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It is unclear how hepatic glucocorticoid receptor (GR) function and hypothalamic-pituitary-adrenal axis tone contribute to the diabetic state and in particular whole-body glucose fluxes. We have previously demonstrated that long-term exposure to hepatic GR inhibition lowers glucose levels in ob/ob mice (J Pharmacol Exp Ther 2005;314:191). The purpose of this study was to determine the effects of a novel GR antagonist (A-348441) on whole-body glucose fluxes in a model of insulin resistance, the Zucker fatty (fa/fa) rat. After an overnight fast, euglycemic-hyperinsulinemic clamp studies were performed 2 hours after single oral dosing as follows: (1) A-348441 at 100 mg/kg or (2) vehicle. Furthermore, effects of 1 week of treatment with either vehicle or A-348441 (3, 10, 30, or 100 mg/kg PO, once per day) were investigated in separate groups of rats fasted overnight and given a final dose of their respective compound, followed 2 hours later by a euglycemic-hyperinsulinemic clamp. One week after catheter implantation, body weight returned to presurgery levels, with no difference between groups. A single, 100-mg/kg dose of A-348441 significantly increased glucose infusion rate 4-fold (P < .05) and reduced endogenous glucose production by 37% (P < .05) but did not change glucose disposal. After 1 week of sub–long-term dosing, fasting glucose levels were reduced dose-dependently with A-348441 vs vehicle (−8%, not significant; −14%, −20%, and −25%, P < .05, at 3, 10, 30, and 100 mg/kg, respectively) with no observed hypoglycemia or change in fasting insulin levels. A-348441 increased the glucose infusion rates after 1-week treatment by 1.3-, 5.7-, 7.3-, and 6.4-fold (P < .05). Endogenous glucose production was decreased (−25%, −44%, −50%, and −61%, P < .05), whereas glucose disposal was increased (29% and 13%, not significant; 23% and 34%, P < .05), with A-348441. In summary, single-dose treatment with the liver-selective GR antagonist A-348441 decreases glucose production with no effect on glucose disposal or fasting glucose levels. After 1 week of treatment with A-348441, (1) there was no effect on body weight, (2) fasting glucose levels decreased, (3) both glucose disposal and glucose infusion rate increased during clamping, and (4) endogenous glucose production was greatly reduced. In addition, hepatic glucose production was highly correlated with fasting glucose levels (r = 0.97). In conclusion, these results indicate that A-348441 increases insulin sensitivity at both the liver and peripheral tissues, leading toward a normalization of the insulin resistant state. Furthermore, with 1-week vs single-dose liver-selective glucocorticoid antagonism, we have determined that the peripheral effect is secondary to the primary event of reduced hepatic glucose production. The approach of inhibiting the hepatic GR may be an advantageous treatment paradigm for individuals with type 2 diabetes mellitus.

An assay using scintillation proximity bead technology has been developed suitable for the quantitation of endothelin (ET) receptor antagonists in preclinical and clinical samples of plasma. The assay measures the competitive inhibition of radiolabelled ET-1 binding to ET(A) receptor membranes bound to wheat germ agglutinin (WGA)-coated scintillation proximity assay (SPA) beads in the presence of plasma containing A-127722, a potent orally active, ET(A) selective ET antagonist. The assay requires as little as 50 microl plasma and no extraction procedure is needed. The SPA methodology eliminates the need for the separation of bound from free ligand. Using this method, A-127722 could be directly quantified in rat plasma with a detection limit of 1 ng/ml.

Tactile allodynia, the enhanced perception of pain in response to normally non-painful stimulation, represents a common complication of diabetic neuropathy. The activation of endothelin ET(A) receptors has been implicated in diabetes-induced reductions in peripheral neurovascularization and concomitant endoneurial hypoxia. Endothelin receptor activation has also been shown to alter the peripheral and central processing of nociceptive information. The present study was conducted to evaluate the antinociceptive effects of the novel endothelin ET(A) receptor-selective antagonist, 2R-(4-methoxyphenyl)-4S-(1,3-benzodioxol-5-yl)-1-(N, N-di(n-butyl)aminocarbonyl-methyl)-pyrrolidine-3R-carboxylic acid (ABT-627), in the streptozotocin-induced diabetic rat model of neuropathic pain. Rats were injected with 75 mg/kg streptozotocin (i. p.), and drug effects were assessed 8-12 weeks following streptozotocin treatment to allow for stabilization of blood glucose levels (>/=240 mg/dl) and tactile allodynia thresholds (

Endothelins, ET-1, ET-2, and ET-3 are potent vasoconstricting and mitogenic 21-amino acid bicyclic peptides, which exert their effects upon binding to the ET A and ET B receptors. The ET A receptor mediates vasoconstriction and smooth muscle cell proliferation, and the ET B receptor mediates different effects in different tissues, including nitric oxide release from endothelial cells, and vasoconstriction in certain vascular cell types. Selective antagonists of endothelin receptor subtypes may prove useful in determining the role of endothelin in various tissue types and disease states, and hence as therapeutic agents for such diseases. The pyrrolidine carboxylic acid A-127722 has been disclosed as a potent and ET A -selective antagonist, and is currently undergoing clinical trials. In our efforts to find antagonists with altered selectivity (ET A -selective, ET B -selective, or nonselective), we investigated the SAR of the 2-substituent on the pyrrolidine. Compounds with alkyl groups at the 2-position possessed ET A selectivity improved over A-127722 (1400-fold selective), with the best of these compounds showing nearly 19,000-fold selectivity.

As a pharmacological class, Endothelin-A receptor (ET A ) antagonists are highly bound (> 98%) to serum albumin. In the presence of physiological concentrations of albumin, their affinities for ET A decrease 10 to 100 fold. We have prepared ET A antagonists which exhibit lower degrees of binding to albumin, while maintaining potency and selectivity for the ET A receptor. The protein induced IC 50 shift is reduced or eliminated in this new series of compounds. The compounds also display altered in vivo and pharmacokinetic profiles which may be consistent with their lower degree of protein binding.

The N-terminal region of skeletal myosin light chain-1 (MLC-1) binds to the C terminus of actin, yet the functional significance of this interaction is unclear. We studied a fragment (MLC-pep; residues 5-14) of the ventricular MLC-1. When added to rat cardiac myofibrils, 10 nM MLC-pep induced a supramaximal increase in the MgATPase activity at submaximal Ca2+ levels with no effect at low and maximal Ca2+ levels. A nonsense, scrambled sequence peptide had no effect at any pCa value. MLC-pep did not affect myosin KEDTA and CaATPase activities or actin-activated MgATPase activities in the absence or presence of tropomyosin. The MLC-pep did not alter the ability of troponin I to inhibit MgATPase activity. Moreover, when troponin I and troponin C were extracted from the myofibrils, the MLC-pep lost its ability to stimulate the ATPase rate. This effect was fully restored upon reconstitution of the extracted myofibrils with troponin I-troponin C complex. Thus, activation of MgATPase activity by the peptide required a full complement of thin filament regulatory proteins. Interestingly, the stimulatory effect occurred at a ratio of 4 peptides to 1 thin filament, suggesting that the peptide engages in a highly cooperative process that may involve activation of the entire thin filament.

The effect of ETA receptor blockade on the bronchopulmonary response to endothelin-1 was determined in the airway of the anesthetized, spontaneously breathing guinea pig. Endothelin-1 administered as an aerosol increased lung resistance and decreased dynamic lung compliance. Delivery of the ETA receptor antagonist, FR139317, 5 min prior to giving endothelin-1 greatly potentiated these changes. A lower dose of endothelin-1 that had no effect on resistance or compliance produced large and significant changes when pretreated with FR139317. In contrast, aerosolized FR139317 had no effect on the bronchopulmonary response to intravenously administered endothelin-1. These data suggest a non-contractile function of ETA receptors accessible from the airways that serve to buffer the constrictor effects of non-ETA receptors.

The endothelin receptor subtypes involved in the vasoconstriction, capsular smooth muscle contraction, prostaglandin E2 and prostacyclin release induced by endothelin-1 have been investigated in the canine isolated perfused spleen using both the endothelin ETA receptor antagonist FR 139317 and the endothelin ETB receptor agonist IRL 1620. The isolated canine spleen was perfused with warmed (37°C) and oxygenated (95% O2/5% CO2) Krebs solution at constant flow with continuous recording of splenic arterial perfusion pressure and spleen weight. Samples of splenic venous effluent were collected to determine the amounts of prostaglandin E2 and prostacyclin, measured by radioimmunoassay. Endothelin-1 (4–200 pmol) and IRL 1620 (20–1000 pmol) dose-dependently increased splenic arterial perfusion pressure but the former was more potent on a molar basis (the molar dose ratio IRL/endothelin-1 required to increase splenic arterial vascular resistance by 25% was approximately 33). The infusion of the nitric oxide inibitor Nω-nitro- l -arginine methyl ester (10 μM), but not of the enantiomer Nω-nitro- l -arginine methyl ester, significantly potentiated the increase in splenic arterial vascular resistance induced by endothelin-1. The infusion of FR 139317 (1 μM) markedly attenuated the increased splenic arterial perfusion pressure induced by endothelin-1 without affecting that evoked by IRL 1620. At the highest dose (200 pmol), endothelin-1 induced a small but significant capsule contraction as reflected by the reduction in the spleen weight. The infusion of FR 139317 (1 μM) abolished this contractile effect. IRL 1620 (in doses up to 1000 pmol) did not significantly affect the capsule tone. The administration of either endothelin-1 (20–200 pmol) or IRL 1620 (20–1000 pmol) caused the release of 6-oxo-prostaglandin F1α (breakdown product of prostacyclin) and prostaglandin E2 into the splenic venous effluent. The amount of both prostanoids released by endothelin-1 was significantly greater than that induced by IRL 1620. FR 139317 (1 μM) significantly reduced (P

A series of 5-membered ring heterocyclic analogs of A-81988 were synthesized in order to determine their activity as angiotensin II antagonists. The activity of these compounds in a rabbit aorta in vitro assay ranged from pA2 values of

Fused ring heterocyclic analogs of A-81080 (pA2= 9.9) were synthesized and their activities in the rabbit aorta in vitro assay were measued. The best compounds (pA2 = 8.6) in series 1 had R1 = Et, R2 = H, W = X = Z = C-H, and Y = C-OMe or C-COOH. In series 2, the best compound (pA2 = 8.3) had R1 = Et, R2 = H, W = N-Me, X = C-H, and Y = N.

Experiments were conducted to characterize the nature of a phosphoramidon-sensitive endothelin-converting enzyme in vivo by evaluating the pressor response to a bolus intravenous (i.v.) injection of endothelin family peptides following administration of phosphoramidon in anesthetized Sprague-Dawley rats. Phosphoramidon given i.v. at 10 mg/kg completely prevented the pressor response to human big endothelin-1-(1-38) (big ET-1). The EC50 for phosphoramidon was determined to be in the range of 1 to 3 mg/kg. The pressor response to big ET-1 60 min after phosphoramidon injection was attenuated by roughly 60% indicating a long inhibitory half-life. Very high doses of big ET-1 (> 20 mg/kg) were capable of over-riding the effect of phosphoramidon and produced characteristic pressor responses suggesting that the inhibition by phosphoramidon can be considered competitive in nature. Human big endothelin-3-(1-41) (big ET-3) produced significant increases in arterial pressure although with less potency and efficacy compared to big ET-1. The pressor response to big ET-3 was also inhibited by phosphoramidon. Phosphoramidon does not act indirectly by interfering with ET-1 receptor-mediated actions since the inhibitor has no effect on the in vivo pressor response to ET-1 and does not antagonize [125I]ET-1 receptor binding or constrictor responses in vitro. These results are consistent with the idea that a phosphoramidon-sensitive endothelin-converting enzyme is capable of cleaving both big ET-1 and big ET-3 to the active peptides in the rat.

Experiments were conducted to determine the importance of the carbohydrate moiety of phosphoramidon in the inhibition of the pressor response to big endothelin-1 in anesthetized rats. Big endothelin-1 produced a 42% increase in mean arterial pressure which was nearly abolished by co-infusion of phosphoramidon. Similarly, when an analog of phosphoramidon lacking the rhamnose group (phosphoryl-leu-trp-OH) was co-infused, a significant attenuation of the pressor response was observed. These findings indicate that the rhamnose moiety of phosphoramidon is not responsible for distinguishing this compound as an inhibitor of the response to big endothelin-1 in the rat.

We compared the hemodynamic effects of continuous i.v. infusion of endothelin-1 and big endothelin-1 in anesthetized rats. Big endothelin-1 was fivefold less potent than endothelin-1 in decreasing cardiac output, heart rate, and stroke volume. However, big endothelin-1 produced a significantly larger increase in mean arterial pressure compared to endothelin-1 at doses that produced identical decreases in cardiac output. These findings support the hypothesis that the hypertensive effects of big endothelin-1 and endothelin-1 are produced by differential effects on systemic vascular resistances.

Endothelin-1 (ET-1) is reported to be the most powerful constrictor of blood vessels known. Atrial natriuretic factor is a potent relaxor of contracted vessels. This study examined the potential interaction between these vasoactive peptides on rabbit aortic rings. ET-1 contracted the rings in a dose-dependent manner with an ec50 (-log M) of 9.78 ± 0.16. Atriopeptin III (APIII) completely relaxed ET-1 (10−9 M)-contracted rings with an EC50 of 9.63 ± 0.07 and methoxamine contracted rings with an EC50 of 9.18 ± 0.09. Pretreatment of aortic rings with APIII (up to 3 × 10−6 M) did not alter the normal ET-1 dose-response curve with respect to potency but did diminish the maximal contraction achieved. An interesting additional finding was that the temporal nature of ET-1-induced contraction is remarkably dose-variable. In conclusion, the potent contractile effects of ET-1 on vascular smooth muscle can be effectively reversed but not prevented by APIII.

An enzyme activity which converts human big endothelin (1-38) to endothelin (1-21) and a C-terminal fragment (CTF, 22-38) was identified in a plasma membrane fraction prepared from rat lung. The conversion activity was optimal at pH 4.0, was inhibited by Pepstatin-A (IC50 = 20 nM), but was not affected by TLCK, Aprotinin, PMSF, E-64, Bestatin, Phosphoramidon or Thiorphan at 40 microM. Metal ions activated the activity by 1.5 - 2.5 fold in the order of Mn+2 greater than Zn+2 = Ca+2 greater than Ba+2. These data suggest that a Pepstatin-A inhibitable, metal ion related aspartic protease may be involved in the conversion of big endothelin to endothelin in rat lung.

This study evaluated the effects of rat ANP(5–28) infusion into the blood-perfused dog gracilis muscle at concentrations ranging from 30 to 10,000 pg/ml. The vasculature of gracilis muscles from anesthetized beagle dogs was isolated and pump-perfused at constant flow with blood utilizing an extracorporeal circuit. Maximal vasodilatory capacity was determined by adenosine injection. ANP was infused into the arterial circuit to produce increasing arterial blood concentrations. Each infusion lasted 10 min. Systemic arterial pressure, central venous pressure, cardiac output and heart rate did not change during ANP infusion into the gracilis vasculature. ANP at arterial blood concentrations up to 10,000 pg/ml did not produce significant vasodilation although the vasculature showed pronounced vasodilation in response to adenosine. In vitro experiments showed that ANP had much less vasorelaxant activity in dog femoral artery and saphenous vein than in rabbit aorta. Therefore, rat ANP(5–28) at concentrations within and well above physiological and pharmacological ranges does not inhibit the basal vascular tone present in the innervated, blood-perfused dog gracilis muscle in situ .