Arylpropanolamines: selective beta3 agonists arising from strategies to mitigate phase I metabolic transformations.

Utilization of N-substituted-4-hydroxy-3-methylsulfonanilidoethanolamines 1 as selective beta(3) agonists is complicated by their propensity to undergo metabolic oxidative N-dealkylation, generating 0.01-2% of a very potent alpha(1) adrenergic agonist 2. A summary of the SAR for this hepatic microsomal conversion precedes presentation of strategies to maintain the advantages of chemotype 1 while mitigating the consequences of N-dealkylation. This effort led to the identification of 4-hydroxy-3-methylsulfonanilidopropanolamines 15 for which the SAR for the unique stereochemical requirements for binding to the beta adrenergic receptors culminated in the identification of the potent, selective beta(3) agonist 15f.

BMS-201620: a selective beta 3 agonist.

A series of N-(4-hydroxy-3-methylsulfonanilidoethanol)arylglycinamides were prepared and evaluated for their human beta3 adrenergic receptor agonist activity. SAR studies led to the identification of BMS-201620 (39), a potent beta3 full agonist (Ki = 93 nM, 93% activation). Based on its favorable safety profile, BMS-201620 was chosen for clinical evaluation.

Beta 3 agonists. Part 1: evolution from inception to BMS-194449.

Screening of the BMS collection identified 4-hydroxy-3-methylsulfonanilidoethanolamines as full beta 3 agonists. Substitution of the ethanolamine nitrogen with a benzyl group bearing a para hydrogen bond acceptor promoted beta(3) selectivity. SAR elucidation established that highly selective beta(3) agonists were generated upon substitution of C(alpha) with either benzyl to form (R)-1,2-diarylethylamines or with aryl to generate 1,1-diarylmethylamines. This latter subset yielded a clinical candidate, BMS-194449 (35).(1)

BMS-196085: a potent and selective full agonist of the human beta(3) adrenergic receptor.

A series of 4-hydroxy-3-methylsulfonanilido-1,2-diarylethylamines were prepared and evaluated for their human beta(3) adrenergic receptor agonist activity. SAR studies led to the identification of BMS-196085 (25), a potent beta(3) full agonist (K(i)=21 nM, 95% activation) with partial agonist (45%) activity at the beta(1) receptor. Based on its desirable in vitro and in vivo properties, BMS-196085 was chosen for clinical evaluation.

Differential effects of pravastatin, simvastatin, and atorvastatin on Ca2+ release and vascular reactivity.

The direct effects of the cholesterol-lowering agents, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG CoA) reductase inhibitors, on vascular smooth muscle responsiveness were examined by incubation of isolated aorta from normocholesterolemic rats with simvastatin, atorvastatin, or pravastatin. The smooth muscle contractions caused by phenylephrine were progressively inhibited with increasing concentrations of simvastatin. Similarly, atorvastatin at the higher concentration caused decreased responses to phenylephrine. In contrast, incubation with pravastatin had no significant effect at all concentrations studied. In Ca2+-free buffer, the transient contraction caused by phenylephrine, which results from intracellular release of Ca2+, also was inhibited by simvastatin and atorvastatin but not by pravastatin. In cultured rat aortic smooth muscle cells loaded with fura-2, increases in intracellular free-Ca2+ concentration ([Ca2+]i) induced by angiotensin II were markedly inhibited in cells incubated with simvastatin and atorvastatin but not pravastatin. The inhibitory effects of simvastatin and atorvastatin were reversed by mevalonate. These findings demonstrate that inhibition of HMG CoA reductase by using simvastatin and atorvastatin, but not pravastatin, has effects on vascular smooth muscle cell responsiveness that involve alteration of Ca2+ homeostasis through a mevalonate-dependent pathway.

A one-million-year-old Homo cranium from the Danakil (Afar) Depression of Eritrea.

One of the most contentious topics in the study of human evolution is that of the time, place and mode of origin of Homo sapiens. The discovery in the Northern Danakil (Afar) Depression, Eritrea, of a well-preserved Homo cranium with a mixture of characters typical of H. erectus and H. sapiens contributes significantly to this debate. The cranium was found in a succession of fluvio-deltaic and lacustrine deposits and is associated with a rich mammalian fauna of early to early-middle Pleistocene age. A magnetostratigraphic survey indicates two reversed and two normal magnetozones. The layer in which the cranium was found is near the top of the lower normal magnetozone, which is identified as the Jaramillo subchron. Consequently, the human remains can be dated at approximately 1 million years before present.

Quantitation of tremor in response to beta-adrenergic receptor stimulation in primates: relationship with hypokalemia.

The primary adverse effect of stimulation of beta2-adrenergic receptors is elicitation of tremor. Tremor measurements in response to beta2-adrenergic receptor stimulation were performed in a quantitative manner using a modified miniature semiconductor accelerometer in African green monkeys. The accelerometer was taped to the middle finger tip of anesthetized monkeys, and recordings of onset, duration and peak tremor responses were obtained. The selective beta2-adrenergic agonist, salbutamol (0.5 mg/kg i.v.), caused a marked increase in tremor which started within 5 min following injection and lasted for approximately 60 min. The finger tremor response was not visible, but was measurable by the accelerometer, and the increase in tremor was significantly greater from baseline within 10 min. Plasma K+ concentrations were markedly decreased within the first 15 min and remained at low steady-state concentrations during the 60-min recordings. The tremor response was abolished by the selective beta2-adrenergic receptor antagonist, ICI-118551 (0.2 mg/kg). ICI-118551 caused a significant reversal of the plasma K+ decrease but the K+ levels remained higher than control levels. These studies demonstrate that stimulation of beta2-adrenergic receptors causes tremor, most likely from entry of K+ into skeletal muscle and that there is a direct correlation between tremor and hypokalemic response.

Dissociation of endothelial cell dysfunction and blood pressure in SHR.

This study was designed to examine the impairment of endothelium-dependent relaxation in spontaneously hypertensive rats (SHR), to determine whether endothelial cell function is normalized by in vivo treatment with a thromboxane A2-prostaglandin endoperoxide (TP)-receptor blocker, and to establish whether endothelial dysfunction contributes to the elevated blood pressure. In isolated aortic rings from SHR, endothelium-dependent relaxations caused by acetylcholine, adenosine diphosphate, and alpha-thrombin were markedly impaired compared with those from Wistar-Kyoto (WKY) normotensive rats. Arachidonic acid-induced contractions were significantly enhanced in aorta from SHR. In contrast, relaxations caused by direct smooth muscle vasodilators, nitroprusside and cromakalim, and contractions caused by U-46619 were not different between SHR and WKY rats. Treatment of SHR with the oral TP-receptor antagonist, ifetroban, at 20 and 50 mg.kg-1.day-1 fully restored endothelium-dependent relaxation toward normal. However, ifetroban produced no effect on blood pressure in SHR. In vitro incubation of aortic rings from SHR with ifetroban also normalized relaxations to acetylcholine but had no effect in aorta from WKY. In contrast, the thromboxane A synthase inhibitor, dazoxiben, only partially improved abnormal acetylcholine-induced relaxations in aorta from SHR. The results demonstrate that endothelial cell dysfunction in hypertension can be restored to normal by selective TP-receptor blockade. Furthermore, endothelial cell dysfunction and TP-receptor activation may not significantly contribute to elevated systemic blood pressure in SHR.

Bradykinin B2 receptor-mediated chronotropic effect of bradykinin in isolated guinea pig atria.

The present study was undertaken to characterize the direct chronotropic effect of bradykinin in isolated spontaneously beating atria of the guinea pig. Bradykinin caused concentration-dependent increases in the beating rate of atria. In contrast, the active metabolite of bradykinin and the typical bradykinin B1 receptor agonist, Des-Arg9-bradykinin, had no effect on the beating rate of atria. Inhibition of converting enzyme or neutral endopeptidase by captopril or SQ-28603, respectively, did not affect beating rate but potentiated bradykinin-induced increase in beating rate. The potent bradykinin B2 receptor antagonist, HOE 140, antagonized bradykinin-induced chronotropic effect. In contrast, the bradykinin B1 receptor antagonist, Lys-[Leu8]Des-Arg9-bradykinin, had no effect. The increase in beating rate caused by bradykinin was not affected by blockade of beta 1-adrenoceptors, cyclooxygenase, or nitric oxide synthesis using atenolol, indomethacin and N omega-nitro-L-arginine, respectively. Unlike bradykinin, angiotensin I and angiotensin II caused very small or no change in beating rate in the presence or absence of captopril and SQ-28603. These results indicate that bradykinin causes a direct positive chronotropic effect which is mediated by activation of bradykinin B2 receptors independently of prostaglandins and beta 1-adrenoceptors.

15-Hydroxyeicosatetraenoic acid and diabetic endothelial dysfunction in rabbit aorta.

We examined the effects of diabetes on eicosanoid metabolism and endothelium-dependent relaxation in isolated aorta from alloxan-induced diabetic rabbits and that from normal rabbits incubated in increased concentrations (44 mM) of glucose in vitro for 6 h. Immunoreactive 15-hydroxyeicosatetraenoic acid (HETE) was assayed in the incubation media of isolated aortic segments. Basal and acetylcholine (ACh)-stimulated release of 15-HETE was significantly greater in aorta of diabetic animals as compared with those of normal rabbits. Incubation of aortic segments from normal rabbits in increased concentrations of glucose caused a significant increase in basal and ACh-stimulated release of 15-HETE; and the release was significantly greater in aortic segments with endothelium than in segments without endothelium. Basal and ACh-stimulated release of 15-HETE was inhibited by indomethacin, a cyclooxygenase inhibitor. 15-HETE caused contractions of aortic rings that were inhibited by the prostaglandin H2 (PGH2) thromboxane A2 (TXA2) receptor blocker SQ-29548, but not by the TXA2 synthase inhibitor carbethoxyhexyl imidazole or indomethacin. Treatment of aortic rings with subthreshold concentrations of 15-HETE impaired ACh-induced relaxation; this was prevented by treatment with SQ-29548. Thus, abnormal release of endothelium-derived 15-HETE may play a role in endothelial cell dysfunction and increased vasoconstriction in diabetes by a mechanism that involves interaction with PGH2/TXA2 receptors.

Enhanced adrenergic neurotransmission in diabetic rabbit carotid artery.

OBJECTIVE: The aim was to examine the effects of diabetes mellitus on adrenergic neurotransmission and smooth muscle responsiveness in the densely innervated carotid artery from six-week alloxan diabetic rabbits. METHODS: Rings of carotid arteries were isolated from normal and diabetic rabbits and isometric tension was measured in response to stimulation of adrenergic nerves, alpha adrenoceptors, and activation by calcium. RESULTS: Basal content and stimulated overflow of endogenous noradrenaline were reduced by approximately 25% in arteries from diabetic as compared to normal rabbits. In contrast, responses to endogenous noradrenaline released from adrenergic nerves by electrical stimulation or tyramine displacement were not different between arteries from normal and diabetic groups. Neuronal uptake blockade using cocaine caused a significantly smaller leftward shift in the contractions produced by electrical stimulation and exogenously applied noradrenaline in arteries from diabetic rabbits. The tonic, but not phasic, contractions caused by phenylephrine were larger in arteries from diabetic rabbits. Calcium-induced contractions caused by readdition of calcium to a calcium-free medium containing potassium (15 mmol.litre-1) were also significantly larger in arteries from diabetic rabbits. BAY K 8644, a calcium channel activator, caused an increase in calcium induced contractions and abolished the difference between the two groups. CONCLUSIONS: Although neurogenic contractions of diabetic carotid artery are normal, there is inefficient or reduced neuronal uptake as well as increased activity of calcium channels in the smooth muscle which increase contractions to alpha adrenoceptor agonists.

Diabetes and vascular disease: functional alterations in adrenergic neurotransmission and endothelium.

Diabetes is characterized by hyperglycemia, a relative lack of insulin, and an inclination to vascular disease and neuropathy. The link between diabetes and vascular disease is not understood, but autonomic dysfunction could partly account for alterations in reactivity of diabetic blood vessels to neurotransmitters and circulating hormones. Changes in local control of vascular tone, such as imbalance in production of relaxing and contracting factors by the endothelium, may be related to the initiation and maintenance of abnormal vascular reactivity characteristically seen in diabetic vascular complications. The emphasis is to discuss functional changes of blood vessel adrenergic neuroeffector mechanisms and endothelial cell dysfunction, together with the complex interrelationship of cyclooxygenase catalysis, protein kinase C activity, sodium-potassium ATPase activity, and flux through the polyol pathway. This review focuses on the common mechanisms by which hyperglycemia causes changes in vascular function.

Thrombin receptor-mediated vascular relaxation differentiated by a receptor antagonist and desensitization.

The vasorelaxant actions of the serine protease, alpha-thrombin, are selectively blocked by the thrombin active site inhibitors, suggesting that proteolytic cleavage is required for alpha-thrombin-induced release of nitric oxide. Whether these relaxations are caused by interaction with a thrombin receptor was evaluated using a prototype thrombin receptor antagonist, a decapeptide analogue of the tethered ligand thrombin receptor [3-mercapto-propionyl-Phe-Cha-Cha-Arg-Lys-Pro-Asn-Asp-Lys-amide (c186-65)]. In rings of pig coronary arteries with endothelium contracted submaximally with U-46619, the relaxation caused by extremely low concentrations of alpha-thrombin were mimicked by the synthetic thrombin receptor-activating peptide (TRAP-7: SFLLRNP). These relaxations were inhibited by C186-65. In contrast, C186-65 had no effect on the relaxations caused by bradykinin and serotonin. Exposure of arteries to alpha-thrombin or TRAP-7 caused heterologous desensitization to subsequent stimulation by alpha-thrombin or TRAP-7 but not by bradykinin. These studies support the hypothesis that alpha-thrombin-induced endothelium-dependent relaxations occur by activation of the cloned "tethered-ligand" thrombin receptor in vascular endothelium.

Beta 1- and beta 2-adrenoceptor antagonist activities of ICI-215001, a putative beta 3-adrenoceptor agonist.

1. The present study was undertaken to characterize the beta 3-adrenoceptor agonist activity of ICI-215001 and to determine whether it exhibits additional activities on beta 1- and beta 2-adrenoceptors in isolated spontaneously beating atrium, trachea and ileum of guinea-pig. 2. In guinea-pig atrium, isoprenaline, a non-selective beta-adrenoceptor agonist, caused concentration-dependent, positive chronotropic effects that were inhibited by atenolol, a selective beta 1-antagonist. ICI-215001 also competitively antagonized the increase in heart rate caused by isoprenaline. 3. ICI-215001 exhibited low intrinsic activity at increasing the beating rate of atrium and no activity on resting or induced tone of tracheal strips. 4. In strips of guinea-pig trachea, contracted submaximally with carbachol, isoprenaline, caused concentration-dependent relaxations. Both ICI-118551, a selective beta 2-adrenoceptor antagonist, and ICI-215001 competitively inhibited the relaxations caused by isoprenaline. 5. In isolated strips of guinea-pig ileum longitudinal smooth muscle contracted with histamine, isoprenaline and ICI-215001 caused relaxations which were inhibited by alprenolol, a beta-adrenoceptor antagonist with modest affinity for beta 3-adrenoceptors, but were resistant to ICI-118551 and atenolol. 6. These results indicate that ICI-215001 exhibits beta 3-adrenoceptor agonist activity as demonstrated by relaxations mediated via atypical beta-adrenoceptors in the longitudinal smooth muscle of guinea-pig ileum. Further, the studies demonstrate that ICI-215001 can act as an antagonist at beta 1- and beta 2-adrenoceptors in situations where its intrinsic agonist activity is low.

Distinct receptors and signaling pathways in alpha-thrombin- and thrombin receptor peptide-induced vascular contractions.

The vasoactive mechanisms of the serine protease alpha-thrombin were examined in isolated coronary arteries from dogs. In resting coronary arteries with endothelium, alpha-thrombin caused concentration-dependent contractions that were characterized by an initial transient relaxation followed by slowly developing sustained contractions. The vascular actions of alpha-thrombin were mimicked by the thrombin receptor-activating peptide (TRAP) SFLLRNP, a synthetic peptide based on the cleaved terminus of the thrombin receptor domain. Treatment of the arteries with N omega-nitro-L-arginine or removal of endothelium abolished the transient relaxations and enhanced the contractions, indicating that the transient relaxations were mediated by the concurrent release of endothelium-derived nitric oxide. alpha-Thrombin that had been catalytically inactivated with the irreversible inhibitor by use of D-Phe-Pro-Arg-chloromethyl ketone did not cause contractions, indicating the requirement of proteolytic cleavage by alpha-thrombin to induce contractions. In contrast to TRAP, alpha-thrombin-induced contractions were blocked by hirudin (a specific thrombin inhibitor), nifedipine and diltiazem (Ca2+ channel blockers), or staurosporine and calphostin C (protein kinase C inhibitors). Unlike alpha-thrombin, which undergoes homologous desensitization, TRAP failed to cause desensitization to subsequent stimulation by alpha-thrombin or TRAP. These observations support the hypothesis that vasoactive actions of alpha-thrombin are mediated by a mechanism that involves cleavage at the active site to expose a new NH2 terminus that activates the thrombin receptor. Further, the dissociation between alpha-thrombin and the synthetic receptor peptide in signal transduction and dissimilar desensitizing properties suggest the existence of distinct thrombin receptor subtypes and/or signaling events in vascular smooth muscle.

Free radicals in diabetic endothelial cell dysfunction.

Several studies have shown impairment of endothelium-dependent relaxations as well as increased release of vasoconstrictor prostanoids in arteries from diabetic animals and humans. This impairment is restored towards normal by prostaglandin (PG) H2/thromboxane A2 receptor blockade or superoxide dismutase, indicating that the PGH2 and/or superoxide anion (O2-.) generated contributes to the abnormality. Of particular note is that PGH2 impairs endothelium-dependent relaxations and causes contractions by a mechanism that involves generation of O2-. in the endothelium. The effects of elevated glucose are exacerbated by increased aldose reductase activity leading to depletion of NADPH and generation of reactive oxidants. Because NADPH is required for generation of nitric oxide from L-arginine, the depletion of NADPH leads to reduced nitric oxide formation. In a manner similar to that observed with elevated glucose, oxygen-derived free radicals or activation of protein kinase C also cause impairment of endothelium-dependent relaxations, smooth muscle contractions, and release constrictor prostanoids, indicating that a common mechanism for the impairment of endothelial cell function may be operative in diabetes. In this review the cumulative effects of oxidative stress on diabetic endothelial cell dysfunction, together with the complex interrelationship of cyclooxygenase catalysis, protein kinase C activity, and flux through the polyol pathway, are considered.

Selective impairment of endothelium-dependent relaxations by prostaglandin endoperoxide.

OBJECTIVE: Studies of aortas from hypertensive and diabetic rats and rabbits have demonstrated impairment of endothelium-dependent relaxations, which were associated with increased release of endothelium-derived thromboxane A2 (TXA2). This implicates enhanced release of TXA2 or its precursor prostanoid, prostaglandin endoperoxide (PGH2), or both, as factors mediating the endothelial cell dysfunction. METHODS: The interaction of vasoconstrictor prostanoids (PGH2, PGF2 alpha and U-46619, a stable thromboxane-receptor agonist) and oxygen-derived free radicals with the release of nitric oxide was examined in isolated aortas from Sprague-Dawley rats. RESULTS: Exogenously applied PGH2 or U-46619 caused concentration-dependent contractions of aortic rings, these contractions being blocked by the newly developed, potent and selective PGH2- and TXA2-receptor antagonist BMS-180291, but not by inhibition of TXA synthase or cyclo-oxygenase (using dazoxiben and indomethacin, respectively). In aortic rings contracted submaximally with phenylephrine, brief exposure to a subthreshold concentration of PGH2 caused impairment of acetylcholine- and ADP-induced, but not of nitroprusside-induced, relaxations. The impairment was restored towards normal by BMS-180291 or by superoxide dismutase (SOD), a superoxide anion scavenger, but not by dazoxiben or indomethacin. In contrast, treatment of aortic rings with U-46619 or PGF2 alpha did not impair the relaxations. Oxygen-derived free radicals generated by xanthine oxidase caused contractions and impaired acetylcholine relaxations which were reversed by SOD but not by BMS-180291. CONCLUSIONS: These findings indicate that activation of PGH2 receptors causes contractions and selective impairment of endothelium-dependent relaxations by a mechanism involving generation of oxygen-derived free radicals in the endothelium.

Involvement of the "tethered ligand" receptor in thrombin-induced endothelium-mediated relaxations.

The mechanisms by which the serine protease, alpha-thrombin, mediates relaxations were examined in isolated dog and pig coronary arteries and dog saphenous veins. In rings of coronary arteries and saphenous veins contracted submaximally with prostaglandin F2 alpha or U46619, alpha-thrombin (0.1-10 nM) caused relaxations that were abolished by treatment with N omega-nitro-L-arginine (L-NNA) or removal of the endothelium, indicating that the relaxations were mediated by endothelium-derived nitric oxide. These relaxations were blocked by the thrombin active site inhibitor, MD-805, indicating the requirement of thrombin's catalytic site to induce the relaxations. The thrombin exosite inhibitor, BMS-180742, decreased the sensitivity to alpha-thrombin without altering maximal relaxations. Indomethacin, a cyclooxygenase inhibitor, had no inhibitory effect on the relaxations caused by alpha-thrombin, indicating that the relaxations were not mediated by cyclooxygenase products. Similar to alpha-thrombin, the thrombin receptor activating peptide (human sequence: SFLLRNP, 1-100 microM) caused relaxations in pig coronary artery and dog saphenous vein but not in dog coronary artery. These relaxations were blocked by L-NNA but not by indomethacin. The results indicate that alpha-thrombin induces endothelium-dependent relaxations by a novel signaling mechanism that involves proteolytic cleavage of the thrombin receptor to expose a new amino terminus that functions as a "tethered peptide ligand" to activate thrombin receptors on the endothelial cells and release nitric oxide.

Reduced Na(+)-K+ pump activity in diabetic rabbit carotid artery: reversal by aldose reductase inhibition.

This study addresses the question of whether a decrease in basal Na+ pump [Na(+)-K(+)-adenosinetriphosphatase (ATPase)] activity occurs in the carotid artery of an alloxan-diabetic rabbit and, if so, whether it is associated with altered 86Rb+ uptake and contractile response to ouabain and K(+)-free solution. Ouabain-sensitive 86Rb+ uptake, an index of Na+ pump activity, was diminished approximately 50% in carotid arteries from diabetic rabbits. Concurrent with this, contractions induced by incubating the carotid arteries in a K(+)-free solution (in the absence of phentolamine) were significantly larger in the diabetic group. Readdition of K+ (1 mM) to arteries contracted with the K(+)-free solution caused relaxations that were slower to occur and of lesser magnitude in diabetic than in control rabbits. In contrast to the contractions caused by the K(+)-free medium, contractions caused by incubation with ouabain (1 mM) in the presence of phentolamine were significantly smaller in the diabetic group. Treatment of diabetic rabbits with an aldose reductase inhibitor, zopolrestat, at both high and low doses restored the alterations in vascular reactivity toward normal. The results indicate that the Na+ pump activity is diminished in the carotid artery of diabetic rabbit, and this is associated with abnormal vascular responsiveness and increased polyol pathway flux.

Aldose reductase inhibition restores endothelial cell function in diabetic rabbit aorta.

A possible relationship between increased aldose reductase activity and abnormal endothelium-dependent relaxation was examined in aorta from alloxan-induced diabetic rabbits. Isolated aorta of diabetic rabbits, contracted submaximally with phenylephrine, showed significantly decreased endothelium-dependent relaxations induced by acetylcholine or adenosine diphosphate compared to those from normal rabbits. Basal and acetylcholine-stimulated levels of cyclic GMP and the relaxations in response to an endothelium-independent vasodilator, sodium nitroprusside, were not significantly different between diabetic and normal rabbits, indicating that nitric oxide release and action on the vascular smooth muscle were unchanged. The release of thromboxane A2 from diabetic vessels was increased, as previously demonstrated. Treatment with an aldose reductase inhibitor, zopolrestat, normalized the elevated red blood cell sorbitol levels in diabetic rabbits. Zopolrestat also restored the abnormal acetylcholine- and adenosine diphosphate-induced relaxations of the aorta. The aldose reductase inhibitor had no effect on the levels of cyclic GMP or on the increased release of thromboxane A2 in diabetic aorta. These findings suggest that increased activity of the aldose reductase pathway in hyperglycemia is responsible for the abnormal endothelium-dependent relaxation in diabetic blood vessels. Significant alterations in endothelial production of neither nitric oxide nor vasoconstrictor prostanoids could be directly implicated in the improvement caused by the drug, suggesting another mechanism of action.