Search

A series of potent, selective, conformationally restricted angiotensin II (AII) receptor antagonists has been discovered. Two classes of conformationally restricted analogues were prepared: triazolone-based and imidazole-based biphenyl derivatives. The most active compound, an imidazole-based analogue, has an IC 50 of 11 nM and a pA 2 of 8.8.

Substituting nitrogen for carbon in the pyridinylphenylmethyl analogs of SC-50560 proved to be detrimental, however, the two phenylpyridinylmethyl analogs of SC-50560 retained their potencies. Of these two analogs, SC-52458 was found to have superior in vivo properties.

The depressor activity of a novel nonpeptidic angiotensin II (AII) receptor antagonist, SC-51316 (2,5-dibutyl-2,4-dihydro-4-[[2-(1H-tetrazol-5-yl)[1,1'-biphenyl]-4 '- yl]-methyl]-3H-1,2,4-triazol-3-one), is described. In anesthetized, ganglion-blocked rats, intravenous administration of SC-51316 inhibited the pressor response to an infusion of AII. To determine antihypertensive efficacy, conscious, spontaneously hypertensive rats were administered SC-51316 (30 mg/kg intragastrically) daily for 5 days. Blood pressure was reduced in a similar manner to that observed with the angiotensin converting enzyme inhibitor enalapril (10 mg/kg intragastrically). SC-51316 had no effect on heart rate. In conscious, sodium-deficient dogs, administration of SC-51316 (30 mg/kg orally) or enalapril (10 mg/kg orally) lowered blood pressure similarly over a 24 h observation period. Thus, SC-51316 antagonizes the activity of AII in vivo and is an orally active, antihypertensive agent.

The surmountable (competitive) N 1 -(2-methylphenyl)-2H-imidazol-2-one angiotensin II receptor antagonist SC-54628 is converted to an insurmountable (noncompetitive) antagonist SC-54629 by the addition of a methyl group at the 6-position of the phenyl ring.

A series of 5-[1-[4-[(4,5-disubstituted-1H-imidazol-1-yl)methyl]- substituted]-1H-pyrrol-2-yl]-1H-tetrazoles and 5-[1-[4-[(3,5-dibutyl-1H-1,2,4-triazol-1-yl)methyl]-substituted]- 1H-pyrrol-2-yl]-1H-tetrazoles were investigated as novel AT1-selective angiotensin II receptor antagonists. Computer-assisted modeling techniques were used to evaluate structural parameters in comparison to the related biphenyl system. New synthetic procedures have been developed to prepare the novel compounds. The best antagonists in this series had IC50 values (rat uterine membrane receptor binding) in the 10(-8) M range and corresponding pA2 in isolated organ assay (rabbit aorta rings). Structure-activity relationships indicate some similarities with the finding in the biphenyl system. Substitution on the pyrrole ring modulates activity. Compound 5 antagonized angiotensin-induced blood pressure increase when administered to conscious rat at 30 mg/kg per os.

We examined the interaction of a non-guanylate cyclase-linked atriopeptin (AP) binding site ligand, SC-46542 (des[Phe106,Gly107,Ala115,Gln116]AP-(103-126], and an endopeptidase 24.11 inhibitor, thiorphan, on mean arterial pressure, urinary sodium excretion, urinary cyclic guanosine monophosphate (cGMP) excretion, plasma cGMP concentration, and plasma AP immunoreactivity (ir) in conscious spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). Compared to vehicle control rats, coadministration of SC-46542 and thiorphan increased urinary sodium excretion in SHR from 2.1 +/- 0.3 to 11.6 +/- 0.7 microEq/min/100 g body weight and in WKY from 1.6 +/- 0.4 to 4.4 +/- 0.4 microEq/min/100 g body weight, and increased urinary cGMP excretion in SHR from 2.7 +/- 0.5 to 79.0 +/- 17.5 pmol/min/100 g body weight and in WKY from 7.0 +/- 3.0 to 72.4 +/- 10.6 pmol/min/100 g body weight. The change in urinary sodium excretion was greater in SHR than WKY. The coadministration of SC-46542 and thiorphan had greater effects on urinary sodium excretion and urinary cGMP excretion than administration of either compound alone. Coadministration of thiorphan and SC-46542 had no effect on glomerular filtration rate or plasma cGMP concentration, suggesting that the urinary cGMP excretion response was nephrogenous. Compared to vehicle control rats, plasma APir was increased during coadministration of SC-46542 and thiorphan in both SHR (998 +/- 76 v 5.10 +/- 116 pg/mL) and WKY (775 +/- 36 v 414 +/- 36 pg/mL).(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclic analogues of angiotensin II (AII) were synthesized by connecting the side chains of residues 3 and 5 via a disulfide bridge. Appropriate conformational constraints afforded an analogue, [Hcy 3,5 ]AII, having high contractile activity (pD 2 = 8.48 vs 8.81 for AII) and excellent binding affinity (IC 50 = 2.1 nM vs 2.2 nM for AII). This type of cyclization was also used to prepare a highly potent AII antagonist, [Sar 1 , Hcy 3,5 , Ile 8 ]AII(pA 2 = 9.09 vs 9.17 for [Sar 1 ,Ile 8 ]AII; IC 50 = 0.9 nM vs 1.9 nM for [Sar 1 ,Ile 8 ]AII). Model building suggests that this ring structure is consistent with a receptor-bound conformation having any of a variety of three-residue turns, including a γ-turn. In contrast, the receptor-bound conformation of AII does not appear to acommodate a β-turn or an α-helix which includes residues 3-5

A series of analogues of the recently reported angiotensin II (AII) antagonist [Sar1]AII-(1-7)-amide or des-Phe8[Sar1]AII (3) have been prepared by solid-phase synthesis and purified by reverse-phase liquid chromatography. The agonist and antagonist properties of these carboxy-truncated analogues of AII were determined in the isolated rabbit aorta assay. In the analogues tested, replacement of aspartic acid in position 1 by sarcosine was found necessary to produce significant antagonist activity. At position 7 of the des-Phe8 analogues, prolinamide could be replaced by proline without significant change in the biological activity. However, substitution of 7-prolinamide by either glycinamide or sarcosinamide provided inactive peptides. Methylation of the 4-tyrosine in [Sar1]AII-(1-7)-NH2 preserved the antagonist potency in isolated rabbit aorta. Deletion of the proline at position 7 resulted in inactive hexapeptides, both in the Asp1 and Sar1 series. However synthesis of the N,N-dimethyl amide at the N-terminus afforded hexapeptide [Sar1]AII-(1-6)-N(CH3)2 (10) with a pA2 value of 7.05. All the antagonistic peptides synthesized were fully reversible, competitive antagonists in vitro. These findings indicate that the structural requirements for receptor blockade by these C-truncated analogues are quite stringent with respect to the nature of the amino acid at positions 1 and 6/7. The analogues 2, 3, 7, 10, 11 (saralasin), and 12 (sarmesin) were tested in vivo in the anesthetized rat and were found to inhibit the AII pressor response. In addition, 3 inhibited angiotensin II stimulated aldosterone release from isolated rat adrenal zona glomerulosa cells and had no agonist activity by itself at the doses tested. Interestingly, analogue 3, when injected intracerebroventricularly in conscious rats, failed to antagonize the dipsogenic response to an angiotensin II icv injection and this reflects some heterogeneity in the AII receptor population. Peptide 3 is the first example of an antagonist that discriminates between peripheral and brain receptor subtypes.

We have previously reported on the basic pharmacologic properties of SC-52458 (5-[(3,5-dibutyl-1H-1,2,4-triazol-1-yl)methyl]-2-[2-(1H-tetrazol-5-ylphenyl)]pyridine), a novel angiotensin (AII) receptor antagonist that binds potently to AT 1 receptors in rat adrenal cortex and blocks AII-mediated contraction in isolated rabbit aorta. In the present study, the ability of SC-52458 to block AII pressor responses in conscious dogs was measured. In addition, we determined whether SC-52458 lowered mean arterial pressure in dogs with 2 kidney/1 clip renal hypertension when given daily for 4 days. In conscious, normotensive dogs, SC-52458 at 30 mg/kg orally, blocked the pressor response to AII (50 ng/kg, intravenously) with maximal inhibition (91%) observed 2 h after dosing. Plasma concentrations of SC-52458 measured by HPLC also were highest at the 2-h time point. After 24 h, the AII pressor response remained inhibited (by 35%) and SC-52458 was still measurable in plasma from treated dogs. In dogs made hypertensive by constriction of the left renal artery, SC-52458 lowered mean arterial pressure compared to vehicle treatment although heart rate was not different in the two groups. The maximal blood pressure lowering achieved with SC-52458 was similar to the maximal effect observed with the angiotensin converting enzyme inhibitor lisinopril. We conclude that SC-52458 blocks AII mediated pressor responses in normotensive, conscious dogs and SC-52458 is an efficacious antihypertensive agent in dogs with 2 kidney/1 clip renal hypertension.

Tissue factor pathway inhibitor (TFPI) is a potent inhibitor of the blood coagulation factor VIIa-tissue factor complex, as well as a direct inhibitor of factor Xa. Intravenously administered TFPI is rapidly cleared from circulation predominantly via liver. We previously reported that the low density lipoprotein receptor-related protein (LRP), a multifunctional endocytic receptor, mediates the uptake and degradation of TFPI in hepatoma cells. This process is inhibited by a 39-kDa receptor-associated protein which binds to LRP and regulates its ligand binding activity. However, a distinct, low affinity binding site (perhaps heparin sulfate proteoglycans, HSPGs) on the endothelium and liver is thought to be responsible for the majority of TFPI cell surface binding. In the current study, we investigated the role of LRP and this second binding site in the clearance of 125I-TFPI in vivo using competitors and inhibitors of the receptors. Mice overexpressing the 39-kDa protein via adenoviral-mediated gene transfer displayed diminished plasma clearance of 125I-TFPI. Blockade of cell surface HSPGs sites by incubation with the positively charged molecule, protamine, inhibited 125I-TFPI binding to the hepatoma cells in vitro. In addition, preadministration of protamine in vivo prolonged the plasma clearance of 125I-TFPI in a dose-dependent manner. However, a dramatic increase of the plasma half-life of 125I-TFPI and virtual elimination of 125I-TFPI clearance was observed in mice overexpressing the 39-kDa protein and administered protamine. Taken together, our results suggest that two receptor mechanisms are involved in the clearance of TFPI in vivo.

Several guanidinium compounds were tested for their ability to inhibit the binding of [ 3 H]idazoxan to the I 2 subtype of the imidazoline site on rabbit kidney basolateral membranes. Phenformin, a biguanide, was the most potent with an IC 50 of 50 ± 3 μ M. Various K + channel modulators were also evaluated for inhibition of [ 3 H]idazoxan binding. 1,2,3,4-Tetrahydro-9-aminoacridine and 4-aminopyridine (IC 50 values of 38 ± 5 μ M and 43 ± 3 μ M, respectively) were the most effective of the K + channel blockers tested. Pinacidil, an ATP-sensitive K + channel opener, inhibited radioligand binding with an IC 50 of 100 ± 10 μ M. The results indicate that I 2 sites are selective in their interaction with guanidinium derivatives and K + channel modulators.

2,5-Dibutyl-2,4-dihydro-4-[[2-(1H-tetrazol-5-yl)[1,1'-biphenyl]-4' - yl]methyl]-3H-1,2,4-triazol-3-one, SC-51316, was synthesized as a potent and orally active angiotensin II (AII) receptor antagonist with a long duration of action. To explore the lipophilic pocket in the AII receptor interacting with the substituent at the 2-position of triazolone-based antagonists, a series of compounds were prepared and evaluated for receptor binding affinity and antagonism of AII-contracted rabbit aortic rings. It has been found that the pocket is very spacious and can accommodate different sizes of lipophilic groups and various functionalities. Acidic groups generally result in a slight decrease in binding affinity. Branched chains are unfavorable. The freedom of rotation around C2-C3 in the flexible side chain is crucial for good binding. The 2-phenylethyl-substituted triazolone analogue exhibits the highest in vitro potency among all compounds that have been synthesized.

The synthesis and in vitro activity of new nonpeptide angiotensin II antagonists is presented. Compared to previously reported biphenyl compounds, the new analogues 8 and 9 have reduced conformational freedom derived from steric hindrance. Methyl 4'-methyl-2',6'-dimethoxy[1,1'-biphenyl]-2-carboxylate 4 has been synthesized by a Von Pechmann condensation of orcinol with oxocyclohexane-2-carboxylate followed by dehydrogenation. This scheme provided the carbon skeleton of the biphenyl potentially substituted on the 2-, 2'-, 4'-, and 6'-positions. Elaboration of the subsituents led to a biphenyl derivative used to alkylate a 2-n-butyl-4-chloro-5-(hydroxymethyl)imidazole. After coupling with the imidazole two regioisomers were separated and identified by 1H NMR. NOESY experiments were useful to establish regiochemistry of the final products that have angiotensin II blocking activity. Their affinity for angiotensin II receptors was established in a binding assay experiment and in an isolated organ test. The presence of 2',6'-dimethoxy substituent on the biphenyl moiety of the antagonist was found to significantly decrease affinity for the receptor.

Publisher Summary This chapter discusses the assays for enzymes that metabolize atrial natriuretic peptide (ANP). ANP is a 28-residue peptide containing a disulfide bond between Cys105 and Cys121 that forms a ring structure. It is produced by the left atria as a 126-residue prohormone and is stored as such in vacuoles until secreted in response to an acute increase in blood pressure. At that time, the C-terminal 28-residue peptide is cleaved off as the active circulating hormone; it causes both an increase in water and sodium excretion by the kidney. In addition, C- and N-terminal shortened analogs are active. This chapter discusses those studies that concentrated on determining which enzymes are involved in the degradation of ANP and the metabolites produced. The elucidation of the specific enzymes involved has been dependent on both the identification of the metabolites and the effect of relatively specific inhibitors, such as thiorphan (endopeptidase 24.11), bestatin (aminopeptidase B and M and leucine aminopeptidase), and captopril (angiotensin-converting enzyme).

We describe the pharmacologic properties of SC-52458, 5-[(3,5-dibutyl-1H-1,2,4-triazol-1-yl)methyl]-2-[2-(1H-tetrazol - 5-ylphenyl)]pyridine, a novel nonpeptide angiotensin II (AII) receptor antagonist. SC-52458 was a potent inhibitor of [125I]AII binding to AT1 receptors in rat adrenal cortex and uterine smooth muscle membranes (IC50 values of 2.8 and 6.9 nM, respectively). Contraction of rabbit aortic rings by AII was antagonized by SC-52458 in a competitive and reversible manner (pA2 of 8.18). SC-52458 had no effect on the activity of angiotensin converting enzyme (ACE) or renin in vitro. In normotensive rats, administration of SC-52458, either intravenously (i.v.) or by gavage, inhibited the pressor response to AII. Daily treatment with SC-52458 at 20, 30, and 50 mg/kg by gavage for 4 days decreased blood pressure (BP) in conscious, spontaneously hypertensive rats (SHR). Further studies in renal-artery ligated rats and sodium-deficient dogs demonstrated that oral administration of SC-52458 decreased BP and that this activity was correlated with significant plasma levels of the compound. SC-52458 is an orally active, competitive AT1-receptor antagonist with antihypertensive properties.

Binding studies revealed the presence of a single class of high affinity binding sites for atriopeptin III on rabbit lung membranes. An apparent dissociation constant (Kd) of 0.32 nM and a binding capacity of 166 fmol/mg protein was determined. Binding was time-dependent and saturable. The relative binding affinities of atrial peptide analogs correlated well with their potencies in eliciting relaxation of norepinephrine-contracted rabbit aorta strips. Unrelated peptide hormones did not compete for the atriopeptin binding site on rabbit lung membranes. The atrial peptide binding data are similar to those obtained for other tissues and indicate the presence of a physiologically relevant atrial peptide receptor in lung.

Analogues of atriopeptin(103-125)amide were prepared having a disulfide bridge at positions different from that found in the natural product. Most of these conformationally perturbed peptides were found to bind selectively to one subclass of binding sites. Binding affinity to a class of specific binding sites that is not associated with any known biological activity (nonvasorelaxant or NVR binding sites) is unaffected or even modestly improved. Affinity for the receptor subclass that is associated with vasorelaxation (VR subclass) decreases in most examples. In several cases, binding to the VR subclass is below the limits of detection for the assay used here. The data demonstrate that binding of atrial peptides to VR receptors requires rigidly defined receptor/ligand interactions. In contrast, the NVR subclass of binding sites appears to tolerate changes in peptide structure quite well.

Computer image analysis programs developed by astronomers for celestial photometry were used for the analysis of autoradiographs of electrophoretic slab gels. Oxytocin-stimulated myosin light chain phosphorylation in mammary myoepithelial cells was studied by incubating cells with [32P]orthophosphate followed by oxytocin addition, polyacrylamide gel electrophoresis of cellular protein, and autoradiography of electrophoretic slab gels. After scanning and digitization of autoradiographs, [32P]phosphate incorporation into the myosin light chain was measured by the determination of radiation flux recorded on the X-ray film. Within seconds after oxytocin addition, the myosin light chain was fully phosphorylated. The large library of astronomical computer programs has applications for the quantitative analysis of both one- and two-dimensional electrophoretic gels.

Atrial natriuretic peptide (ANP) contains a disulfide which is generally considered to be required for biological activity. A truncated linear ANP analog, des-Cys105,Cys121-ANP-(104-126) (referred to as analog I), that lacks the 2 cysteine residues of the parent peptide was synthesized. In competition binding studies using rabbit lung membranes, ANP-(103-126) and analog I displaced bound 125I-ANP-(103-126) from specific ANP binding sites 100 and 73%, respectively. The concentrations of ANP-(103-126) and analog I that produced 50% inhibition of radioligand binding to the membranes were 0.26 +/- 0.07 and 0.31 +/- 0.09 nM, respectively. Radioiodinated ANP-(103-126) and analog I were chemically cross-linked to binding sites on rabbit lung membranes, and the labeled membrane proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. 125I-Analog I specifically labeled a 65,000-dalton protein and a 135,000-dalton protein which, under reducing conditions, dissociated into 65,000-dalton subunits. In contrast, 125I-ANP-(103-126) labeled specifically a nonreducible 135,000-dalton protein, in addition to the 65,000-dalton species and the reducible 135,000-dalton species. ANP-(103-126) (100 nM) stimulated rabbit lung particulate guanylate cyclase activity, whereas analog I, at the same concentration, had no effect on cyclic GMP production and did not antagonize the effect of ANP-(103-126). From these observations, we conclude that analog I is a selective ligand which binds to approximately 73% of the total ANP binding sites present in rabbit lung membranes. Unlike ANP-(103-126), analog I does not bind to the remaining 27% of the binding sites and does not activate guanylate cyclase. Binding to the cyclase-linked ANP receptor correlates with the specific labeling by 125I-ANP-(103-126) of the nonreducible 135,000-dalton membrane protein.

A linear decapeptide, [cyclohexylalanine 106]ANP-(105-114)NH2 (1), where ANP is atrial natriuretic peptide, was prepared by solid phase synthesis and purified by reverse-phase liquid chromatography. This novel peptide was found to bind to ANP receptors in rabbit lung membranes, to stimulate cGMP production in various tissues, and to fully relax precontracted rabbit aorta in a dose-dependent fashion. The potency of 1 in the various in vitro assays varies between one-twentieth and one-eightieth of the potency of the reference peptide, the 24-mer rat ANP-(103-126). The linear decapeptide 1, which encompasses amino acid residues from the rat ANP sequence (105-114), features a cyclohexylalanine residue instead of the phenylalanine 106 residue in the hormone sequence, a free sulfhydryl function at the N-terminal cysteine 105, and a carboxamide C terminus. Its disulfide dimer 6 was active in the rabbit aorta assay while the S-methyl cysteine 7 analogue was not active in the same assay at similar concentrations. The decapeptide 1 is of particular significance because it is the shortest analogue reported to date endowed with agonistic activity at the guanylate cyclase-coupled ANP receptor. In particular, it is interesting to compare its structure to the structures of other short linear analogues of ANP which are totally devoid of the ability to stimulate particulate guanylate cyclase activity.

Atrial natriuretic peptides (ANPs) are degraded rapidly by renal brush border membranes in vitro. Here, we report that thiorphan, a specific inhibitor of endopeptidase 24.11, afforded almost complete protection against inactivation of ANPs by a renal brush border membrane preparation. The diastereoisomers of [3-(N-hydroxy)carboxamido-2-benzylpropanoyl]-L-alanine (HCBA) are potent inhibitors of endopeptidase 24.11 and were also tested for their abilities to inhibit ANP-(103-126) degradation. The (S,S)-diastereoisomer was more effective than the (R,S)-diastereoisomer (kelatorphan), but both were less potent than thiorphan. To determine if endopeptidase inhibitors could decrease ANP metabolism in in vivo, thiorphan and (S,S)-HCBA were given to rats with or without a continuous infusion of ANP-(103-126). Both inhibitors induced rapid increases in plasma ANP concentration in rats administered exogenous ANP-(103-126), but had no effect on endogenous ANP levels. Thus, specific inhibitors of endopeptidase 24.11 decrease the degradation of ANPs in vitro, and are effective in reducing the metabolism of ANP-(103-126) in vivo.

Oxytocin (10 nM) stimulated the phosphorylation of the 20,000 mol wt myosin light chain in rat mammary myoepithelial cells from a basal level of 0.17 to 0.85 mol phosphate/mol light chain within 30 sec. Of the smooth muscle stimulants tested, oxytocin appears to be the only normal physiological stimulus for myosin phosphorylation in these cells. The roles of cAMP, cGMP, and calcium ions were investigated in the mode of action of oxytocin and the regulation of myosin phosphorylation. Although oxytocin had no effect on cGMP metabolism, there was an increase in the cAMP content of the treated myoepithelial cells. Further investigation suggested that the increase in cAMP levels in response to oxytocin was not directly involved in the regulation of myosin phosphorylation. Various agents known to interfere with calcium ion transport were used to study the role of calcium ions in the action of oxytocin and the regulation of myosin phosphorylation. The results indicate that the duration of the cellular response to oxytocin depends on an influx of extracellular calcium through calcium-specific channels in the plasma membrane.

Publisher Summary This chapter discusses the experimental results that should be useful to the medicinal chemist for designing atrial natriuretic peptide (ANP) analogs with increased potency or extended duration of action. ANP has vasorelaxant properties and through its interactions with other hormonal systems, inhibits steroidogenesis and the secretion of renin and vasopressin. Several biological properties have been ascribed to ANP, including effects on fluid, electrolyte, and blood pressure homeostasis. ANP is the most potent diuretic substance known and exhibits a ceiling for natriuresis that is similar to hydrochlorothiazide. ANP binding proteins have been isolated from bovine adrenocortical cells, bovine aortic smooth muscle cells, and bovine lung and appear to be disulfide-linked dimers of approximately 125-140 kDa with a subunit M r of 60-70 kDa. In contrast, other ANP binding proteins have been isolated that copurify with guanylate cyclase activity. ANP contains a 17-amino acid ring, formed by a disulfide bridge between Cys105 and Cys121. Several laboratories have investigated the requirement for this ring in the interaction of ANP with its receptor and in eliciting biological responses. Early studies demonstrated that ANP lost its natriuretic and vasorelaxant roperties when the cyclic conformation was destroyed by reduction and carboxymethylation of the disulfide bridge or by cleavage of the Asp111–Arg112 peptide bond within the ring. This chapter describes the design of analogs that are selective for the noncyclase-linked receptor that has provided a useful tool for discriminating between the two types of ANP receptor. Efforts to sequence both types of the ANP receptors continue and the complete sequence of at least one of these receptor proteins was expected.

Atrial natriuretic factor (ANF), a 28-amino-acid peptide secreted from the mammalian heart, is known to be cleared rapidly from the circulation. In vitro and in vivo studies implicate the kidney as an important site for clearance and subsequent degradation of atrial natriuretic factor. We have observed that atrial natriuretic factor is inactivated rapidly by rabbit kidney brush-border membranes. The rate of degradation of ANF measured by the loss of bioactivity followed a similar time-course to the decrease in peptide peak area measured by high-performance liquid chromatography. Interestingly, inactivation of ANF produced only a single major degradation product, which was isolated and purified. Sequence analysis revealed that the product had the same sequence of amino acids as ANF with the Cys-7-Phe-8 bond cleaved and the disulfide bridge between Cys-7 and Cys-23 remaining intact. As the renal brush border contains an abundance of proteolytic activities, it is surprising that this peptide is cleaved primarily at a single peptide bond.

Atriopeptin (AP) 24, containing amino acids Ser103—Tyr126 of the carboxy-terminal portion of the atrial natriuretic peptide prohormone, was degraded rapidly by rabbit kidney brush border membranes. The rate of degradation of AP24 measured by the loss of vasorelaxant activity followed a similar time course to the decrease in peptide peak area measured by high-performance liquid chromatography. Inactivation of AP24 produced peptide fragments which were separated by HPLC. The major products were purified individually and their peptide sequences determined. Results indicate that AP24 was proteolytically cleaved at three peptide bonds: Ser103-Ser104, Cys105-Phe106 and Ser123-Phe124. des-Ser103-AP24 had similar vasorelaxant activity to AP24, while AP24 cleaved at Cys105-Phe106 was inactive. Regarding the proteolytic cleavage at Ser123-Phe124, there was an accumulation of the C-terminal tripeptide, Phe-Arg-Tyr, only at the later time points of the incubation. Degradation experiments were repeated with an amino- and carboxy-terminal protected peptide, acetyl-AP24-amide. Peptide sequence analysis of the major degradation products of this peptide revealed that the critical peptide bond cleaved was Cys105-Phe106. We conclude that the Cys-Phe peptide bond renders atrial peptides highly susceptible to proteolysis by renal brush border membranes, resulting in inactivation.

The response of mammary myoepithelial cells to oxytocin was studied by monitoring the level of phosphorylation of the 20,000 mol wt light chain of myosin. Myoepithelial cells were obtained by collagenase dispersion of involuted rat mammary tissue. The cells were equilibrated with [32P]orthophosphate, and then stimulated with oxytocin. Phosphorylated proteins were separated by polyacrylamide gel electrophoresis, and incorporation of 32P into the proteins was detected by autoradiography. Nanomolar concentrations of oxytocin caused a 3- fold increase in the level of phosphorylation of the myosin light chain within 0.5 min. When the cells were incubated with oxytocin in a calcium-free medium, there was only a transient phosphorylation of myosin. However, readdition of calcium to these cells resulted in phosphorylation of the myosin light chain. The results suggest that calcium is involved in the intracellular events following stimulation of the cells with oxytocin.

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.

Conformationally restricted analogues of atriopeptin(103-125)amide were prepared by synthesizing novel bicyclic peptides in which a second disulfide bridge linking residues 108 and 117 was introduced. These syntheses were shown to proceed with no significant scrambling of the disulfide bonds and demonstrated that structurally defined bicyclic analogues of atrial peptides could be easily prepared. The conformationally restrained analogues described here were found to be biologically active with potencies (EC50s) ranging from 0.05 to 3 microM. In addition, these bicyclic peptides (and many of the monocyclic precursors) were found to bind selectively to a class of specific tissue binding sites that have not been shown to be associated with any known second messenger system (NVR binding sites). Since affinity for the receptor class linked to vasorelaxation was negatively affected by the conformational restrictions described here, binding of atrial peptides to this class of receptors appears to have more specific conformational requirements than does binding to the NVR sites.

The structure-activity relationships for affinity and selective binding of atrial natriuretic peptide (ANP) and analogues to guanylate cyclase coupled (CC) and non-cyclase coupled (NC) receptors in rabbit lung membranes are described. We have designed a series of peptides to try to identify the minimal sequence involved in specific recognition of each receptor subtype. The affinity of the peptides was determined from competitive binding experiments. Several peptides derived from the rat ANP sequence, e.g., des-[Phe106, Gly107, Ala115, Gln116]ANP-(103-125)NH2 (4), des-[Cys105,121]ANP-(104-126) (5), and [Acm-Cys105]ANP-(105-114)NH2 (9) have high affinity and selectivity for the noncoupled site. Peptide 4 was the most selective ligand with an affinity superior to that of ANP-(103-126). This compound does not displace the radiolabeled ligand from the guanylate cyclase coupled receptor at the highest concentration tested (100 nM). The structure-activity relationship for affinity and selectivity is discussed. Comparison of the peptide sequences suggests that the structural feature responsible for recognition of the NC site resides in a single sequence of seven contiguous amino acids from the cyclic core of the hormone. The corresponding heptapeptide retains affinity to the guanylate cyclase uncoupled binding site and is proposed to encompass the minimal sequence for specific recognition of the non-guanylate cyclase coupled ANP receptor.