Dynorphin and vasopressin: common localization in magnocellular neurons.

The opioid peptide dynorphin is widely distributed in neuronal tissue of rats. By immunocytochemical methods, it was shown previously that dynorphin-like immunoreactivity is present in the posterior pituitary and the cells of the hypothalamic neurosecretory magnocellular nuclei which also are responsible for the synthesis of oxytocin, vasopressin, and their neurophysins. By using an affinity-purified antiserum to the non-enkephalin part of the dynorphin molecule it has now been demonstrated that dynorphin and vasopressin occur in the same hypothalamic cells of rats, whereas dynorphin and oxytocin occur in separate cells. Homozygous Brattleboro rats (deficient in vasopressin) have magnocellular neurons that contain dynorphin separate from oxytocin. Thus dynorphin and vasopressin, although they occur in the same cells, appear to be under separate genetic control and presumably arise from different precursors.

Specific binding of endothelin on human vascular smooth muscle cells in culture.

Endothelin is a newly discovered, potent vasoconstrictor peptide secreted by endothelial cells. The binding of endothelin was studied on cultured human vascular smooth muscle cells obtained from umbilical veins. A single specific binding site for 125I-endothelin was identified, with an apparent Kd of 126 pM and a maximal binding capacity of approximately 10,000 sites per smooth muscle cell. At room temperature the binding was saturable, reached equilibrium at 2 h (using 20 pM endothelin), and was slowly and only partially reversed by unlabeled endothelin. The calcium antagonists nifedipine, nicardipine, and diltiazem did not compete for the same binding site. Conditioned medium from cultured human umbilical vein endothelial cells inhibited the binding of 125I-endothelin dose dependently. This effect was antagonized by anti-endothelin antiserum. We conclude that human umbilical vein smooth muscle cells possess specific binding sites for endothelin, and that human endothelial cells secrete an endothelinlike material.

Temporal evolution of endothelial dysfunction in a rat model of chronic heart failure.

OBJECTIVES: The goal of this study was to investigate the evolution of endothelial dysfunction and plasma renin activity in a rat model of heart failure. BACKGROUND: Endothelial dysfunction has been demonstrated in heart failure and may play a significant role in this pathophysiologic process. Studies have also suggested a physiologic interaction between the renin-angiotensin system and endothelium-derived relaxing factor. However, the evolution of endothelial dysfunction and plasma renin activity in heart failure has not been studied to date. METHODS: Endothelium-dependent and -independent relaxations were studied at 1, 4 and 16 weeks after coronary artery ligation in a rat model of heart failure. Thoracic aortic rings were placed in isolated organ baths and acetylcholine and sodium nitroprusside concentration response curves were generated. Plasma renin activity was assessed at each time point. RESULTS: Aortic rings from rats with heart failure demonstrated no evidence of endothelial dysfunction at week 1, although progressive rightward shifts in the acetylcholine curves and decreasing maximal relaxation over time compared with findings in sham-operated control rats were evident at weeks 4 and 16. The sodium nitroprusside curves were not different between rats with heart failure and sham-operated rats. Plasma renin activity was elevated at week 1 and progressively increased through week 16, even though it correlated poorly with endothelial dysfunction. CONCLUSIONS: This study suggests that endothelial dysfunction in heart failure is a progressive time-dependent process that probably plays a minor role early in heart failure. Although plasma renin activity increased significantly in rats with heart failure, it was poorly predictive of endothelial dysfunction.

Renin inhibition by substituted piperidines: a novel paradigm for the inhibition of monomeric aspartic proteinases?

BACKGROUND: The aspartic proteinase renin catalyses the first and rate-limiting step in the conversion of angiotensinogen to the hormone angiotensin II, and therefore plays an important physiological role in the regulation of blood pressure. Numerous potent peptidomimetic inhibitors of this important drug target have been developed, but none of these compounds have progressed past clinical phase II trials. Limited oral bioavailability or excessive production costs have prevented these inhibitors from becoming new antihypertensive drugs. We were interested in developing new nonpeptidomimetic renin inhibitors. RESULTS: High-throughput screening of the Roche compound library identified a simple 3, 4-disubstituted piperidine lead compound. We determined the crystal structures of recombinant human renin complexed with two representatives of this new class. Binding of these substituted piperidine derivatives is accompanied by major induced-fit adaptations around the enzyme's active site. CONCLUSIONS: The efficient optimisation of the piperidine inhibitors was facilitated by structural analysis of the renin active site in two renin-inhibitor complexes (some of the piperidine derivatives have picomolar affinities for renin). These structural changes provide the basis for a novel paradigm for inhibition of monomeric aspartic proteinases.

Comparative effects of three different potent renin inhibitors in primates.

The goal of the present study was to compare the effects of three potent reference renin inhibitors (remikiren, CGP 38560A, and enalkiren) in sodium-depleted normotensive squirrel monkeys. In these monkeys, arterial pressure was measured in the conscious state with a telemetry system. Oral and intravenous maximal effective doses of the three renin inhibitors were compared in parallel groups of monkeys. In additional experiments, remikiren was given on top of either CGP 38560A or enalkiren in the same animals. Finally, the three drugs were compared with the angiotensin converting enzyme inhibitor cilazapril. The effects of the three drugs on the plasma components of the renin-angiotensin system (plasma renin activity, immunoreactive renin, and immunoreactive angiotensin II concentrations) were also measured. Our results show that remikiren was as effective as cilazapril and markedly more effective than CGP 38560A or enalkiren in reducing arterial pressure in our monkey model. Interestingly, these differences in arterial pressure could not be explained by differences of in vitro potency or different biochemical changes of the plasma components of the renin-angiotensin system, because the inhibitors all reduced immunoreactive angiotensin II to similarly low levels. One possible explanation is that, in our model, remikiren in contrast to CGP 38560A and enalkiren is able to inhibit renin in a functionally important extraplasmatic compartment.

Effects of renin-angiotensin system blockade in guinea pigs.

The goal of the present study was to compare the hemodynamic and biochemical effects of the renin inhibitor Ro 42-5892, the angiotensin converting enzyme inhibitor cilazapril, and the angiotensin II receptor blocker EXP132, the aldehyde derivative of DuP 753. The three drugs were evaluated in guinea pigs, previously treated with furosemide, using their maximal effective doses. Cilazapril decreased arterial blood pressure more than Ro 42-5892 and EXP132. In contrast, Ro 42-5892 and EXP132 had similar effects. The larger decrease of arterial pressure induced by cilazapril was not due to a larger decrease of angiotensin II in plasma and was not influenced by cyclooxygenase inhibition with indomethacin or by bradykinin antagonism with Hoe 140. After binephrectomy, most of the blood pressure-lowering effect of Ro 42-5892 disappeared. In contrast, cilazapril was still markedly effective, pointing to extrarenal effects. We conclude that in furosemide-treated guinea pigs, as opposed to previously published animal models, the decrease of arterial pressure induced by angiotensin converting enzyme inhibitors may be partly due to extrarenal effects not related to the renin-angiotensin system.

Vascular renin in the guinea pig. Suppression by the renin inhibitor remikiren.

Angiotensin I and II are generated by the vascular wall. Whether this generation depends on renin or on other enzymes is debated. We tested the hypothesis that remikiren, a highly specific inhibitor of human and guinea pig renin, may inhibit the vascular renin-angiotensin system. Isolated hindquarters from guinea pigs were perfused with an artificial medium, and angiotensin I and II release was measured by high-performance liquid chromatography and radioimmunoassay. Guinea pig hindquarters released angiotensin I (23.8 +/- 5.6 fmol/30 min; n = 13) and angiotensin II (95.2 +/- 19 fmol/30 min; n = 13) spontaneously. Inhibition of the angiotensin I-converting enzyme by captopril (10 nmol/mL) suppressed angiotensin II by 85% and increased angiotensin I by 352% (n = 5, P < .05). Infusion of remikiren (1.6 nmol/mL) in addition to captopril decreased angiotensin I release by 68% (P < .05 versus captopril alone, n = 5 each). We conclude that renin generates angiotensin I in an isolated guinea pig resistance vessel bed. Our study demonstrates that renin rather than nonrenin enzymes is responsible for the major part of vascular angiotensin formation.

Ro 42-5892 is a potent orally active renin inhibitor in primates.

The goal of the present study was to characterize the new renin inhibitor Ro 42-5892 in vitro and in vivo. In vitro, Ro 42-5892 inhibited purified human renin and human plasma renin specifically with an IC50 of 0.7 nM and 0.8 nM, respectively. In vivo, Ro 42-5892 reduced mean arterial blood pressure in sodium-depleted marmosets and squirrel monkeys with as low a dose as 0.1 mg/kg orally. Higher doses reduced pressure by 30-35 mm Hg in both species. The duration of blood pressure decrease with 3 mg/kg orally was more than 24 hours. Maximal changes of plasma renin activity, immunoreactive angiotensin I, and immunoreactive angiotensin II were observed at 15 minutes. Renin was reduced by 74 +/- 31%, angiotensin I by 85 +/- 14%, angiotensin II by 89 +/- 17%, and immunoreactive active renin was increased by 70 +/- 39%. However, unlike pressure, these maximal effects were only transient with complete recovery of renin at 60 minutes under still reduced levels of angiotensin I (61 +/- 24%) and angiotensin II (71 +/- 38%) and increased concentrations of active renin (86 +/- 30%). The blood pressure lowering was due to specific renin inhibition as exemplified by the influence of the kidney, sodium status, species, or stereoselectivity. Moreover, the reduction of arterial blood pressure was similar to the action of the angiotensin converting enzyme inhibitor cilazapril and was not associated with reflex tachycardia in contrast to the pure vasodilator minoxidil. We conclude that Ro 42-5892 is a potent orally active renin inhibitor acting mainly by inhibition of renin in an extraplasmatic compartment.

Ciprokiren (Ro 44-9375). A renin inhibitor with increasing effects on chronic treatment.

The present study characterizes the new transition-state renin inhibitor ciprokiren (Ro 44-9375) in squirrel monkeys. Arterial blood pressure was monitored by telemetry in freely moving, chronically instrumented conscious animals. In vitro at pH 7.4, ciprokiren inhibited human renin in buffer and human plasma with an IC50 of 0.07 and 0.65 nmol/L, respectively. It was equipotent against primate plasma renin and also inhibited plasma renin from dog and guinea pig in the nanomolar range (IC50, 29 and 65 nmol/L, respectively). After acute oral administration it reduced arterial blood pressure dose dependently in normotensive sodium-depleted and cyclosporin-induced hypertensive squirrel monkeys, starting with the minimal oral dose of 3 micrograms/kg. Daily oral doses of 1 microgram/kg showed a progressive blood pressure decrease, with a maximal response reached after 1 week. The drug could also be applied transdermally with similar hemodynamic effects without any decrease of plasma renin activity or plasma immunoreactive angiotensin II. Thus, ciprokiren is characterized in squirrel monkeys as a renin inhibitor with high in vivo potency that might act mainly in the tissular compartment.

Effects of human renin in the vasculature of rats transgenic for human angiotensinogen.

Transgenic rats, which express the human angiotensinogen gene, provide a unique model for studying local vascular effects of human renin. We examined the cleavage of human angiotensinogen to angiotensin I (Ang I) by human renin and its inhibition by a human renin inhibitor in an isolated perfused hindlimb preparation from such rats. Perfusion resulted in the sustained release of human angiotensinogen, which decreased from 19.4 to 11.8 pmol/mL over 45 minutes. Active human renin at doses of 3, 10, and 30 ng/mL perfusate for 15 minutes increased Ang I release from undetectable levels (mean +/- SEM) to 31.9 +/- 3.3, 147.1 +/- 26.2, and 206.4 +/- 17.1 fmol/mL, respectively, by 9 minutes. In separate experiments aimed at the quantification of renin-induced vasoconstriction, captopril decreased the perfusion pressure and lowered Ang II concentrations to nondetectable levels, whereas Ang I values increased sharply. When renin (30 ng/mL) was infused for 15 minutes, renin values in the perfusate decreased to barely detectable levels within minutes after termination of the infusion. However, Ang I values remained high for at least 30 minutes thereafter. The addition of a human renin inhibitor during renin infusion caused Ang I values to promptly decrease within minutes to undetectable levels. Hindlimbs from non-transgenic control rats released no detectable amounts of Ang I, with or without human renin. Finally, by in situ hybridization we documented the presence of human angiotensinogen message in the vessels of the hindlimb. We conclude that renin acts on angiotensinogen at a site in the vascular wall. The cleavage depends on renin and not on other lysosomal proteases.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of human prorenin in rats transgenic for human angiotensinogen.

The physiological role of prorenin is unknown; however, the possibility that prorenin inhibits renin locally has been suggested. We tested the hypothesis that prorenin may be an endogenous competitor for renin uptake in the tissue. We also investigated whether prorenin can be activated to active renin and affect mean arterial pressure (MAP). Isolated perfused hindquarters of rats transgenic for human angiotensinogen were infused with human renin and/or prorenin. The plateau phase of angiotensin (Ang) I release 15 minutes after cessation of infusions was used as a parameter for renin uptake. Renin (10 ng/mL for 15 minutes) caused sustained release of Ang I (153+/-16 fmol/mL). Coinfusion with a 15-fold excess of prorenin did not affect local Ang I formation (153+/-19 fmol/mL). Prorenin infusion alone showed no activation to active renin. In addition, we investigated MAP and plasma Ang II levels after injection of saline (DeltaMAP, -1+/-2 mm Hg; 40+/-5 fmol/mL Ang II), 9 ng renin (DeltaMAP, +37+/-3 mm Hg; 378+/-39 fmol/mL), and 144 ng prorenin (DeltaMAP, +10+/-5 mm Hg; 61+/-5 fmol/mL) and the coinjection of renin and prorenin (DeltaMAP, +41+/-4 mm Hg; 305+/-23 fmol/mL) in anesthetized rats. The data show that prorenin was not activated to active renin and did not affect MAP in short-term experiments. Renin-induced Ang formation was not affected by prorenin. Renin may have been taken up specifically because of its physical and chemical properties or because of nonspecific sequestration in the extravascular space. We conclude that prorenin does not act as an endogenous antagonist for the long-lasting effects of renin in the vascular wall. Moreover, prorenin does not affect acute renin-related effects on blood pressure.

Inhibitors of the Plasmodium falciparum parasite aspartic protease plasmepsin II as potential antimalarial agents.

Malaria is a very serious infectious disease against which the currently available drugs are loosing effectiveness. The main problem is the emergence and the spreading of resistant parasite strains. New treatments are needed in order to regain control over the disease. Drug discovery efforts towards this goal are likely to be more successful, if they focus towards novel mechanisms of action. Such efforts will result in drugs that are functionally and structurally different from the existing drugs and therefore will overcome existing resistances. Here we focus on the aspartic protease plasmepsin II, which is a promising new drug target. We review the drug discovery efforts that were published in the literature on this enzyme, and we present the compounds synthesized at Actelion Pharmaceuticals Ltd.

A comparison of the effects of renin inhibition and angiotensin converting enzyme inhibition upon bradykinin potentiation.

OBJECTIVE: The goal of the present study was to show that, in contrast to an angiotensin converting enzyme (ACE) inhibitor, Ro 42-5892, a new renin inhibitor, can block the renin-angiotensin system without potentiating skin reactions induced by bradykinin. DESIGN: Potentiation of skin reaction to i.d. injections of bradykinin and histamine was evaluated in guinea pigs in the presence and absence of the drug (placebo, Ro 42-5892 or cilazapril). The elimination rate of radioactive bradykinin in blood was measured in other groups of guinea pigs treated with the same drugs. Maximal effective doses of each drug were used. METHODS: Measurements of erythema area induced by bradykinin and histamine injection were performed using a digital planimeter. Radioactive bradykinin was measured in blood by high-performance liquid chromatography and followed over 40 min. RESULTS: The ACE inhibitor cilazapril increased the area of erythema induced by bradykinin but not that induced by histamine. In contrast, Ro 42-5892 did not potentiate the effect of bradykinin. In addition, cilazapril did not change the elimination rate of i.v. radioactive bradykinin in blood. CONCLUSION: These results suggest that potentiation of bradykinin-induced skin reaction by cilazapril is due to a tissular (and not systemic) inhibition of ACE and does not occur with Ro 42-5892. Thus, side effects such as rash, angioneurotic edema or cough, which have been attributed to bradykinin accumulation by ACE inhibitors, may not occur with the use of specific renin inhibitors such as Ro 42-5892.

Effects of the blockade of the renin-angiotensin system in cyclosporin-induced hypertension.

OBJECTIVE: To compare the acute hypotensive effects of three different methods of inhibiting the renin-angiotensin system in a primate model of cyclosporin-induced hypertension. DESIGN: The effects of maximally effective doses of an angiotensin I converting enzyme inhibitor, antihuman renin antibodies or a renin inhibitor (Ro 42-5892) on arterial pressure were evaluated in cyclosporin-treated monkeys. METHODS: Squirrel monkeys were made hypertensive by a 4-week treatment with oral cyclosporin (30 mg/kg) and were equipped with a telemetry system in order to measure arterial blood pressure in the conscious state. RESULTS: Each inhibitor induced a complete suppression of plasma angiotensin II 1 h after administration. The renin antibodies did not decrease blood pressure. Cilazapril decreased blood pressure and Ro 42-5892 was more effective than cilazapril. Moreover, when the renin inhibitor was administered after injection of renin antibodies (rabbit antiserum, 0.4 ml intravenously) or after cilazapril (10 mg/kg orally), it induced a supplementary fall in blood pressure. CONCLUSIONS: These data demonstrate that in this experimental model of hypertension the three different methods of maximal inhibition of the renin-angiotensin system do not lead acutely to the same blood pressure decrease. The results also suggest that renin inhibition might be more effective than angiotensin converting enzyme inhibition in certain forms of hypertension.

Age-dependent hypertension in Mpv17-deficient mice, a transgenic model of glomerulosclerosis and inner ear disease.

The mutant mouse strain Mpv17-/-, carries a retroviral germline integration that inactivates the Mpv17 gene. Mpv17-deficient mice develop progressive glomerulosclerosis and sensineural deafness at early age. Characteristic basement membrane alterations are found in both sites of pathology. Mpv17 is a peroxisomal protein involved in the metabolism of reactive oxygen species, yet its molecular function is unknown. Dysregulation of antioxidant enzymes and basal membrane components has been established in this model and successful therapeutic intervention with antioxidants prove the causal role of reactive oxygen species in the development of the disease phenotype. We here investigated if the Mpv17-/- mice might be hypertensive. Indeed, our study revealed that Mpv17-/- mice developed significant systemic hypertension and tachycardia between 4 weeks and 5 months of age, accompanied by polyuria and elevated natriuresis. Judging from serum and urine parameters, the hypertensive condition develops concomitantly with the renal disease. Biochemical and pharmacological studies that used the endothelin receptor antagonist bosentan and the angiotensin converting enzyme inhibitor cilazapril indicated no involvement of the endothelin and renin-angiotensin systems in this hypertension, suggesting a potential novel mechanism of blood pressure regulation in this new murine hypertension model. Thus, Mpv17-/- mice unravel an intriguing new association between a defect in reactive oxygen metabolism and the age-dependent development of hypertension.

Nonparallel effects of renin inhibitor treatment on plasma renin activity and angiotensins I and II in hypertensive subjects. An assay-related artifact.

Nonparallel effects of renin inhibitor treatment on plasma renin activity (PRA) and the plasma levels of angiotensins (ANG), as well as on blood pressure, have been observed in subjects with hypertension. This study addresses the possibility that renin inhibitors may show a high degree of plasma protein binding in vivo and that displacement of protein-bound inhibitor during the assay of PRA in vitro may lead to overestimation of renin inhibition. Indeed, with the ultrafiltration technique it was found that 96% of the novel renin inhibitor Ro 42-5892, when added to EDTA plasma, was bound to protein. The angiotensinase inhibitors phenylmethylsulfonyl fluoride (PMSF) and 8-hydroxy-quinoline sulfate (8-OHQ), which are currently used in PRA assays, caused a displacement of protein-bound inhibitor, thereby increasing its free concentration. This displacement was sufficient to explain the reduction in IC 50 of Ro 42-5892, which was seen in the PRA assay when PMSF and 8-OHQ were added to plasma. Such reductions in IC 50 were also seen with the renin inhibitors CGP 29-287, CGP 38-560A, and SR 43-845. When Ro 42-5892 was given, 1 mg/kg intravenously in 10 min, to subjects with hypertension, it appeared that plasma ANG I and II returned to baseline after 6-8 h, whereas PRA measured in the presence of PMSF and 8-OHQ was still suppressed. However, when PRA was measured without these angiotensinase inhibitors, the inhibition of PRA was parallel to the suppression of ANG I and II.(ABSTRACT TRUNCATED AT 250 WORDS)

Specific receptors for endothelin on membranes from human placenta. Characterization and use in a binding assay.

High-affinity binding sites for endothelin have been found in a human placenta membrane preparation. 125I-endothelin bound to placenta membranes at 20 degrees C with an association half-time of 30 min, whereas the binding was only slowly reversed with a dissociation half-time of 250 min. In saturation experiments, a single class of high-affinity binding sites was identified with an apparent dissociation constant (KD) of 24 pM and a maximal density of 240 fmol per mg of protein. The binding of 125I-endothelin was half-maximally inhibited by cold endothelin at a concentration (IC50) of 140 pM. In contrast, no inhibition was found at 10(-4) M for a variety of vasoactive peptides such as angiotensin II, vasopressin, neuropeptide Y, substance P, CGRP, bradykinin, leucine enkephalin or dynorphin A. Similarly, the binding was modulated neither by the calcium channel blockers nifedipine, verapamil or diltiazem, nor by the calcium channel agonist Bay k 8644. There was also no effect with the structurally-related bee venom apamin. Using this membrane preparation, endothelin-like activity could be measured in the medium of cultured human endothelial cells by competition binding technique.

Major role of the renin angiotensin system in the neointima formation after vascular injury in guinea pigs.

ACE inhibition has been shown to prevent neointima formation after vascular injury. However, it is not known if this effect is due to a specific inhibition of the renin angiotensin system or to another mechanism such as the accumulation of bradykinin. In order to answer this question we compared the effects of maximal effective doses of cilazapril, an angiotensin converting enzyme (ACE) inhibitor, and ciprokiren, a new renin inhibitor, in guinea pigs. Vascular injury was induced by endothelial denudation of the right carotid artery of guinea pigs treated either by saline (control group), cilazapril (30 mg/kg/day) or ciprokiren (24 mg/kg/day). Twelve days after the ballooning, the guinea pigs were sacrificed, the carotid arteries were perfused fixed and neointima formation was evaluated by quantitative morphometry. Both, ciprokiren and cilazapril prevented neointima formation to the same extent (inhibition by 42 and 49%, respectively, p < 0.05). These results suggest that, in guinea pigs, renin inhibition prevents neointima formation to a similar extent as ACE inhibition. Therefore, ACE inhibitors seem to act in this model by inhibiting the renin angiotensin system and not by other effects such as accumulation of bradykinin.

Anantin--a peptide antagonist of the atrial natriuretic factor (ANF). I. Producing organism, fermentation, isolation and biological activity.

Anantin, a peptide binding to the receptor of the atrial natriuretic factor (ANF) was isolated from a strain of Streptomyces coerulescens. The molecule consists of 17 natural L-amino acids which form a peptidic ring system. It has a MW of 1,871.0. The chemical composition is C90H111N21O24. The compound was found to bind competitively to ANF-receptors from bovine adrenal cortex (Kd = 0.61 microM). Furthermore, it dose-dependently inhibited the ANF-induced intracellular cyclic guanosine monophosphate accumulation in bovine aorta smooth muscle cells. At the same concentration no agonistic effects were detectable in these cells. Thus, anantin is considered to be the first microbially produced antagonist of the cardiac hormone, ANF.

Piperidine renin inhibitors: from leads to drug candidates.

Non-peptidomimetic renin inhibitors of the piperidine type represent a novel structural class of compounds potentially free of the drawbacks seen with peptidomimetic compounds so far. Synthetic optimization in two structural series focusing on improvement of potency, as well as on physicochemical properties and metabolic stability, has led to the identification of two candidate compounds 14 and 23. Both display potent and long-lasting blood pressure lowering effects in conscious sodium-depleted marmoset monkeys and double transgenic rats harboring both the human angiotensinogen and the human renin genes. In addition, 14 normalizes albuminuria and kidney tissue damage in these rats when given over a period of 4 weeks. These data suggest that treatment of chronic renal failure patients with a renin inhibitor might result in a significant improvement of the disease status.