Angiotensin converting enzyme 2 (ACE2) activity in fetal calf serum: implications for cell culture research.

Cell culture experiments often employ the use of culture media that contain fetal calf serum (FCS). The angiotensin peptides angiotensin II and angiotensin 1-7 have opposing effects with angiotensin converting enzyme 2 (ACE2) being the enzyme predominantly responsible for generating angiotensin 1-7 from angiotensin II. The effect of FCS on angiotensin peptides has not previously been described. We have shown that FCS has ACE2 enzyme activity capable of degrading angiotensin II and generating angiotensin 1-7. Researchers should be aware that FCS possesses ACE2 activity and that heat-treating FCS to 56 degrees C only partially inhibits this enzyme activity, whereas heat-treating to 70 degrees C completely abolishes ACE2 activity.

Localization of angiotensin-converting enzyme in the human prostate: pathological expression in benign prostatic hyperplasia.

Benign prostatic hyperplasia (BPH) is the most common hyperplastic disease in man and it is characterized by increased cellular growth (stromal and epithelial hyperplasia) and enhanced local sympathetic tone, both of which are known to be augmented by activation of the renin-angiotensin system (RAS) in other tissues. Angiotensin-converting enzyme (ACE) is an integral component of the RAS that is responsible for the production of the active peptide angiotensin II from the inactive precursor angiotensin I. The present study was undertaken to map the anatomical localization of ACE protein and messenger ribonucleic acid (mRNA) in the normal human prostate and to establish whether their expression is pathologically altered in BPH. Human prostate samples were obtained at post-mortem and histologically defined as normal or hyperplastic. ACE protein binding/expression was determined by in vitro autoradiography and immunohistochemistry using the ACE-specific radioligand [125I]-MK351A and a mouse anti-ACE polyclonal antibody, respectively, whereas the spatiotemporal distribution of ACE mRNA was determined by in situ hybridization using 35S-labelled oligonucleotide probes. ACE protein was localized to the glandular epithelium in the human prostate. ACE binding and immunostaining were increased in BPH compared with normal (non-hyperplastic) prostate specimens [X-ray film autoradiography: normal 873+/-48 dpm/mm2 (n=8) vs. BPH 1631+/-274 dpm/mm2 (n=6), p<0.05; emulsion autoradiography: normal 3.1+/-0.5 grains/mm2 (n=6) vs. BPH 32.8+/-8.6 grains/mm2 (n=5), p<0.01]. ACE mRNA was also localized to glandular epithelial cells in the human prostate with a significant increase in ACE mRNA expression in BPH compared with the normal prostate [normal 11.04+/-2.03 grains/cell (n=220 cells total) vs. BPH 22.29+/-1.34 grains/cell (n=198 cells total), p<0.05]. The findings of the present study suggest that ACE is localized to the glandular epithelium of the human prostate and that its expression, at both protein and mRNA level, is aberrantly increased in BPH. These data support the concept that hyperactivity of the local RAS in the prostate may be involved in the pathogenesis of BPH.

Angiotensin AT(4) receptors in the normal human prostate and benign prostatic hyperplasia.

The cellular localisation and expression of angiotensin AT(4) receptors was examined in the normal human prostate and benign prostatic hyperplasia (BPH) by quantitative in vitro autoradiography using [(125)I]-Ang IV. In the normal human prostate, AT(4) receptors were localised to the glandular epithelium. Interestingly, specific AT(4) receptor binding was significantly reduced in BPH compared to the normal prostate, as quantitated macroscopically (normal: 5038+/-476 dpm/mm(2), n=6 vs BPH: 2701+/-176 dpm/mm(2), n=6, P<0.001) and microscopically (normal: 7.28+/-0.36 grains/mm(2), n=6 vs BPH: 2.50+/-0.47 grains/mm(2), n=6, P<0.001). The findings of the present study demonstrate the presence of AT(4) receptors in the human prostate, being localised to the glandular epithelium, which suggest that the Ang IV/AT(4) system may play a role in the regulation of ionic transport and glandular secretion in the human prostate. The observation that AT(4) receptors appear reduced in BPH suggests that the AT(4) receptor may undergo agonist-induced receptor internalisation, possibly due to increased local tissue levels of Ang IV in BPH.

Effect of glycerol-induced hyperhydration on thermoregulation and metabolism during exercise in heat.

This study examined the effect of glycerol ingestion on fluid homeostasis, thermoregulation, and metabolism during rest and exercise. Six endurance-trained men ingested either 1 g glycerol in 20 ml H2O x kg(-1) body weight (bw) (GLY) or 20 ml H2O x kg(-1) bw (CON) in a randomized double-blind fashion, 120 min prior to undertaking 90 min of steady state cycle exercise (SS) at 98% of lactate threshold in dry heat (35 degrees C, 30% RH), with ingestion of CHO-electrolyte beverage (6% CHO) at 15-min intervals. A 15-min cycle, where performance was quantified in kJ, followed (PC). Pre-exercise urine volume was lower in GLY than CON (1119 +/- 97 vs. 1503 +/- 146 ml x 120 min(-1); p < .05). Heart rate was lower (p < .05) throughout SS in GLY, while forearm blood flow was higher (17.1 +/- 1.5 vs. 13.7 +/- 3.0 ml x 100 g tissue x min(-1); p < .05) and rectal temperature lower (38.7 +/- 0.1 vs. 39.1 +/- 0.1 degrees C; p < .05) in GLY late in SS. Despite these changes, skin and muscle temperatures and circulating catecholamines were not different between trials. Accordingly, no differences were observed in muscle glycogenolysis, lactate accumulation, adenine nucleotide, and phosphocreatine degradation or inosine 5'-monophosphate accumulation when comparing GLY with CON. Of note, the work performed during PC was 5% greater in GLY (252 +/- 10 vs. 240 +/- 9 kJ; p < .05). These results demonstrate that glycerol, when ingested with a bolus of water 2 hours prior to exercise, results in fluid retention, which is capable of reducing cardiovascular strain and enhancing thermoregulation. Furthermore, this practice increases exercise performance in the heat by mechanisms other than alterations in muscle metabolism.

In-vitro and in-vivo inhibition of rat neutral endopeptidase and angiotensin converting enzyme with the vasopeptidase inhibitor gemopatrilat.

OBJECTIVES: Vasopeptidase inhibitors are single molecules that simultaneously inhibit neutral endopeptidase (NEP) and angiotensin converting enzyme (ACE). The aim of this study was to characterize in-vitro and in-vivo inhibition of NEP and ACE in the rat with the vasopeptidase inhibitor gemopatrilat. DESIGN AND METHODS: In-vitro NEP and ACE inhibition was studied by radioinhibitory binding assay using rat renal membranes and the specific NEP inhibitor radioligand 125I-RB104 and the specific ACE inhibitor radioligand 125I-MK351A, respectively (n = 3 per curve). In-vivo NEP and ACE inhibition was studied using in-vitro autoradiography in rats that received oral gemopatrilat (1, 3, 10 mg/kg; n = 4 per dose) and were killed 1 h later, or received oral gemopatrilat (3, 10 mg/kg) and were killed at time points 1, 2, 4, 8, 18, 24 and 48 h (n = 4 per time point). RESULTS: Gemopatrilat caused a concentration-dependent displacement of specific radioligands from renal membrane NEP (IC50 305 +/- 5.4 nmol/I) and ACE (IC50 3.6 +/- 0.02 nmol/). In the dose-response study gemopatrilat (1, 3 and 10 mg/kg) caused significant inhibition of plasma ACE (P< 0.01), and renal ACE and NEP (3, 10 mg/kg, P < 0.01). In the time course experiment, gemopatrilat (10 mg/kg) increased plasma renin activity for 8 h (P< 0.01) and inhibited plasma ACE (P< 0.05), renal NEP (P< 0.01) and renal ACE (P< 0.05) for 48 h. CONCLUSIONS: Gemopatrilat is a potent in-vitro vasopeptidase inhibitor that also causes prolonged inhibition of circulating and renal ACE and renal NEP after a single oral dose. The data suggest that gemopatrilat may be a useful addition to existing vasopeptidase inhibitors in the treatment of cardiovascular disease.

Identification, distribution, and expression of angiotensin II receptors in the normal human prostate and benign prostatic hyperplasia.

The tissue distribution, cellular localization, and level of expression of angiotensin II (Ang II) receptors were examined in the normal human prostate and benign prostatic hyperplasia (BPH) by in vitro autoradiography, immunohistochemistry, and radioligand binding studies. In the normal human prostate, Ang II receptors were of the AT(1) subtype and localized predominantly to periurethral stromal smooth muscle. The AT(1) receptor antagonist losartan totally displaced specific [(125)I]-[Sar(1),Ile(8)]Ang II binding, in a concentration-dependent manner, whereas the AT(2) receptor antagonist PD123319 was without effect. There was no significant difference in receptor affinity, but AT(1) receptor density was markedly reduced in BPH compared with that in normal prostate. In rat prostate, Ang II (0.01-1 microM) produced a concentration-dependent increase in [(3)H]-noradrenaline release from sympathetic nerves. The findings of the present study suggest that angiotensin AT(1) receptors predominate in the human prostate. The high concentration of AT(1) receptors in the periurethral region suggests a role for Ang II in modulating cell growth, smooth muscle tone, and possibly micturition. Furthermore, down-regulation of AT(1) receptors in BPH may be due to receptor hyperstimulation by increased local levels of Ang II in BPH. Finally, Ang II may play a functional role in modulating sympathetic transmission in the prostate. These data support the novel concept that activation of the renin-angiotensin system may be involved in the pathophysiology of BPH.

Comparative in vivo effects of irbesartan and losartan on angiotensin II receptor binding in the rat kidney following oral administration.

We examined the ability of the new non-peptide angiotensin II receptor antagonist irbesartan to inhibit AT(1) receptors in vivo in the rat kidney following oral administration, compared with the prototype drug losartan. Male Sprague-Dawley rats (250-300 g) were gavaged with either irbesartan or losartan at doses of 1, 3, 10, 30 or 100 mg/kg, or with corresponding vehicle. Rats were killed at 0, 1, 2, 8, or 24 h after drug administration, trunk blood was collected and the kidneys were removed. The effects of irbesartan and losartan on angiotensin II receptor binding were determined by quantitative in vitro autoradiography using the specific radioligand (125)I-[Sar(1),Ile(8)]angiotensin II. High levels of angiotensin II receptor binding in the rat kidney were demonstrated in the glomeruli and inner stripe of the outer medulla, which was attributed to AT(1) receptors. At 1 h after dosing, irbesartan (1-100 mg/kg) and losartan (1-30 mg/kg) significantly inhibited AT(1) receptor binding in all anatomical areas of the kidney, in a dose-dependent manner, with a maximal effect at 100 mg/kg and 30 mg/kg respectively. For a 10 mg/kg dose, inhibition of AT(1) receptor binding was maximal around 1-2 h after oral administration of losartan, whereas maximal binding occurred between 2 and 8 h for irbesartan; both drugs produced persistent tissue blockade at 24h. In radioligand binding studies, irbesartan, losartan and EXP3174 (1x10(-10) to 1x10(-5) M) displaced (125)I-[Sar(1),Ile(8)]angiotensin II binding from renal AT(1) receptors in a concentration-dependent manner, with a rank order of potency of irbesartan>EXP3174>losartan. The concentration required to displace 50% of radioligand binding (IC(50)) by irbesartan, EXP3174 and losartan was 1.00+/-0.2 nM, 3.5+/-0.4 nM and 8.9+/-1.1 nM respectively. In conclusion, the findings of the present study suggest that irbesartan and losartan produce effective and sustained inhibition of AT(1) receptors in vivo in the kidney following oral administration. However, irbesartan appears less potent, with respect to dosage, than losartan in vivo, despite having a higher affinity for AT(1) receptors in vitro. The reason for this apparent discrepancy is unclear, but it may reflect the slower onset of action of irbesartan and its rate of tissue accessibility. Inhibition of angiotensin II receptors in target tissues such as the kidney may represent an important action of AT(1) receptor antagonists, which may contribute to the beneficial effects of these agents in the clinical setting.

In vivo inhibition of angiotensin receptors in the rat kidney by candesartan cilexetil: a comparison with losartan.

The present study examined the in vivo effects of candesartan cilexetil compared with losartan on angiotensin II (Ang II) receptor binding in the rat kidney after oral administration. Male Sprague-Dawley rats (250 to 300 g) were gavaged with candesartan cilexetil or losartan in doses of 0.1, 0.3, 1, 3, 10, or 30 mg/kg, or corresponding vehicle. Rats were killed at 0, 1, 2, 8, or 24 h after drug administration, trunk blood collected, and kidneys removed. The effects of candesartan cilexetil and losartan on Ang II receptor binding were determined by quantitative in vitro autoradiography using the radioligand [125I]-[Sar1,Ile8] Ang II. Ang II receptor binding in the kidney was mainly due to AT1 receptors with high levels of binding localized to the inner stripe of the outer medulla and glomeruli in cortical regions. Candesartan cilexetil (0.1 to 30 mg/kg) inhibited Ang II receptor binding to all anatomical sites of the kidney, in a dose-dependent manner. Losartan (0.1 to 30 mg/kg) also produced dose-dependent inhibition of Ang II receptor binding but was approximately 10- to 30-fold less potent than candesartan cilexetil. Inhibition of Ang II receptor binding was near maximal about 1 h after administration of candesartan cilexetil (10 mg/kg) or losartan (10 mg/kg), with both drugs producing persistent blockade at 24 h despite plasma renin activity and plasma drug concentrations returning to near normal levels. In vitro, candesartan, losartan, and EXP3174 (1 x 10(-10) to 1 x 10(-5) mol/L) displaced [125I]-[Sar1,Ile8] Ang II binding from AT1 receptors in the kidney in a concentration-dependent manner with a rank order of potency of candesartan > EXP3174 > losartan. The concentration required to displace 50% of radioligand binding (IC50) by candesartan, EXP3174, and losartan was 0.9+/-0.1 nmol/L, 3.4+/-0.4 nmol/L, and 8.9+/-1.1 nmol/L, respectively. In conclusion, the findings of the present study suggest that candesartan cilexetil is more potent than losartan in antagonizing AT1 receptors in the kidney in vivo. Nonetheless, both candesartan cilexetil and losartan produce rapid, complete, and sustained blockade of AT1 receptors in the rat kidney. Tissue blockade of Ang II receptors in target organs, such as the kidney, may contribute to the beneficial effects of Ang II receptor antagonists as antihypertensive agents.

O-glycosylation delays the clearance of human IGF-binding protein-6 from the circulation.

OBJECTIVE: The actions of insulin-like growth factors (IGF-I and IGF-II) are modulated by a family of six structurally related, high-affinity binding proteins (IGFBPs 1-6). IGFBP-6, an O-linked glycoprotein, preferentially binds IGF-II and inhibits its actions. The aim of this study was to investigate whether O-glycosylation modulates the pharmacokinetics of IGFBP-6. DESIGN AND METHODS: The pharmacokinetic profiles of (125)I-labelled glycosylated (g) and non-glycosylated (n-g) recombinant human IGFBP-6 were studied following intravenous bolus administration in anaesthetised rats. RESULTS: The redistribution half-life of gIGFBP-6 was 2.3-fold greater than that of n-gIGFBP-6 (14.4+/- 1.2 vs 6.3+/-1.5 min, P=0. 006). The elimination half-life of gIGFBP-6 was 21-fold greater than that of n-gIGFBP-6 (584.2+/-130.2 vs 28.0+/-4.2 min, P=0.019). The effect of O-glycosylation on IGFBP-6 pharmacokinetics was not due to inhibition of intravascular proteolysis. Radioactivity was found in stomach, kidneys, lung, spleen, heart and liver but not brain 4h after injection of g or n-gIGFBP-6. CONCLUSIONS: O-glycosylation delays the clearance of IGFBP-6 from the circulation and may therefore contribute to its role as a circulating inhibitor of IGF-II actions.

Increased renal expression of vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 in experimental diabetes.

It has been suggested that the cytokine vascular endothelial growth factor (VEGF) has an important role in the pathogenesis of diabetic retinopathy, but its role in nephropathy has not been clearly demonstrated. Assessment of VEGF, 125I-VEGF binding, and vascular endothelial growth factor receptor-2 (VEGFR-2) in the kidney was performed after 3 and 32 weeks of streptozotocin-induced diabetes. Gene expression of both VEGF and VEGFR-2 was assessed by Northern blot analysis and the localization of the ligand and receptor was examined by in situ hybridization. VEGF and VEGFR-2 protein were also evaluated by immunohistochemistry. Binding of the radioligand 125I-VEGF was evaluated by in vitro and in vivo autoradiography. Diabetes was associated with increased renal VEGF gene expression. VEGF mRNA and protein were localized to the visceral epithelial cells of the glomerulus and to distal tubules and collecting ducts in both diabetic and nondiabetic rats. Renal VEGFR-2 mRNA was increased after 3 weeks of diabetes but not in long-term diabetes. In situ hybridization and immunohistochemical studies revealed that glomerular endothelial cells were the major site of VEGFR-2 expression. In addition, VEGFR-2 gene expression was detected in cortical and renomedullary interstitial cells and on endothelial cells of peritubular capillaries. There was an increase in 125I-VEGF binding sites after 3 but not 32 weeks of diabetes. The major VEGF binding sites were in the glomeruli. 125I-VEGF binding was also observed in medullary rays and in the renal papillae. These studies indicate an early and persistent increase in renal VEGF gene expression in association with experimental diabetes. In addition, an early and transient increase in renal VEGF receptors was also observed in diabetic rats. These findings are consistent with a role for VEGF in mediating some of the changes observed in the diabetic kidney.

Inhibition of kidney neutral endopeptidase after administration of the neutral endopeptidase inhibitor candoxatril: quantitation by autoradiography.

Inhibition of neutral endopeptidase (NEP) in the kidney was studied ex vivo after oral administration of candoxatril (UK79300), an NEP inhibitor, to rats to study the time course and dose response by quantitative in vitro autoradiography by using the NEP inhibitor 125I-SCH47896 as a radioligand. In control rats, high NEP binding was demonstrated in the deep proximal tubule. After oral administration of candoxatril (10 mg/kg), kidney NEP binding was rapidly decreased and recovered gradually over a period of 24 h. The inhibition was maximal at 1 h (13.3 +/- 2.5% of control). Increasing doses of candoxatril administered to rats produced progressive inhibition of NEP binding in the kidney. A dose of 100 mg/kg inhibited kidney NEP binding to 2.6 +/- 0.2% of the control value at 1 h after administration. Candoxatrilat (UK73967), an active metabolite of candoxatril, given intravenously inhibited kidney NEP binding also in a time- and dose-dependent manner. This inhibition of NEP activity at the tissue level may be important in the actions of NEP inhibitors.

Hypotensive effect of ZD7155, an angiotensin II receptor antagonist, parallels receptor occupancy in two-kidney, one-clip Goldblatt hypertensive rats.

OBJECTIVE: To examine the effect of ZD7155, an angiotensin II receptor antagonist, on blood pressure, heart rate and occupancy of tissue angiotensin II receptor in two-kidney, one-clip Goldblatt hypertensive rats. METHODS: Goldblatt hypertension was produced in Sprague-Dawley rats. ZD7155 was administered orally at 3 mg/kg and blood pressure and heart rate were monitored for up to 48 h. In a second series of experiments, the rats were administered increasing doses of ZD7155 and monitored for 1 h. For each time and dose, rats were killed and their blood and tissues were collected. Tissue angiotensin II receptor binding was assessed by quantitative autoradiography in vitro. For a separate group of rats, the pressor response to 0.1 microg/kg angiotensin II was monitored before and after the administration of 3 mg/kg ZD7155. RESULTS: After oral administration of ZD7155, blood pressure was rapidly lowered and this lowering was sustained for up to 48 h. This effect was accompanied by sustained inhibition of angiotensin II receptor binding in the aorta, kidney and adrenal gland, together with an increase in plasma renin activity. Increasing doses of ZD7155 dose-dependently reduced blood pressure and inhibited angiotensin II receptor binding in the tissues. Degrees of inhibition varied among the different tissues and had different time courses. ZD7155 inhibited the pressor response to angiotensin II remarkably for 24 h. CONCLUSIONS: ZD7155 is a potent antihypertensive agent in two-kidney, one-clip Goldblatt hypertensive rats and this effect is accompanied by sustained inhibition of tissue angiotensin II binding.

Effect of chronic neutral endopeptidase inhibition on cardiac hypertrophy after experimental myocardial infarction.

Candoxatril is an inhibitor of neutral endopeptidase, a membrane-bound enzyme that degrades atrial natriuretic peptide. The effects of candoxatril on hemodynamic parameters and cardiovascular hypertrophy were evaluated in the rat model of myocardial infarction. Myocardial infarction was induced by left coronary artery ligation in rats and they were treated either with candoxatril (10mg/kg per day) or a vehicle for up to 4 weeks. Systolic blood pressure and body weight did not change for up to 4 weeks between the 2 groups. At the end of treatment, hemodynamic parameters were measured, and then plasma, heart, lungs and kidneys were collected. Kidney neutral endopeptidase, as measured by the quantitative autoradiographic method, was significantly inhibited in candoxatril-treated rats compared with that in controls (66.6+/-3.2% of control, p<0.001). On the contrary, there were no significant differences in right atrial pressure, left ventricular end-diastolic pressure, systemic pressure, and plasma level of atrial natriuretic peptide between the 2 groups. There were also no significant differences in cardiac weight and lung weight. These data indicate that inhibition of neutral endopeptidase by candoxatril at a dose of 10 mg/kg per day did not oppose cardiac hypertrophy in the rat model of myocardial infarction in spite of significant neutral endopeptidase inhibition.

Angiotensin-converting enzyme inhibition and salt in experimental myocardial infarction.

It is well known that angiotensin-converting enzyme inhibitors attenuate progressive ventricular enlargement or hypertrophy after myocardial infarction and that cardiac angiotensin-converting enzyme activity is increased in the rat model of myocardial infarction. In this study, to determine whether the beneficial effects of angiotensin-converting enzyme inhibition on cardiac hypertrophy after myocardial infarction are due to a reduction in ventricular afterload or to inhibition of cardiac angiotensin-converting enzyme, we used sodium loading during angiotensin-converting enzyme inhibition. The rat model of myocardial infarction was treated with a vehicle, 1% saline, as drinking fluid, perindopril (2 mg/kg/day), or 1% saline as drinking fluid plus perindopril (2 mg/kg/day) for 6 weeks. Perindopril reduced blood pressure, prevented cardiac hypertrophy, and inhibited cardiac angiotensin-converting enzyme. The effects of perindopril on blood pressure and cardiac hypertrophy were abolished by sodium loading, which did not alter the degree of cardiac angiotensin-converting enzyme inhibition. Thus the actions of perindopril on cardiac hypertrophy depend more on blood pressure reduction than on cardiac angiotensin-converting enzyme inhibition in the rat model of myocardial infarction.

Angiotensin II, sodium, and cardiovascular hypertrophy in spontaneously hypertensive rats.

Angiotensin II (Ang II) may cause cardiovascular hypertrophy as a consequence of increased blood pressure or possibly by direct trophic actions. To dissociate Ang II and blood pressure in young spontaneously hypertensive rats (SHR), we used sodium loading during angiotensin converting enzyme inhibitor treatment. Animals were treated between 6 and 10 weeks of age with perindopril to lower Ang II and blood pressure, or with perindopril and 1% saline drinking fluid or perindopril and aldosterone infusion to lower Ang II but maintain high blood pressure. Blood pressure, heart weight, and media/lumen ratio of mesenteric resistance arteries were studied while rats were on treatment at 10 weeks of age and 15 weeks after treatment at 25 weeks of age. Perindopril lowered blood pressure and inhibited the development of cardiovascular hypertrophy. Saline or aldosterone restored high blood pressure during perindopril treatment and resulted in increased heart weight/body weight and resistance artery media/lumen ratios in direct proportion to the elevation of blood pressure. Because increased structure occurred despite perindopril treatment, we conclude that direct trophic actions of Ang II are not essential for the development of cardiovascular hypertrophy in young SHR and that the antitrophic actions of angiotensin converting enzyme inhibitors depend more on changes in blood pressure than on Ang II. However, restoration of blood pressure and structure by sodium during perindopril treatment raises the possibility that the design of the cardiovascular system and blood pressure may depend indirectly on Ang II through effects on sodium metabolism.

The effects of Ca(2+)-free perfusion and the calcium paradox on [125I] endothelin-1 binding to rat cardiac membranes.

The binding characteristics of [125I]endothelin-1 (ET-1) to cardiac membranes isolated from rat hearts subjected to Ca(2+)-free perfusion or the Ca2+ paradox were examined. The effect of treatment with 2, 3 butanedione monoxime (BDM), which inhibits the tissue damage associated with the calcium paradox, was also investigated. Membranes from rat hearts perfused under control conditions bound [125I]ET-1 to a single population of sites with a Bmax of 107.7 +/- 3.7 fmol/mg protein and an affinity (KD) of 153 +/- 12 pM. Ten minutes of Ca(2+)-free perfusion resulted in a significant (P < 0.01) increase in Bmax to 167.5 +/- 8.3 fmol/mg protein without change in KD. Ca2+ repletion following Ca(2+)-free perfusion tended to increase further the Bmax (180.6 +/- 10.4 fmol/mg protein) without change in KD. Treatment with BDM attenuated but did not prevent the rise in Bmax following Ca(2+)-free perfusion. Following Ca2+ repletion, however, Bmax returned to control levels in the BDM treated group. These changes were not associated with changes in the ability of ET-1 and ET-3 to inhibit [125I]ET-1 binding. The results demonstrate that Ca(2+)-free perfusion is associated with an increase in the binding site density of [125I]ET-1 which is maintained or further increased upon Ca2+ repletion. If, however, the tissue damage associated with the Ca2+ paradox is prevented with BDM, Ca2+ repletion is associated with a reversal of the increase due to Ca(2+)-free perfusion.

Effect of amlodipine pretreatment on ischaemia-reperfusion-induced increase in cardiac endothelin-1 binding site density.

Endothelin-1 (ET-1) may be implicated in the pathophysiology of myocardial ischaemia. To determine whether the long-acting calcium antagonist amlodipine attenuates the ischaemia- and reperfusion-induced increase in cardiac ET-1 binding sites, hearts from rats pretreated with amlodipine (0.25 or 0.5 mg/kg) 2 or 5 h before they were killed were made ischaemic for 20 or 40 min, reperfused, and subfractionated. Twenty- and 40-min ischaemia caused a time-dependent increase in ET-1 binding site density (Bmax) identified with [125I]ET-1. Amlodipine pretreatment attenuated this increase in a time- and dose-dependent manner. 0.25 and 0.5 mg/kg amlodipine also suppressed the reperfusion-induced increase in [125I]ET-1 binding site density, even when the 0.5-mg/kg pretreatment series reperfusion was administered after 40-min ischaemia.

Localization and characterization of neutral metalloendopeptidase (EC 3.4.24.11), the degradative enzyme for atrial natriuretic peptide, in rat kidney using a radioiodinated neutral metalloendopeptidase inhibitor.

Atrial natriuretic peptide (ANP) is rapidly degraded by neutral metalloendopeptidase (EC 3.4.24.11, NEP), with the kidney being a major site of ANP clearance. NEP has been anatomically localized in the rat kidney by in vitro autoradiography and the active site studied by a radioinhibitor binding assay (RIBA) using a newly developed radioinhibitor as a radioligand. SCH47896 is a phenolic derivative of SCH39370, a potent specific inhibitor of NEP, which can be radioiodinated with 125I. NEP catalytic activity in the rat kidney was inhibited by SCH47896 and its di-iodo analog SCH48446. Specific binding of [125I]SCH47896 to renal plasma membranes fitted a single-site model with Kd = 43.3 nM and maximal binding site density = 13.8 pmol/mg protein. Thus, [125I]SCH47896 retains full enzymatic inhibitory activity and full binding to the active site of the NEP. Autoradiographs using [125I]SCH47896 demonstrated maximal binding to deep proximal renal tubules. This binding was displaced in a dose-dependent manner by NEP inhibitors. Renal NEP was inhibited by SCH39370. Inhibition of ANP degradation by NEP in the kidney by the new NEP or atriopeptidase inhibitors may explain their natriuretic and diuretic effect in the absence of changes in plasma ANP levels. These studies will allow investigation of the regulation of NEP and the role inhibition of tissue NEP plays in the actions of the new atriopeptidase inhibitors. Furthermore, this method of radioinhibitor binding is applicable to any enzyme, provided a suitable radioligand can be constructed.

Effect of age on endothelin-1 binding sites in rat cardiac ventricular membranes.

To establish whether the density, affinity, or selectivity of endothelin-1 (ET-1) binding sites in cardiac ventricular membranes varies with age, membranes were harvested from 5- to 7-day-, 20-day-, and 8- to 9-week-old Sprague Dawley rats and labeled with [125I]ET-1. Selectivity was established by using cold ET-1, ET-2, ET-3, big ET-1, and (+)PN200-110 to inhibit specific binding of [125I]ET-1. Over the age span studied, selectivity and affinity of the [125I]ET-1 binding sites was unchanged, but density (Bmax) decreased from 209.7 +/- 18.4 at 5-7 days to 154.0 +/- 8.9 (p less than 0.02) at 20 days, and to 89.7 +/- 5.2 (p less than 0.01) fmol/mg protein at 8-9 weeks. These age-dependent differences in Bmax were not accompanied by a change in membrane yield and occurred at a time when the specific binding of (+)[3H]PN200-110 increased.

The inhibitory effect of [D-Arg1,D-Phe,D-Try7,9,Leu11] substance P on endothelin-1 binding sites in rat cardiac membranes.

The specific binding of [125I]ET-1 to rat cardiac membrane fragments was inhibited by [D-Arg1,D-Phe, D-Try7,9,Leu11] substance P [substance P(D)], a potent bombesin antagonist. This inhibitory effect required high concentrations (greater than 3X10(-6)M) of substance P(D) and was accompanied by a steep increase in non-specific binding, and not a decrease in total binding. Such results indicate that substance P(D) does not competitively inhibit the specific binding of [125I]ET-1 to rat cardiac membrane fragments.