Gillian M. Olins

Inactivation of atrial natriuretic factor by the renal brush border.

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.

Specific inhibitors of endopeptidase 24.11 inhibit the metabolism of atrial natriuretic peptides in vitro and in vivo

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.

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

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

Central and peripheral actions of a nonpeptidic angiotensin II receptor antagonist.

Nonpeptidic imidazole derivatives were recently reported to be angiotensin II receptor antagonists with acute blood pressure-lowering activity. In the present study, we characterized the angiotensin II receptor antagonist properties of one such derivative, 4′-{[2-butyl-4-chloro-5-(hydroxymethyl)- lH-imidazol-l-yl]methyl}-[l,l′-biphenyl]-2-carboxylic acid (IMI). In receptor binding studies, IMI displaced bound [125I] angiotensin II from rat uterine membranes with an IC50 of 0.17 μM. In isolated rabbit aortic rings, IMI shifted the angiotensin II concentration-response curve to the right in a parallel and concentration-dependent manner. A Schild plot of these data indicated a pA2o of 7.13±0.16 and a slope of 0.94±0.06. In rat kidney slices, IMI shifted the concentration-response curve for angiotensin II-induced inhibition of renin release to the right Antagonism of the angiotensin II pressor response by IMI was dose dependent and reversible in ganglion-blocked, anesthetized rats. The water intake and pressor responses to intracerebroventricular angiotensin II (100 pmol) were inhibited by intracerebroventricular IMI (25 or 50 nmol) in conscious Sprague-Dawley rats. Similarly, the drinking and pressor responses to intravenous angiotensin II were blocked by intravenous IMI in conscious rats. IMI alone had no effects on mean arterial pressure or drinking when administered either intravenously or intracerebroventricularly. IMI decreased mean arterial pressure throughout 5 days of infusion in spontaneously hypertensive rats. In summary, IMI was a full competitive antagonist without partial agonist activity in peripheral tissues and the central nervous system. Moreover, the chronic administration of this angiotensin II receptor antagonist was antihypertensive in spontaneously hypertensive rats.

Specific receptors for atriopeptin III in rabbit lung.

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.

Pharmacology of SC-52458, an orally active, nonpeptide angiotensin AT1 receptor antagonist

Summary 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 (AH) receptor antagonist. SC-52458 was a potent inhibitor of [125I]AII binding to AT, 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 AH. 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.

Interaction of guanidinium compounds and K+ channel modulators with imidazoline binding sites in rabbit kidney

Several guanidinium compounds were tested for their ability to inhibit the binding of [3H]idazoxan to the I2 subtype of the imidazoline site on rabbit kidney basolateral membranes. Phenformin, a biguanide, was the most potent with an IC50 of 50 +/- 3 microM. Various K+ channel modulators were also evaluated for inhibition of [3H]idazoxan binding. 1,2,3,4-Tetrahydro-9-aminoacridine and 4-aminopyridine (IC50 values of 38 +/- 5 microM and 43 +/- 3 microM, respectively) were the most effective of the K+ channel blockers tested. Pinacidil, an ATP-sensitive K+ channel opener, inhibited radioligand binding with an IC50 of 100 +/- 10 microM. The results indicate that I2 sites are selective in their interaction with guanidinium derivatives and K+ channel modulators.

Discovery of nonpeptide potent conformationally restricted angiotensin II receptor antagonists

Abstract 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.

1H-1,2,4-triazole angiostensin II receptor antagonists: “qC-linked”q analogs of SC-50560

Abstract Novel 1H-1,2,4-triazole analogs in which the biphenylmethyl group is attached to carbon and the butyl group is attached to the adjacent nitrogen were found to be potent angiotensin II receptor antagonists. Additional substitution at the carbon bearing the biphenyl group proved to be very detrimental to potency. The in vivo properties of the dibutyl analog SC-51757 were found to be similar to SC-50560.

Chapter 11. Atrial Natriuretic Peptide

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.