, and -24 +/- 3 mm Hg, respectively). Finally, equidepressor doses of SQ 29,072 and ANP 99-126 both increased urine volume as well as sodium and cyclic GMP excretion in conscious DOCA/salt hypertensive rats. In conclusion, the profile of depressor and renal activities produced by SQ 29,072 was consistent with potentiation of endogenous ANP by inhibition of NEP in conscious SHR and DOCA/salt hypertensive rats.     

Cyclooxygenase-1 derived prostaglandins are involved in the maintenance of renal function in rats with cirrhosis and ascites

1. The maintenance of renal function in decompensated cirrhosis is highly dependent on prostaglandins (PGs). Since PG synthesis is mediated by cyclooxygenase-1 and -2 (COX-1 and COX-2), the present study was designed to examine which COX isoform is involved in this phenomenon. 2. Renal COX-1 and COX-2 protein expression and distribution were analysed by Western blot and immunohistochemistry in nine rats with carbon tetrachloride-induced cirrhosis and ascites and 10 control animals. The effects of placebo and selective COX-1 (SC-560) and COX-2 (celecoxib) inhibitors on urine flow (V), urinary excretion of sodium (U(Na)V) and PGE(2) (U(PGE2)V), glomerular filtration rate (GFR), renal plasma flow (RPF), the diuretic and natriuretic responses to furosemide and renal water metabolism were assessed in 88 rats with cirrhosis and ascites. 3. COX-1 protein levels were found to be unchanged in kidneys from cirrhotic rats. In contrast, these animals showed enhanced renal COX-2 protein expression which was focally increased in the corticomedullary region. Although U(PGE2)V was equally reduced by SC-560 and celecoxib, only SC-560 produced a significant decrease in U(Na)V, GFR and RPF and a pronounced impairment in the diuretic and natriuretic responses to furosemide in rats with cirrhosis and ascites. Neither SC-560 nor celecoxib affected renal water metabolism in cirrhotic rats. 4. These results indicate that despite abundant renal COX-2 protein expression, the maintenance of renal function in cirrhotic rats is mainly dependent on COX-1-derived prostaglandins.     

莨菪亭解热作用机理实验研究

目的:研究莨菪亭降低正常和内毒素致热家兔体温的作用机理。方法:用放射免疫法和比色法测定正常和内毒素致热家兔中枢和外周与体温调节密切相关的PGE2、cAMP、cGMP、Na+-K+-ATPase、PLA2水平或活性。结果:莨菪亭对正常家兔的PGE2、cAMP、cGMP、PLA2水平或活性无显著影响,仅对Na+-K+-ATPase的活性有显著抑制作用;能显著降低内毒素致热家兔的PGE2、cAMP水平,升高cGMP水平,并能显著抑制Na+-K+-ATPase、PLA2的活性,与阳性药比较,莨菪亭高剂量的作用明显强于复方氨基比林注射液。结论:莨菪亭降低正常家兔体温的作用可能是通过抑制Na+-K+-ATPase的活性实现的;而降低内毒素致热家兔体温是通过降低或抑制PGE2、cAMP、Na+-K+-ATPase、PLA2水平或活性,升高cGMP水平等多途径实现的。     

The effect of thiorphan on release of sensory neuropeptides from guinea-pig cerebral venous sinuses

The effect of peptidase inhibitors on neuropeptide release from peripheral endings of capsaicin-sensitive sensory neurons was studied in cerebral superior sagittal and transverse sinuses of guinea-pig. Capsaicin (1 microM)-evoked release of substance P-like immunoreactivity (SP-LI) was increased in a concentration-dependent manner by thiorphan (0.1-10 microM). Captopril (10 microM) or a mixture of bestatin (10 microM), leupeptin (10 microM) and bacitracin (10 microM) did not affect the capsaicin-evoked SP-LI release. Thiorphan (10 microM) increased also the capsaicin-evoked release of neurokinin A-like immunoreactivity (TK-LI) and calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) by 228% and 172%, respectively, while captopril (10 microM) was without effect. Thiorphan (10 microM), but not captopril (10 microM), enhanced by 239% CGRP-LI release induced by bradykinin (10 microM). In the cerebral venous vessels neutral endopeptidase (EC 3.4.24.11, NEP)-like activity was 58.8 +/- 6.1 pmol/mg protein/min, while angiotensin converting enzyme-like activity was below the detection limit of the assay. A thiorphan-sensitive mechanism, putatively attributable to NEP, plays a major role in the inactivation of peptides released from or acting on capsaicin-sensitive sensory fibres of cerebral venous sinuses of guinea-pig.     

Inhibition of neutral endopeptidase by thiorphan does not modify coronary vascular responses to angiotensin I, angiotensin II and bradykinin in the isolated guinea pig heart

Both angiotensin-converting enzyme (ACE) and neutral endopeptidase (NEP) are involved in the regulation of renin-angiotensin and kallikrein-kinin systems. The aim of the present study was to assess the role of NEPand ACE in the regulation of vascular responses to angiotensin I (Ang I), angiotensin II (Ang II) and bradykinin (Bk) in the coronary circulation. For this purpose we used typical inhibitors of ACE and NEP, perindoprilate (1 microM) and thiorphan (1 micromM and 10 microM), respectively, and analyzed their effects on the coronary vasoconstrictor responses to Ang I and Ang II and coronary vasodilator responses to Bk in the isolated guinea pig heart. Perindoprilate abolished coronary vasoconstriction induced by Ang I and potentiated coronary vasodilation evoked by Bk. Thiorphan at a concentration of 1 muM slightly reduced response to Ang I without a significant effect on the responses to Ang II and Bk. However, thiorphan at a concentration of 10 muM abolished coronary vasoconstrictor response to Ang I and enhanced Bk-induced vasodilation. Importantly, in the presence of perindoprilate, addition of thiorphan (10 microM) did not modify further either responses to Ang I, Ang II or to Bk. In conclusion, vascular responses induced by Ang I, Ang II and Bk in the isolated guinea pig heart are regulated by ACE but not by NEP. Moreover, thiorphan is not a perfect tool to asses functional role of NEP as it displays ACE inhibitory activity.     

Mechanisms underlying the contractile response to endothelin-1 in the rat renal artery

We assessed the functional response and the mechanisms following receptor stimulation of endothelin-1 (ET-1) in the rat renal artery. In this study, isometric tension was recorded in renal artery rings without endothelium. Cumulative application of ET-1 from 0.1 to 100 nmol/l induced a sustained concentration-dependent contraction in the renal artery. Submaximal contraction induced by 10 nmol/l ET-1 in 2.5 mmol/l Ca(2+) and in the absence of inhibitors was used as control response (100%). The relative contribution of different sources of Ca(2+) in ET-1-induced contraction was evaluated. The contractile response to 10 nmol/l ET-1 in 2.5 mmol/l Ca(2+ )(1.2 +/- 0.2 g) was significantly inhibited either in Ca(2+)-free solution containing 100 micromol/l ethylene glycol bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (0.6 +/- 0.1 g) or after depletion of intracellular Ca(2+) stores (0.62 +/- 0.05 g). The contribution of phospholipase C and protein kinase C was evaluated by using their inhibitors 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate (NCDC) and [1-(5-isoquinolinesulfonyl)-2-methylpiperazine] (H-7), respectively. The contractile response to 10 nmol/l ET-1 was inhibited by 10 micromol/l NCDC (to 80 +/- 6%) and 30 micromol/l H-7 (to 76.6 +/- 6.5%). We found that 1 micromol/l nifedipine inhibited the ET-1-induced contraction (to 48.7 +/- 6.9%), indicating the contribution of Ca(2+) influx through voltage-gated L-type Ca(2+) channels to this response. Further, the inhibitory effect of nifedipine was to a greater extent as compared with NCDC or H-7. Additive inhibition of ET-1-induced contraction was not observed in the presence of both nifedipine and NCDC. We also evaluated the role of the ionic transport system in the ET-1-induced response by using 20 nmol/l 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), an inhibitor of Na(+)-H(+) exchange, or 100 micromol/l ouabain, an inhibitor of Na(+)-K(+)-ATPase. The response to ET-1 was decreased by both EIPA (to 61.6 +/- 8.4%) and ouabain (to 62.1 +/- 8.6%). The contribution of Na(+)-Ca(2+) exchange to ouabain action was tested using the inhibitor dimethyl amiloride HCl (10 micromol/l). The decrease in ET-1-induced contraction by the combination of ouabain and dimethyl amiloride HCl was similar to that observed with ouabain alone. In view of these observations, both extra- and intracellular sources of Ca(2+) contribute to the contractile response induced by ET-1 in the renal artery. Our findings also revealed the importance of Ca(2+) influx through voltage-gated L-type Ca(2+) channels in mediating contraction to ET-1 in the renal artery, whereas a minor role of phospholipase C and protein kinase C was observed. Na(+)-H(+) exchange and Na(+)-K(+)-ATPase also play a role in the ET-1-induced contraction in renal artery. Moreover, the contribution of Na(+)-K(+)-ATPase in ET-1 contraction is not an Na(+)-Ca(2+) exchange-related process.     

Altered regulation of renal Acid base transporters in response to ammonium chloride loading in rats

The role of the kidney in combating metabolic acidosis has been a subject of considerable interest for many years. The present study was aimed to determine whether there is an altered regulation of renal acid base transporters in acute and chronic acid loading. Male Sprague-Dawley rats were used. Metabolic acidosis was induced by administration of NH(4)Cl for 2 days (acute) and for 7days (chronic). The serum and urinary pH and bicarbonate were measured. The protein expression of renal acid base transporters [type 3 Na(+)/H(+) exchanger (NHE3), type 1 Na(+)/HCO(3) (-) cotransporter (NBC1), Na-K(+) ATPase, H(+)-ATPase, anion exchanger-1 (AE-1)] was measured by semiquantitative immunoblotting. Serum bicarbonate and pH were decreased in acute acid loading rats compared with controls. Accordingly, urinary pH decreased. The protein expression of NHE3, H(+)-ATPase, AE-1 and NBC1 was not changed. In chronic acid loading rats, serum bicarbonate and pH were not changed, while urinary pH was decreased compared with controls. The protein expression of NHE3, H(+)-ATPase was increased in the renal cortex of chronic acid loading rats. These results suggest that unaltered expression of acid transporters combined with acute acid loading may contribute to the development of acidosis. The subsequent increased expression of NHE3, H(+)-ATPase in the kidney may play a role in promoting acid excretion in the later stage of acid loading, which counteract the development of metabolic acidosis.     

Effects of the selective neutral endopeptidase inhibitor, retrothiorphan, on renal function and blood pressure in conscious normotensive Wistar and hypertensive DOCA-salt rats.

Atrial natriuretic peptide (ANP) is degraded by neutral endopeptidase (NEP) mainly in the proximal tubule of the kidneys. We studied the effects of retrothiorphan, a potent and highly specific NEP inhibitor on renal function and blood pressure (BP). A 25-mg/kg bolus injection (group bolus), or bolus injection plus infusion 25 mg/kg + 25 mg/kg/h (group infusion), was given to conscious normotensive Wistar and hypertensive DOCA-salt rats. Bolus and infusion produced increases in diuresis (110 +/- 15 vs. 103 +/- 15 vs. 42 +/- 9 microliters/min) and natriuresis (10.6 +/- 3.0 vs. 7.0 +/- 1.0 vs. 5.4 +/- 1.0 mumol/min) in normotensive rats, with a maximum change at 30 min. Change in kaliuresis was not significant. These renal effects were associated with nonsignificant increases in urinary cyclic GMP and ANP. Arterial pressure and heart rate (HR) were not affected. Bolus or infusion of retrothiorphan also induced increases in diuresis (92 +/- 16 vs. 124 +/- 13 vs. 38 +/- 6 microliters/min) and natriuresis (10.3 +/- 2.0 vs. 12.5 +/- 1.0 vs. 5.0 +/- 1.0 mumol/min) in DOCA-salt hypertensive rats, with a maximum change at 30 min. The changes in diuresis and natriuresis induced by retrothiorphan were correlated with a significant increase in urinary cyclic GMP excretion (r = 0.89, p < 0.001 and r = 0.91, p < 0.001). Urinary ANP did not change in controls but significantly increased in the treated rats; urinary immunoreactive bradykinin (BK) also tended to increase. Plasma ANP and hematocrit did not change after retrothiorphan, but plasma cyclic GMP increased significantly after infusion. Only infusion caused a decrease in arterial pressure in DOCA-salt rats (-20 mm Hg at 120 min). Renal clearance studies in DOCA-salt rats showed that retrothiorphan has a transient effect on renal hemodynamics, with increases in glomerular filtration and renal blood flow (RBF) and a decrease in renal vascular resistance (RVR). Its renal action was also tubular, with an increase in fractional sodium excretion. We also compared the effects of retrothiorphan in normotensive Brown-Norway kininogen-deficient rats (BN-Kat) and DOCA-salt hypertensive kininogen-deficient rats. The NEP inhibitor induced increases in diuresis and natriuresis in both groups, with increased urinary cyclic GMP. Urinary immunoreactive BK did not change significantly in normotensive or DOCA-salt hypertensive kininogen-deficient rats.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of the neutral endopeptidase 24.11 in the conversion of big endothelins in guinea-pig lung parenchyma.

1. We have studied the conversion of big endothelin-1 (big ET-1), big endothelin-2 (big ET-2) and big endothelin-3 (big ET-3) and characterized the enzyme involved in the conversion of the three peptides in guinea-pig lung parenchyma (GPLP). 2. Endothelin-1 (ET-1), endothelin-2 (ET-2) and endothelin-3 (ET-3) (10 nM to 100 nM) caused similar concentration-dependent contractions of strips of GPLP. 3. Big ET-1 and big ET-2 also elicited concentration-dependent contractions of GPLP strips. In contrast, big ET-3, up to a concentration of 100 nM, failed to induce a contraction of the GPLP. 4. Incubation of strips of GPLP with the dual endothelin converting enzyme (ECE) and neutral endopeptidase (NEP) inhibitor, phosphoramidon (10 microM), as well as two other NEP inhibitors thiorphan (10 microM) or SQ 28,603 (10 microM) decreased by 43% (P < 0.05), 42% (P < 0.05) and 40% (P < 0.05) the contractions induced by 30 nM of big ET-1 respectively. Captopril (10 microM), an angiotensin-converting enzyme inhibitor, had no effect on the contractions induced by big ET-1. 5. The incubation of strips of GPLP with phosphoramidon (10 microM), thiorphan (10 microM) or SQ 28,603 (10 microM) also decreased by 74% (P < 0.05), 34% and 50% (P < 0.05) the contractions induced by 30 nM big ET-2 respectively. As for the contractions induced by big ET-1, captopril (10 microM) had no effect on the concentration-dependent contractions induced by big ET-2. 6. Phosphoramidon (10 microM), thiorphan (10 microM) and SQ 28,603 (10 microM) significantly potentiated the contractions of strips of GPLP induced by both ET-1 (30 nM) and ET-3 (30 nM). However, the enzymatic inhibitors did not significantly affect the contractions induced by ET-2 (30 nM) in this tissue. 7. These results suggest that the effects of big ET-1 and big ET-2 result from the conversion to ET-1 and ET-2 by at least one enzyme sensitive to phosphoramidon, thiorphan and SQ 28,603. This enzyme corresponds possibly to EC 3.4.24.11 (NEP 24.11) and could also be responsible for the degradation of ETs in the GPLP.