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Plasticity of phenotypic traits refers to an organism's ability to change in response to environmental stimuli. As a result, the response may alter an organism's physiological state, morphology, behavior, and phenotype. Phenotypic plasticity in cancer cells describes the considerable ability of cancer cells to transform phenotypes through non-genetic molecular signaling activities that promote therapy evasion and tumor metastasis via amplifying cancer heterogeneity. As a result of metastable phenotypic state transitions, cancer cells can tolerate chemotherapy or develop transient adaptive resistance. Therefore, new findings have paved the road in identifying factors and agents that inhibit or suppress phenotypic plasticity. It has also investigated novel multitargeted agents that may promise new effective strategies in cancer treatment. Despite the efficiency of conventional chemotherapeutic agents, drug toxicity, development of resistance, and high-cost limit their use in cancer therapy. Recent research has shown that small molecules derived from natural sources are capable of suppressing cancer by focusing on the plasticity of phenotypic responses. This systematic, comprehensive, and critical review analyzes the current state of knowledge regarding the ability of phytocompounds to target phenotypic plasticity at both preclinical and clinical levels. Current challenges/pitfalls, limitations, and future perspectives are also discussed., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Humans have a remarkable ability to predict the actions of others. To address what information enables this prediction and how the information is modulated by social context, we used videos collected during an interactive reaching game. Two participants (an "initiator" and a "responder") sat on either side of a plexiglass screen on which two targets were affixed. The initiator was directed to tap one of the two targets, and the responder had to either beat the initiator to the target (competition) or arrive at the same time (cooperation). In a psychophysics experiment, new observers predicted the direction of the initiators' reach from brief clips, which were clipped relative to when the initiator began reaching. A machine learning classifier performed the same task. Both humans and the classifier were able to determine the direction of movement before the finger lift-off in both social conditions. Further, using an information mapping technique, the relevant information was found to be distributed throughout the body of the initiator in both social conditions. Our results indicate that we reveal our intentions during cooperation, in which communicating the future course of actions is beneficial, and also during competition despite the social motivation to reveal less information.

Mineralocorticoid receptor (MR) antagonism has proven to effectively attenuate the pathophysiological effects of aldosterone in clinical and experimental settings of hypertension and heart failure. MR activates transcription of target genes upon aldosterone binding, and eplerenone selectively binds to MR and blocks aldosterone- mediated activation. In this review, we summarize the preclinical and clinical evidence supporting the beneficial effects of eplerenone (INSPRA), a selective aldosterone blocker, in the treatment of hypertension and heart failure. We also review the current status in understanding the molecular mechanisms of action of the MR and its ligand. In addition, we compare the effects of eplerenone and spironolactone, a nonselective aldosterone blocker, on the transcriptional activity of MR and provide a molecular explanation for the improved side-effect profile of eplerenone compared with spironolactone.

The classical mineralocorticoid effect of aldosterone on unidirectional transepithelial sodium transport in the kidney was long thought to be the predominant effect of this hormone. However, there is convincing evidence for additional extrarenal actions of aldosterone that are mediated via activation of mineralocorticoid receptors (MRs) in the heart, vasculature and brain. It is now postulated that many of the detrimental effects of aldosterone are mediated through MR activation in these nonclassical target organs. The selective aldosterone blocker, eplerenone (Inspra), is under development for human therapeutic use for treatment of hypertension and heart failure post-myocardial infarction (MI). Clinical and preclinical studies have linked elevated aldosterone to hypertension, left ventricular and vascular remodeling, cardiac, renal, and cerebral vascular inflammation and injury, and increased risk of mortality in heart failure patients. Multiple studies in experimental models of hypertension and heart failure demonstrate that selective blockade of aldosterone by eplerenone effectively preserves cardiac function, attenuates maladaptive left ventricular remodeling and tissue and vascular injury in part by reducing vascular inflammation in aldosterone target organs.

Since the isolation and purification of aldosterone from adrenal extracts 50 years ago (Experientia 9 (1953) 33), scientists have learned a great deal about how and where aldosterone acts, the factors that control its release, what is its role in the pathophysiology of cardiovascular disease, how to make and study aldosterone antagonists, and for what medical purposes these agents are useful. In this paper, we will discuss the evolution of aldosterone antagonists from the relatively nonselective spironolactone (Aldactone), to the highly selective eplerenone (Inspra). Eplerenone represents a molecule with improved steroid receptor selectivity and pharmacokinetic properties in man compared to spironolactone. Recent clinical results have demonstrated that these improvements translate into tolerability and efficacy in patients with cardiovascular disease.

Aldosterone classically promotes unidirectional transepithelial sodium transport, thereby regulating blood volume and blood pressure. Recently, both clinical and experimental studies have suggested additional, direct roles for aldosterone in the cardiovascular system. To evaluate aldosterone activation of cardiomyocyte mineralocorticoid receptors, transgenic mice overexpressing 11beta-hydroxysteroid dehydrogenase type 2 in cardiomyocytes were generated using the mouse alpha-myosin heavy chain promoter. This enzyme converts glucocorticoids to receptor-inactive metabolites, allowing aldosterone occupancy of cardiomyocyte mineralocorticoid receptors. Transgenic mice were normotensive but spontaneously developed cardiac hypertrophy, fibrosis, and heart failure and died prematurely on a normal salt diet. Eplerenone, a selective aldosterone blocker, ameliorated this phenotype. These studies confirm the deleterious consequences of inappropriate activation of cardiomyocyte mineralocorticoid receptors by aldosterone and reveal a tonic inhibitory role of glucocorticoids in preventing such outcomes under physiological conditions. In addition, these data support the hypothesis that aldosterone blockade may provide additional therapeutic benefit in the treatment of heart failure.

Background: We evaluated the role of aldosterone as a mediator of renal inflammation and fibrosis in a rat model of aldosterone/salt hypertension using the selective aldosterone blocker, eplerenone., Methods: Unnephrectomized, Sprague-Dawley rats were given 1% NaCl (salt) to drink and randomized to receive treatment for 28 days: vehicle infusion (control); 0.75 microg/hour aldosterone subcutaneous infusion; or aldosterone infusion + 100 mg/kg/day oral dose of eplerenone. Blood pressure and urinary albumin were measured and kidneys were evaluated histologically. Renal injury, inflammation, and fibrosis were assessed by immunohistochemistry, in situ hybridization, and reverse transcription-polymerase chain reaction (RT-PCR)., Results: Aldosterone/salt induced severe hypertension compared to controls (220 +/- 4 mm Hg vs. 131 +/- 4 mm Hg, P < 0.05), which was partially attenuated by eplerenone (179 +/- 4 mm Hg, P < 0.05). In aldosterone/salt treated rats, renal histopathologic evaluation revealed severe vascular and glomerular sclerosis, fibrinoid necrosis and thrombosis, interstitial leukocyte infiltration, and tubular damage and regeneration. Aldosterone/salt increased circulating osteopontin (925.0 +/- 80.2 ng/mL vs. 53.6 +/- 6.3 ng/mL) and albuminuria (75.8 +/- 10.9 mg/24 hours vs. 13.2 +/- 3.0 mg/24 hours) compared to controls and increased expression of proinflammatory molecules. Treatment with eplerenone reduced systemic osteopontin (58.3 +/- 4.2 ng/mL), albuminuria (41.5 +/- 7.2 mg/24 hours), and proinflammatory gene expression: osteopontin (OPN), monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), and interleukin-1beta (IL-1beta)., Conclusion: These findings indicate that aldosterone/salt-induced renal injury and fibrosis has inflammatory components involving macrophage infiltration and cytokine up-regulation. Attenuation of renal damage and inflammation by eplerenone supports the protective effects of aldosterone blockade in hypertensive renal disease.

Activation of the renin-angiotensin-aldosterone system is associated with unsatisfactory outcomes in patients with hypertension and heart failure in that activation of this system is correlated strongly with both the incidence and extent of end-organ damage. Despite the availability of the angiotensin converting enzyme inhibitors (ACEi) and the angiotensin receptor blockers (ARB), unblocked aldosterone levels remain an important risk factor for cardiovascular disease progression. New preclinical data generated over the last few years strongly supports the hypothesis that aldosterone has important deleterious effects on the cardiovascular system independent of the classical action of this hormone on renal epithelial cells. The new selective aldosterone blocker, eplerenone, has been shown to produce significant cardioprotective and renoprotecive effects in experimental models of cardiovascular disease. Early clinical studies suggests that eplerenone may have important therapeutic benefit in the treatment of hypertension and heart failure post-myocardial infarction (post-MI).

Background: In heart failure (HF), aldosterone has been implicated in the formation of reactive interstitial fibrosis, a maladaptation that contributes to left ventricular (LV) remodeling. Eplerenone is a novel selective aldosterone blocker. The present study examined the effects of long-term monotherapy with eplerenone on the progression of LV dysfunction and remodeling in dogs with chronic HF., Methods and Results: HF was produced in 14 dogs by intracoronary microembolizations that were discontinued when LV ejection fraction (EF) was between 30% and 40%. Two weeks after the last embolization, dogs were randomized to 3 months of oral therapy with eplerenone (10 mg/kg twice daily, n=7) or no therapy at all (control, n=7). Hemodynamic measurements were made just before randomization and were repeated at the end of 3 months of therapy. In control dogs, LV end-diastolic and end-systolic volume increased significantly (62+/-4 versus 68+/-4 mL, P<0.001, and 38+/-3 versus 47+/-3 mL, P<0.001, respectively), and EF decreased significantly (38+/-1% versus 31+/-2%, P<0.001). In contrast, end-diastolic volume, end-systolic volume, and EF remained unchanged during the 3 months of treatment in eplerenone-treated dogs. LV end-diastolic wall stress increased significantly in control dogs but decreased significantly in eplerenone-treated dogs. Compared with control, eplerenone was associated with a 28% reduction in cardiomyocyte cross-sectional area, a 37% reduction of volume fraction of reactive interstitial fibrosis, and a 34% reduction of volume fraction of replacement fibrosis., Conclusions: Our results indicate that long-term therapy with eplerenone prevents progressive LV dysfunction and attenuates LV remodeling in dogs with chronic HF.

Heart failure is a complex multifactorial disease resulting in a myriad of progressive changes at the molecular, cellular, and physiological level. To better understand the mechanisms associated with the development of congestive heart failure, a comprehensive examination of the aging lean male spontaneously hypertensive, heart failure-prone rat (SHHF) was conducted. Myocardial function and structural integrity progressively diminished as evidenced by decreased ejection fraction and increased left ventricular volume measured using echocardiography. Functional and structural changes were accompanied by elevations in circulating inflammatory markers, including tumor necrosis factor-alpha (TNF-alpha), IL-6, and TNF receptors type 1 and 2. Increased systemic inflammatory marker levels were consistent with age-dependent changes in the expression pattern of genes that contribute to stress, inflammation, and the extracellular matrix in SHHF animals analyzed from age 4 to 18 mo. In summary, the SHHF rat shares many hallmark features of the human disease state and represents a key experimental model for the dissection of complex human heart failure pathophysiology.

Vascular inflammation was examined as a potential mechanism of aldosterone-mediated myocardial injury in uninephrectomized rats receiving 1% NaCl-0.3% KCl to drink for 1, 2, or 4 wk and 1) vehicle, 2) aldosterone infusion (0.75 microg/h), or 3) aldosterone infusion (0.75 microg/h) plus the selective aldosterone blocker eplerenone (100 mg. kg(-1). day(-1)). Aldosterone induced severe hypertension at 4 wk [systolic blood pressure (SBP), 210 +/- 3 mmHg vs. vehicle, 131 +/- 2 mmHg, P < 0.001], which was partially attenuated by eplerenone (SBP, 180 +/- 7 mmHg; P < 0.001 vs. aldosterone alone and vehicle). No significant increases in myocardial interstitial collagen fraction or hydroxyproline concentration were detected throughout the study. However, histopathological analysis of the heart revealed severe coronary inflammatory lesions, which were characterized by monocyte/macrophage infiltration and resulted in focal ischemic and necrotic changes. The histological evidence of coronary lesions was preceded by and associated with the elevation of cyclooxygenase-2 (up to approximately 4-fold), macrophage chemoattractant protein-1 (up to approximately 4-fold), and osteopontin (up to approximately 13-fold) mRNA expression. Eplerenone attenuated proinflammatory molecule expression in the rat heart and subsequent vascular and myocardial damage. Thus aldosterone and salt treatment in uninephrectomized rats led to severe hypertension and the development of a vascular inflammatory phenotype in the heart, which may represent one mechanism by which aldosterone contributes to myocardial disease.

Although multiple advancements have been made in the treatment of heart failure (HF), mortality rates remain alarmingly high. The accepted arsenal of therapeutics includes a diuretic, digitalis, a beta-blocking agent and an inhibitor of the renin-angiotensin-aldosterone system. Despite the employment of a vast array of agents, nearly 300,000 patients in the US die annually with HF as a primary or contributory cause of death. Additional molecular targets are being evaluated in preclinical and clinical settings including vasopeptidase inhibitors, endothelin-1 receptor antagonists, arginine vasopressin antagonists, selective aldosterone blockers, TNF-alpha blockers and matrix metalloproteinase inhibitors. Although these approaches hold promise as viable therapeutics, a thorough evaluation of clinical benefit from these agents requires additional trials. Future disease-modifying approaches will also undoubtedly include cell transplantation and gene therapy. It is likely that notable advances in HF treatment will come from agents that attenuate myocardial remodelling. Indeed, maintenance or improvement of cardiac structure can attenuate HF development and improve mortality.

Activation of the renin-angiotensin-aldosterone system is associated with unsatisfactory outcomes in patients with hypertension and congestive heart failure, in that activation of this system is correlated strongly with both the incidence and extent of end-organ damage. Despite the availability of the angiotensin-converting enzyme inhibitors and the AT1 receptor antagonists, unblocked aldosterone levels remain an important risk factor for cardiovascular disease progression. New preclinical data generated over the past few years strongly support the hypothesis that aldosterone has important deleterious effects on the cardiovascular system independent of the classical action of this hormone on renal epithelial cells. The new selective aldosterone receptor antagonist eplerenone has been shown to produce significant cardioprotective effects in experimental models of cardiovascular disease. Early clinical testing suggests that eplerenone may have important therapeutic benefit in the treatment of hypertension and heart failure.

Insulin resistance can be induced in vivo by intravenous infusion of glucosamine or in cells by incubation with glucosamine. However, a publication (Hresko, R. C., et al. (1998) J. Biol. Chem. 273, 20658-20668) suggests a trivial explanation of glucosamine-induced insulin resistance whereby intracellular ATP pools are depleted presumably due to the phosphorylation of glucosamine to glucosamine 6-phosphate, a hexosamine pathway intermediate. The reduced ATP level impaired insulin receptor (IR) autophosphorylation and tyrosine kinase activity toward substrates. The present work describes the development and comparison of two methods for inducing insulin resistance, by treating 3T3-L1 adipocytes overnight using either 25 mM glucose/5 nM insulin or 2 mM glucosamine. Under these conditions basal glucose transport rates were comparable with controls. Insulin-stimulated 2-deoxyglucose uptake, however, was reduced by approximately 45% in response to both high glucose/insulin and glucosamine treatment, relative to control cells. The total relative amounts of the insulin-responsive glucose transporter, Glut4, remained constant under both treatment conditions. The relative phosphotyrosine (Tyr(P)) contents of the insulin receptor and its substrate 1 (IRS-1) were assessed in whole cell homogenates. With both methods to induce insulin resistance, IR/IRS-1 Tyr(P) levels were virtually indistinguishable from those in control cells. Insulin-stimulated phosphorylation of Akt on Ser(473) was not impaired in insulin-resistant cells. Furthermore, the relative Tyr(P) content of the PDGF receptor was comparable in high glucose/insulin- or glucosamine-treated 3T3-L1 adipocytes upon subsequent challenge with PDGF. Finally, the relative amounts of glutamine:fructose-6-phosphate amidotransferase and O-linked N-acetylglucosamine transferase, two important hexosamine pathway enzymes, were similar in both treatments when compared with controls. Thus, 3T3-L1 adipocytes can be used as a model system for studying insulin resistance induced by increased influx of glucose. Under appropriate experimental conditions, glucosamine treatment can mimic the effects of increased glucose flux without impairment of tyrosine phosphorylation-based signaling., (Copyright 2000 Academic Press.)

Lithium has been shown to increase glucose uptake in skeletal muscle and adipose tissues. The therapeutic effect of lithium on bipolar disease is thought to be mediated by its inhibitory effect on myo-inositol-1-monophosphatase (IMPase). We tested the hypothesis that the stimulatory effect of lithium on glucose uptake results from inhibition of IMPase and the resultant accumulation of inositol monophosphates (IP1) by comparing the effects of lithium and a selective IMPase inhibitor, L-690,488, on isolated rat adipocytes. Insulin produced a concentration-dependent stimulation of 2-deoxy-D-[14C]glucose (2-DG) transport (10 microU/ml caused half-maximal activation). Acute exposure to lithium stimulated basal glucose transport activity in a concentration-dependent manner, with a threefold stimulation at 30 mM lithium. Lithium also potentiated insulin-stimulated 2-DG transport. Lithium produced a concomitant increase in IP1 accumulation. In contrast, L-690,488 increased IP1 to levels comparable to those of lithium without stimulatory effects on 2-DG transport. These results demonstrate that stimulatory effects of lithium on glucose transport are not mediated by the inhibition of IMPase and subsequent accumulation of IP1 in rat adipocytes.

Oxygen radical generation can be measured when blood flow is restored to previously ischemic tissue. Although several studies have suggested oxygen radicals contribute to lethal injury of myocardium after ischemia, other studies have failed to confirm this implication. Antioxidants, such as N-(2-mercaptoptopionyl)-glycine (MPG) and superoxide dismutase, have had inconsistent effects on lethal myocardial injury in animal models of ischemia and reperfusion. Many variables influence lethal myocardial injury in these models: time of ischemia, time of reperfusion, dose of antioxidant, myocardial oxygen demand, area at risk, collateral blood flow, and body core temperature. The purpose of this study is to test the effects of infusion of MPG on lethal reperfusion injury in a canine model of ischemia and reperfusion with these variables tightly controlled. The left anterior descending coronary artery of anesthetized dogs was ligated for 90 minutes and reperfused for 4 hours. MPG was infused (100 mg/kg/h) 15 minutes before the end of ischemia and throughout reperfusion. Core body temperature was closely monitored, and infarct size was adjusted to transmural myocardial blood flow during ischemia. MPG had no effect on infarct size or infarct size adjusted for changes in collateral blood flow. These data reinforce a general difficulty in demonstrating the effects of antioxidant therapies on lethal injury, even when closely monitoring covariates known to impact infarct size.

Enhanced glucose flux via the hexosamine biosynthetic pathway has been implicated in insulin resistance. We measured products of this pathway, UDP-N-acetyl hexosamines (UDP-HexNAc), and activity of the rate-limiting enzyme L-glutamine:D-fructose-6-phosphate amidotransferase (GFAT) in tissues of ob/ob mice and lean controls. Ob/ob mice were obese, hyperglycemic, and hyperinsulinemic. Resistance to the effect of insulin on glucose transport was demonstrated in isolated soleus muscles, although total GLUT-4 concentration was mildly increased in muscles from ob/ob mice. UDP-HexNAc concentrations in hindlimb muscles decreased between 8 and 17 wk but were always higher in ob/ob vs. controls (P < 0.001, mean increase 67%). Concentrations of UDP-hexoses and GDP-mannose were similar in ob/ob and control muscles. Muscle GFAT activity declined with age but was increased in ob/ob vs. controls at each age examined (P < 0.001, mean increase 108%). UDP-HexNAc concentrations and GFAT activity were similar in livers of ob/ob and controls. These data suggest that glucose flux via the hexosamine pathway is selectively increased in muscle but not liver of ob/ob mice and may contribute to muscle insulin resistance in this model of non-insulin-dependent diabetes mellitus.

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 AT1 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.

Prolongation of action potential duration (APD) and the effective refractory period (ERP) is one mechanism to prevent reentrant atrial and ventricular arrhythmias. Because arrhythmias are usually associated with an elevated sympathetic tone and increase of circulating catecholamines, the potential influence of catecholamines on antiarrhythmic effects of an agent are critical to predicting the potential clinical response. In this study the effects of three different antiarrhythmic agents, bidisomide, flecainide and dofetillide, each of which prolongs atrial ERP, were compared before and after treatment with isoproterenol, a beta-adrenergic agonist. Standard intracellular microelectrode recording techniques were used to record action potentials from isolated canine atrial tissue. Bidisomide and flecainide elicited a 20 to 27 msec increase in APD and ERP that was independent of stimulation frequency (1-5 Hz). Dofetilide prolonged APD and ERP at 1 Hz (40 and 37 msec, respectively) but was completely ineffective at 5 Hz. After equilibration with the anti-arrhythmic agent, tissues were additionally exposed to isoproterenol. Only bidisomide prolonged APD and ERP in the presence of isoproterenol. In the converse series of experiments after treatment with isoproterenol, which caused a 20 to 30% reduction in APD and ERP, only bidisomide completely reversed the effect of 1 microM isoproterenol. Bidisomide was inactive in a functional beta-adrenergic antagonism assay, thus ruling out beta-adrenergic blockade as a potential mechanism. These results indicate that bidisomide, unlike flecainide and dofetilide was able to prolong APD and ERP in isolated canine atrium even at high stimulation frequencies and in the presence of isoproterenol. These data suggest that bidisomide would be effective in the presence of elevated sympathetic tone and on the agents studied, only bidisomide possessed a unique and desirable antiarrhythmic profile.

SC-56525 is a nanomolar inhibitor of plasma renin activity in human, cynomolgus monkey, dog, guinea pig, Yucatan micropig, and rabbit but is less active in rat. The oral bioavailability of SC-56525 in conscious dogs at doses of 5 mg/kg IV and 30 mg/kg PO was 66.1 +/- 16.4%. Oral dosing with SC-56525 at 3, 10, and 30 mg/kg in salt-depleted dogs induced a dose-dependent reduction in mean arterial pressure and inhibition of plasma renin activity with no significant effect on heart rate. In two-kidney, one clip renal hypertensive dogs, SC-56525 given orally at 10, 30, and 60 mg/kg daily for 4 days lowered blood pressure significantly. In conscious dogs monitored in their home cages via radiotelemetry, no significant changes in heart rate occurred in response to large drops in blood pressure in both renal hypertensive and salt-depleted dogs with the renin inhibitor SC-56525. SC-56525 is a nanomolar, orally active inhibitor of renin and effectively lowers blood pressure in both salt-depleted and renal hypertensive dogs.

SC-54628 [1-(2-methylphenyl)-4-butyl-1,3-dihydro-3-[[6-[2-(1H- tetrazol-5-yl)phenyl]-3-pyridinyl]methyl]-2H-imidazol-2-one] and its 1-(2,6-dimethylphenyl)-2H-imidazol-2-one derivative SC-54629 were potent inhibitors of 125I-angiotensin II (125I-AII) binding to rat adrenal cortex angiotensin type 1 (AT1) receptors. SC-54628 and SC-54629 antagonized AII-induced contraction of rabbit vascular smooth muscle in a surmountable fashion and an insurmountable fashion, respectively. Binding experiments with SC-54629 were undertaken to determine the nature of receptor interaction, which might explain the insurmountable mode of antagonism of SC-54629. The presence of a high concentration of SC-54629 did not affect the dissociation of membrane-bound 125I-AII induced by an excess of unlabeled AII, indicating that the antagonist binds to the agonist binding site and not an allosteric domain. Incubation of adrenal cortex membranes with SC-54629 decreased the density of 125I-AII binding sites. When incubation of the SC-54629-treated membranes with radiolabeled AII was prolonged, the SC-54629-induced decrease in AT1 receptor density was attenuated, suggesting that binding of the antagonist is slowly reversible. Furthermore, the dissociation of [3H]SC-54629 was 5-fold slower than that of 125I-AII bound to AT1 receptors. These results suggest that the insurmountable antagonism of AII by SC-54629 is most likely due to the slow reversibility of SC-54629 binding to the AT1 receptor.

Infusion of radiocontrast agents in vivo results in renal artery constriction and subsequent renal hypoperfusion. To examine the role of endothelin and of prostaglandins in radiocontrast-mediated renal vasoconstriction, rats were treated with an endothelin receptor antagonist, CP170687, and with indomethacin. The dose of CP170687 utilized was sufficient to reverse endothelin1-mediated constriction of isolated aortic rings and of renal blood flow in intact rats. In normal rats there was a transient drop in renal blood flow to 80% of baseline following sodium iothalamate injection, an effect which was not prevented by CP170687. In rats first given indomethacin, the drop in renal blood flow was more pronounced (to 63% of baseline) and was sustained. In this instance, CP170687 fully reversed the sustained decrease of renal perfusion. CP170687 also diminished the rise in systemic blood pressure seen following iothalamate injection. In the absence of indomethacin, iothalamate increased urinary prostaglandin E2 to a maximum of sevenfold above baseline values. In summary, injection of radiocontrast results in an immediate decrease in renal blood flow that is counteracted by an increase in renal prostaglandin formation. When prostaglandin synthesis is inhibited, prolonged endothelin-mediated renal vasoconstriction is observed that is reversed by an endothelin receptor antagonist.

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.

We reported previously that the endothelin converting enzyme (ECE) inhibitor phosphoramidon lowers mean arterial pressure (MAP) when infused in conscious, spontaneously hypertensive rats (SHRs). In this study we determined the dose-response relationship for this action in SHRs and in a high-renin hypertensive model, the renal artery-ligated rat. We also determined whether the ETA receptor antagonist BQ-123 (cyclo [D-Trp-D-Asp-Pro-D-Val-Leu]) might lower MAP in hypertensive rats. Phosphoramidon lowered MAP by 9 +/- 4, 31 +/- 4, and 40 +/- 4 mm Hg after 5 h when infused in SHRs at 10, 20, and 40 mg/kg/h. This lowering of MAP was associated with dose-related inhibition of the pressor response to a bolus intravenous injection of big ET (1-39) at 1 nmol/kg. BQ-123 also lowered MAP in SHRs (by 25 +/- 3 mm Hg), but only at a very high dose (50 mg/kg/h for 5 h). At this dose, BQ-123 blocked the pressor response to a bolus intravenous injection of ET-1 (1 nmol/kg), but the blockade was incomplete. Phosphoramidon infused in conscious, renal hypertensive rats lowered MAP by 31 +/- 9, 46 +/- 8, and 54 +/- 1 mm Hg after 5 h at 10, 20, and 40 mg/kg/h, respectively. This lowering of MAP was associated with blockade of the pressor response to big ET (1-39). BQ-123 did not lower MAP in renal hypertensive rats when infused at 30 mg/kg/h for 5 h.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

To investigate the mechanism by which angiotensin-converting enzyme (ACE) inhibition attenuates atherogenesis, we have studied the effects of a non-sulfhydryl ACE inhibitor, enalapril, and an angiotensin receptor antagonist, SC-51316, in cholesterol-fed rabbits. After 3 mo of enalapril treatment (10 mg/kg per d, p.o.) the percent plaque areas in the thoracic aortas of treated animals were significantly reduced (controls: 86.8 +/- 3.5%; treated: 31.1 +/- 8%, P < 0.001). Aortic cholesterol content was also reduced (controls: 31.4 +/- 3.2 mg/g tissue; treated: 7.4 +/- 1.8 mg/g, P < 0.001). Enalapril had no significant effect on plasma lipid levels or conscious blood pressure. In a second study, the angiotensin II receptor antagonist SC-51316 was administered at a dose equivalent to enalapril at blocking angiotensin pressor effects in vivo (30 mg/kg per d, p.o.). Evaluation after 3 mo indicated no significant attenuation of aortic atherosclerosis. These results demonstrate that: (a) enalapril attenuates atherogenesis without affecting either blood pressure or plasma lipid levels; (b) antioxidant activity, found with sulfhydryl-containing ACE inhibitors, is not necessary for reducing plaque formation; and (c) the attenuation of atherogenesis by ACE inhibition may not be due to blockade of the renin-angiotensin system. Alternatively, one must consider the multiple effects of ACE inhibition on other hormone systems, such as bradykinin, or the possibility that alternate angiotensin II receptors may be involved in atherosclerosis.

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.

The present study was designed to determine whether an endothelinA (ETA)-receptor antagonist BQ-123 (cyclo[Dtrp, Dasp, pro-D-Val-Leu]) or an ET-converting enzyme inhibitor phosphoramidon may prevent development of cerebral vasospasm after subarachnoid hemorrhage (SAH). A "double hemorrhage" canine model of the disease was used (n = 17 dogs), and the degree of vasospasm of the basilar artery was assessed by angiography. Mongrel dogs of either sex were divided into three experimental groups: animals treated with daily intracisternal injections of BQ-123 (10(-4) M; n = 6) or phosphoramidon (2 x 10(-4) M; n = 6) and control animals treated with saline solution (n = 5). Diameter of basilar arteries in animals treated with saline solution was reduced by SAH to 56 +/- 7% of control diameter. BQ-123 and phosphoramidon did not significantly affect SAH-induced vasospasm (diameters were 62 +/- 0% and 56 +/- 10% of control diameters for BQ-123 and phosphoramidon, respectively). In contrast, in isolated canine basilar arteries BQ-123 (10(-5) M) selectively inhibited concentration-dependent contractions to ET-1 (10(-11)-3 x 10(-8) M; n = 5). Levels of immunoreactive ET in plasma and cerebrospinal fluid were not affected by development of vasospasm. These results suggest that intracisternal injections of ETA-receptor antagonist or phosphoramidon cannot prevent SAH-induced cerebral vasospasm and that ET-1 may not be the major mediator responsible for the decrease in cerebral arterial diameter associated with SAH.

The properties of a novel nonpeptidic angiotensin II (AII) receptor antagonist, 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), are described. SC-51316 inhibited [125I]AII binding selectively to the AT1 receptor with IC50 values of 3.6 and 5.1 nM in rat adrenal cortical and rat uterine membrane preparations, respectively. The compound was a competitive and reversible antagonist of AII-mediated contraction of rabbit aortic rings with a pA2 of 8.86. In addition, SC-51316 inhibited AII-induced aldosterone release from rat adrenal zona glomerulosa cells and blocked inhibition of renin release by AII from rat kidney slices with pA2 values of 8.62 and 8.9, respectively. The agent (0.1 mM) did not inhibit angiotensin-converting enzyme or plasma renin activity. These data demonstrate that SC-51316 is a potent AII receptor antagonist which may prove to be useful as a pharmacologic tool for studying the role of the renin-angiotensin system in cardiovascular diseases.

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.

The present investigation was undertaken to define the hemodynamic mechanisms by which the selective cyclic GMP phosphodiesterase inhibitor zaprinast (M&B 22,948; 2-o-propoxy-phenyl-8-azapurin-6-one) lowers mean arterial pressure (MAP). Anesthetized rats were instrumented with electromagnetic or pulsed-Doppler flow probes to measure cardiac output or regional blood flow, respectively, and catheters to record MAP, left ventricular pressure and administer drugs. Zaprinast (0.33-2.0 mg.min-1.kg-1) produced dose-dependent decreases in MAP (maximum = -39 +/- 6 mm Hg) and total peripheral resistance (maximum = -45 +/- 5%) when infused i.v. The greatest reductions in regional vascular resistance were observed in the mesenteric bed, with smaller decreases in the hindquarters and renal beds. Cardiac output increased (maximum = 22 +/- 7%) during the infusion of zaprinast; however, heart rate was only minimally affected. These increases in cardiac output were still evident in animals pretreated with beta adrenergic and muscarinic receptor antagonists. At the lowest dose, zaprinast tended to increase maximal first derivative of left ventricular pressure, whereas, at higher doses maximal first derivative of left ventricular pressure was depressed or unchanged. Additional studies were performed to examine the effect of the cyclic GMP phosphodiesterase inhibitor on the pressor and vasoconstrictor activity of phenylephrine and angiotensin II. The increases in MAP and total peripheral resistance produced by these agents were attenuated markedly during continuous infusion of zaprinast (1 mg.min-1.kg-1). These data suggest that zaprinast lowers MAP by decreasing peripheral vascular resistance and by antagonizing the actions of endogenous neurohumoral vasoconstrictor systems.

In porcine aortic endothelial cells, the 21-amino acid peptide endothelin-1 (ET-1) is formed from a 39-amino acid intermediate called "big endothelin-1" (big ET-1) by a putative ET-converting enzyme (ECE) that cleaves the 39-mer at the bond between Trp-21 and Val-22. Since big ET-1 has only 1/100-1/150th the contractile activity of ET-1, inhibition of ECE should effectively block the biological effects of ET-1. Big ET-1 injected intravenously into anesthetized rats produces a sustained pressor response that presumably is due to conversion of big ET-1 into ET-1 by ECE. We determined the type of protease activity responsible for this conversion by evaluating the effectiveness of protease inhibitors in blocking the pressor response to big ET-1 in ganglion-blocked anesthetized rats. The serine protease inhibitor leupeptin, the cysteinyl protease inhibitor E-64, and the metalloprotease inhibitors captopril and kelatorphan were all ineffective at blocking the pressor response to big ET-1. However, the metalloprotease inhibitors phosphoramidon and thiorphan dose-dependently inhibited the pressor response to big ET-1, although phosphoramidon was substantially more potent than thiorphan. None of the inhibitors blocked the pressor response to ET-1 and none had any effect on mean arterial pressure when administered alone. In a rabbit lung membrane preparation, ECE activity was identified that was blocked by the metalloprotease inhibitors phosphoramidon and 1,10-phenanthroline in a concentration-dependent manner. This enzyme converted big ET-1 to a species of ET that comigrated on HPLC with ET-1 and produced an ET-like contraction in isolated rat aortic rings. Our results suggest that the physiologically relevant ECE is a metalloprotease.

In porcine aortic endothelial cells, the 21-amino acid peptide endothelin-1 (ET-1) is formed from a 39-amino acid intermediate big endothelin (big ET) by a putative endothelin-converting enzyme (ECE) that cleaves the 39-mer at the Trp21-Val22 bond. Because big ET has less than 1% of the contractile activity of ET-1, inhibition of ECE should effectively block the biological effects of big ET. Big ET injected intravenously into anesthetized rats produces a sustained pressor response that presumably is due to conversion of big ET to ET-1 by ECE. We determined the type of protease activity responsible for this conversion by evaluating the effectiveness of protease inhibitors in blocking the pressor response to big ET in ganglion-blocked anesthetized rats. The serine protease inhibitor leupeptin, the cysteinyl protease inhibitor E-64, and the metalloprotease inhibitors captopril and kelatorphan were ineffective at blocking the pressor response to big ET. However, the metalloprotease inhibitors phosphoramidon and thiorphan both dose-dependently inhibited the pressor response to big ET, although phosphoramidon was substantially more potent than thiorphan. None of the inhibitors blocked the pressor response to ET-1 and none had any effect on blood pressure when administered alone as an i.v. bolus to the ganglion-blocked anesthetized rat. However, phosphoramidon infused intravenously at 20 mg/kg/h for 4 h lowered the mean arterial pressure (MAP) in conscious spontaneously hypertensive rats (SHRs) whereas kelatorphan at the same dose did not. Our results suggest that ECE is a novel metalloprotease and that ECE inhibitors could have therapeutic potential for the treatment of hypertension.

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, [Hcy3,5]AII, having high contractile activity (pD2 = 8.48 vs 8.81 for AII) and excellent binding affinity (IC50 = 2.1 nM vs 2.2 nM for AII). This type of cyclization was also used to prepare a highly potent AII antagonist, [Sar1,Hcy3,5,Ile8]AII (pA2 = 9.09 vs 9.17 for [Sar1, Ile8]AII; IC50 = 0.9 nM vs 1.9 nM for [Sar1,Ile8]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 gamma-turn. In contrast, the receptor-bound conformation of AII does not appear to accommodate a beta-turn or an alpha-helix which includes residues 3-5.

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)-1H-imidazol-1-yl]methyl)- [1,1'-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 microM. 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 pA2 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.

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 investigated the role of altered vascular calcium handling in the development of aldosterone-salt hypertension in the rat. The calcium sensitivity of isometric force in response to 50 mM KCl was compared in aortic rings from control and aldosterone-hypertensive rats. Over the entire range of calcium concentrations studied, responses in aortas from the hypertensives were significantly depressed compared to controls [ED50: aldosterone-hypertensive rats (n = 6), 0.739 +/- 0.137; controls (n = 7), 0.141 +/- 0.021 mM; P less than 0.001]. However, calcium sensitivity in response to 1 microM norepinephrine was similar in aortas from both hypertensives and controls [ED50: aldosterone-hypertensive rats (n = 7), 0.196 +/- 0.022; controls (n = 7), 0.180 +/- 0.024 mM]. The calcium sensitivity of Triton X-100 skinned aortic rings from aldosterone-hypertensive rats was likewise not significantly different from sensitivity in controls [ED50: aldosterone-hypertensive rats (n = 9), 3.61 X 10(-7) +/- 0.57; controls (n = 8), 3.89 X 10(-7) +/- 0.64 M]. Therefore, the observed decrease in calcium sensitivity in response to membrane depolarization in aortas from aldosterone-hypertensive rats probably is not due to a change in calcium sensitivity of the contractile system itself. The time course for development of changes in calcium handling in vessels from the aldosterone-hypertensive rats was found to be quite different from the time course for changes in monovalent ion metabolism. Whereas increases in monovalent ion permeability reportedly appear as early as one week after the start of aldosterone-salt treatment, significant alterations in calcium handling were not apparent until after four weeks of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Aortas from aldosterone-salt treated hypertensive rats and vehicle-infused normotensive controls were compared with respect to mechanical properties, metabolism, and energy turnover. Passive wall stress at any given circumference was slightly higher in aortas from the hypertensive rats, whereas active isometric force normalized to cross-sectional area was similar in vessels from the two groups at their respective optimal circumference for active tension developments [hypertensive rats: (n = 14), 19.73 +/- 1.98; controls: (n = 13), 22.57 +/- 2.13 mN/mm2). Metabolic parameters were measured with the aortas held at optimal circumference for active tension development, the optimal length for tension development (n = 6), 5.67 +/- 0.17 and (n = 7), 5.29 +/- 0.18 mm for control and aldosterone-salt groups, respectively. Basal oxygen consumption rate was elevated significantly in aortas from the hypertensive rats [(n = 14), 0.457 +/- 0.026 vs. (n = 13), 0.267 +/- 0.028 mumol/min per g; P less than 0.001]. Under resting conditions, lactate production rate similar in aortas from the two groups [hypertensive rats: (n = 20), 0.129 +/- 0.010; controls: (n = 21), 0.112 +/- 0.008 mumol/min per g]. Upon activation with added KCl, the rate of oxygen consumption and lactate production increased with tension development in both groups, but the stimulated rate of oxygen consumption was higher in hypertensives compared to controls [(n = 14), 0.580 +/- 0.031 vs. (n = 13), 0.441 +/- 0.049 mumol/min per g; P less than 0.025]. The stimulated rates of lactate production were similar in the two groups [hypertensive rats: (n = 20), 0.172 +/- 0.009; controls: (n = 21), 0.118 +/- 0.009].(ABSTRACT TRUNCATED AT 250 WORDS)

The vasorelaxant effects of the atriopeptins, AP-(103-126) and AP-(103-123) were compared in human internal mammary artery (IMA) rings which had been contracted with norepinephrine. AP-(103-126) completely relaxed the IMA with an IC50 within the physiologically relevant range (15 nM). However, relaxation with AP-(103-123) was greatly reduced compared to AP-(103-126), with complete relaxation observed in only half of the vessels tested and only at concentrations of 10-50 microM. These results indicate that the atriopeptin (AP) receptor in human arteries may be more similar to the AP receptor in rabbit rather than rat arteries.

We investigated whether big endothelin (porcine 1-40) had contractile activity in isolated rat aorta or pressor activity when injected intravenously into the anesthetized rat. When isolated rat aorta was exposed to a 100 nM concentration of big endothelin, 4.8% of a maximal KCl contraction was observed, compared to 131% of KClmax when paired aortic rings were exposed to an equivalent concentration of synthetic endothelin. Likewise, big endothelin had very weak pressor activity when injected intravenously into anesthetized, ganglion-blocked rats at 10 nmol/kg. When big endothelin was incubated with chymotrypsin, native endothelin and other peptide fragments were formed. Chymotrypsin-treated big endothelin produced an endothelin-like contraction when applied to isolated rat aortic rings, and a characteristic endothelin-like effect on blood pressure in vivo. Our results indicate that the biological activity of endothelin could be effectively blocked by inhibiting the enzyme which converts big endothelin to endothelin.

Chronic infusion of d-aldosterone into uninephrectomized rats on high sodium intake resulted in a significant increase in systolic blood pressure. The steady state efflux of 36Cl in aorta and femoral artery of hypertensive rats was significantly elevated compared to that in arteries of control rats. These elevations in chloride efflux persisted in the presence of the Cl-HCO3 exchange inhibitor, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), and in Cl-HCO3 free solution. The Cl-HCO3-dependent component of the 36Cl efflux was also higher under some conditions. DIDS (100 mumol/l), also significantly reduced the increase in 36Cl efflux caused by norepinephrine (NE) in one of four groups, but had no significant effect on the NE stimulated 42K efflux. Contractile responses to both NE and KCl were unaffected by DIDS treatment. Therefore, it appears that Ca influx and release mechanisms function normally in the presence of DIDS. Similarly, DIDS had either a small or no effect on 42K movements under basal conditions. A slight reduction in the passive efflux of 24Na was observed in aortic smooth muscle exposed to DIDS with no significant effect on active Na transport. These findings indicate that DIDS is reasonably selective for Cl exchange sites in vascular smooth muscle during an exposure of 15 min or less and has little effect on cationic transport processes. The application of this blocker as well as anion substitution indicates that an elevation in the 36Cl leak efflux and related Cl permeability of arterial smooth muscle is associated with aldosterone-salt hypertension.

Studies have been conducted to determine which, if any, of the parameters governing active Na-K transport (K and/or ouabain sensitive) are altered in vascular smooth muscle during mineralocorticoid-salt hypertension. Rats with one kidney removed were treated with either aldosterone or deoxycorticosterone acetate plus saline for 3-4 wk. Ion transport was measured in arteries incubated in a physiological salt solution for periods of 4-6 h. Increased active Na efflux was observed in femoral arteries and aortas from the hypertensive group. This alteration resulted primarily from an elevation in the saturation or maximal capacity of the active transport mechanism, which operated with no significant change in cell Na concentration. The transport parameters related to ion selectivity, cooperativity, and temperature dependence were not significantly altered. Measures of active Na efflux and K influx in the same aortic strips indicated that both fluxes were elevated in the hypertensive group. The ratio of Na to K for active transport was significantly greater than one but was unchanged in the hypertensive rats. These studies provide evidence for the electrogenic operation of the Na-K pump in arterial smooth muscle and for the operation of the pump at a higher level during mineralocorticoid-salt hypertension. This alteration may result from increased turnover of individual sites or possibly the incorporation of more sites into the membrane as a result of mineralocorticoid-salt treatment.

We examined the effects of an acute infusion of M&B 22,948 (2-o-propoxyphenyl-8-azapurin-6-one), a (cGMP)-selective phosphodiesterase inhibitor, on mean arterial pressure (MAP) and urinary sodium excretion in anesthetized rats. M&B 22,948 (at doses of 0.34-2.72 mg/kg/min for 30 min) lowered MAP in a dose-dependent manner, with a 60 mm Hg fall in pressure produced at the highest dose. Despite large decreases in MAP, a profound natriuresis was observed at all doses. Plasma concentrations of cGMP increased in parallel with the depressor action of M&B 22,948, whereas increases in the urinary excretion of cGMP temporally correlated with the natriuresis. The concentration of cyclic AMP in plasma increased transiently in rats treated with M&B 22,948 but the urinary excretion of cyclic AMP was not elevated in these animals. Because changes in cGMP correlated with the physiological effects of M&B 22,948, and the increase in cyclic AMP did not, it is likely that the depressor and natriuretic actions of M&B 22,948 are mediated by increases in cGMP. M&B 22,948 administered chronically at an oral dose of 200 mg/kg/day normalized MAP in spontaneously hypertensive rats; whereas MAP in vehicle-treated spontaneously hypertensive rats remained at hypertensive levels. cGMP-selective phosphodiesterase inhibitors (like M&B 22,948) could be more effective antihypertensive drugs than currently available vasodilators because, when administered acutely, M&B 22,948 simultaneously lowers blood pressure and promotes sodium excretion in the anesthetized rat.

Atrial muscles of the heart are known to produce polypeptide hormones called atrial natriuretic factors (ANF) which have potent diuretic and hypotensive action. These hormones are synthesized as a larger protein precursor called pro atrial natriuretic factor or proANF which contains the biologically active ANF sequences at its C-terminus. Rat proANF (representing amino acids -1 to 128 of the coding sequence) was expressed in a soluble form in Escherichia coli. A simple purification procedure was developed which consists of boiling E. coli cell extracts in 1 M acetic acid and subjecting the supernatant to reversed-phase HPLC. The effect of intravenous administration of the purified recombinant proANF on mean arterial blood pressure was examined. The displacement dose-response curves obtained demonstrated that proANF exhibits similar, albeit less potent, physiological activity than ANF.

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.

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 present study was undertaken to determine the extent to which cyclic nucleotide-induced relaxation in the intact rat aorta is mediated at the level of the contractile system. The relaxant effects of the cyclic nucleotide analogues [8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) and dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP)] and forskolin were examined in both the intact vessel and a Triton X-100-skinned preparation of rat thoracic aorta. Relaxation of a norepinephrine-induced contraction was essentially complete 30 min after the addition of 50 microM 8-BrcGMP [% relaxation = 87.2 +/- 4.4% (n = 4)], 100 microM DBcAMP [98.2 +/- 1.2% (n = 4)], and 1 microM forskolin [107.0 +/- 3.3% (n = 5)]. These same doses were ineffective in relaxing precontracted skinned rat aortic rings compared with the relaxation achieved in the intact vessel. The largest relaxation in the skinned aortas was achieved with the addition of 1 microM forskolin [17.4 +/- 1.5% (n = 4)]. The addition of catalytic subunit of cAMP-dependent protein kinase had no effect on isometric tension in the precontracted skinned aorta. Preincubation with the cyclic nucleotide analogues or forskolin in a low-Ca2+ solution (pCa less than 8) was also ineffective in inhibiting subsequent isometric tension development. Our results suggest that only a very small fraction of the relaxation with cyclic nucleotides and forskolin in the intact rat aorta is due to the action of these agents at the level of the contractile system.