Palosuran inhibits binding to primate UT receptors in cell membranes but demonstrates differential activity in intact cells and vascular tissues.

BACKGROUND AND PURPOSE: The recent development of the UT ligand palosuran (1-[2-(4-benzyl-4-hydroxy-piperidin-1-yl)-ethyl]-3-(2-methyl-quinolin-4-yl)-urea sulphate salt) has led to the proposition that urotensin-II (U-II) plays a significant pathological role in acute and chronic renal injury in the rat. EXPERIMENTAL APPROACH: In the present study, the pharmacological properties of palosuran were investigated further using a series of radioligand binding and functional bioassays. KEY RESULTS: Palosuran functioned as a 'primate-selective' UT ligand in recombinant cell membranes (monkey and human UT K(i) values of 4 +/- 1 and 5 +/- 1 nM), lacking appreciable affinity at other mammalian UT isoforms (rodent and feline K(i) values >1 microM). Paradoxically, however, palosuran lost significant (10- to 54-fold) affinity for native and recombinant human UT when radioligand binding was performed in intact cells (K(i) values of 50 +/- 3 and 276 +/- 67 nM). In accordance, palosuran also exhibited diminished activity in hUT (human urotensin-II receptor)-CHO (Chinese hamster ovary) cells (IC50 323 +/- 67 nM) and isolated arteries (K(b)>10 microM in rat aorta; K(b)>8.5 microM in cat arteries; K(b)>1.6 microM in monkey arteries; K(b) 2.2 +/- 0.6 microM in hUT transgenic mouse aorta). Relative to recombinant binding K(i) values, palosuran was subjected to a 392- to 690-fold reduction in functional activity in monkey isolated arteries. Such phenomena were peculiar to palosuran and were not apparent with an alternative chemotype, SB-657510 (2-bromo-N-[4-chloro-3-((R)-1-methyl-pyrrolidin-3-yloxy)-phenyl]-4,5-dimethoxybenzenesulphonamide HCl). CONCLUSIONS AND IMPLICATIONS: Collectively, such findings suggest that caution should be taken when interpreting data generated using palosuran. The loss of UT affinity/activity observed in intact cells and tissues cf. membranes offers a potential explanation for the disappointing clinical efficacy reported with palosuran in diabetic nephropathy patients. As such, the (patho)physiological significance of U-II in diabetic renal dysfunction remains uncertain.

Expression of monocyte chemotactic protein-3 mRNA in rat vascular smooth muscle cells and in carotid artery after balloon angioplasty.

Monocyte chemotactic protein-3 (MCP-3) is a CC chemokine that functions in chemoattraction and activation of monocytes, T lymphocytes, eosinophils, basophils, natural killer cells and dendritic cells. The activation of the target cells by MCP-3 is via specific chemokine receptors CCR2 and CCR3, of which CCR2 is shared with MCP-1. MCP-1 and CCR2 have been implicated in vascular diseases including atherosclerosis and restenosis, that are known to be involved in inflammation (accumulation of T lymphocytes and monocytes) and smooth muscle cell (SMC) activation (proliferation, migration and matrix deposition). To investigate a potential role of MCP-3 in vascular injury, the present work examined its mRNA expression in rat aortic SMCs stimulated with various inflammatory stimuli including LPS, TNF-alpha, IL-1beta, IFN-gamma and TGF-beta. A time- and concentration-dependant induction of MCP-3 mRNA in SMCs was observed by means of Northern analysis. A strikingly similar expression profile was observed for MCP-3 and MCP-1 mRNA in SMCs. Furthermore, MCP-3 mRNA expression was induced in rat carotid artery after balloon angioplasty. A significant induction in MCP-3 mRNA was observed in the carotid artery at 6 h (41-fold increase over control, P<0.001), 1 day (13-fold increase, P<0.001) and 3 days (6-fold increase, P<0.01) after balloon angioplasty as quantitated by reverse transcription and polymerase chain reaction. These data provide evidence for the cytokine-induced expression of MCP-3 in SMCs and in carotid artery after balloon angioplasty, suggesting a potential role of MCP-3 in the pathogenesis of restenosis and atherosclerosis.

Selective estrogen receptor modulator idoxifene inhibits smooth muscle cell proliferation, enhances reendothelialization, and inhibits neointimal formation in vivo after vascular injury.

BACKGROUND: Idoxifene (ID) is a tissue-selective estrogen receptor modulator (SERM). The pharmacological profile of ID in animal studies suggests that it behaves like an estrogen receptor (ER) agonist in bone and lipid metabolism while having negligible ER activity on the reproductive system. It is unknown whether ID retains the vascular protective effects of estrogen. METHODS AND RESULTS: In cultured vascular smooth muscle cells (VSMCs), ID inhibited platelet-derived growth factor-induced DNA synthesis and mitogenesis with IC(50) values of 20.4 and 27.5 nmol/L, respectively. Treatment with ID resulted in S-phase cell cycle arrest in serum-stimulated VSMCs. ID 1 to 100 nmol/L significantly protected endothelial cells from tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis in vitro. Virgin Sprague-Dawley rats ovariectomized 1 week before the study were treated with ID (1 mg x kg(-1) x d(-1)) or vehicle by gavage for 3 days before balloon denudation in carotid artery. The SMC proliferation in injured vessels was determined by immunostaining for proliferating cell nuclear antigen (PCNA). The number of PCNA-positive SMCs was reduced by 69%, 82%, and 86% in the media at days 1, 3 and 7, respectively, and by 78% in the neointima at day 7 after injury in ID- versus vehicle-treated group (P:<0.01). ID significantly enhanced reendothelialization in the injured carotid arteries as determined by Evans blue stain and immunohistochemical analysis for von Willebrand factor. In the former assay, the reendothelialized area in injured vessels was 43% in ID-treated group versus 24% in the vehicle group (P:<0.05); in the latter assay, the numbers of von Willebrand factor-positive cells per cross section increased from 24. 8 (vehicle) to 60.5 (ID) (P:<0.01) at day 14 after injury. In addition, the production of nitric oxide from excised carotid arteries was significantly higher in ID-treated than the vehicle group (8.5 versus 2.7 nmol/g, P:<0.01). Finally, ID treatment reduced neointimal area and the ratio of intima to media by 45% and 40%, respectively (P:<0.01), at day 14 after balloon angioplasty. CONCLUSIONS: The results indicate that ID beneficially modulates the balloon denudation-induced vascular injury response. Inhibition of VSMC proliferation and acceleration of endothelial recovery likely mediate this protective effect of ID.

In vivo myocardial protection from ischemia/reperfusion injury by the peroxisome proliferator-activated receptor-gamma agonist rosiglitazone.

BACKGROUND: Diabetes is associated with increased risk of mortality as a consequence of acute myocardial infarction. This study determined whether rosiglitazone (ROSI) could reduce myocardial infarction after ischemia/reperfusion injury. METHODS AND RESULTS: Male Lewis rats were anesthetized, and the left anterior descending coronary artery was ligated for 30 minutes. After reperfusion for 24 hours, the ischemic and infarct sizes were determined. ROSI at 1 and 3 mg/kg IV reduced infarct size by 30% and 37%, respectively (P<0.01 versus vehicle). Pretreatment with ROSI (3 mg. kg(-1). d(-1) PO) for 7 days also reduced infarct size by 24% (P<0.01). ROSI also improved ischemia/reperfusion-induced myocardial contractile dysfunction. Left ventricular systolic pressure and positive and negative maximal values of the first derivative of left ventricular pressure (dP/dt) were significantly improved in ROSI-treated rats. ROSI reduced the accumulation of neutrophils and macrophages in the ischemic heart by 40% and 43%, respectively (P<0.01). Ischemia/reperfusion induced upregulation of CD11b/CD18 and downregulation of L-selectin on neutrophils and monocytes; these effects were significantly attenuated in ROSI-treated animals. Likewise, intercellular adhesion molecule-1 expression in ischemic hearts was markedly diminished by ROSI, as was the ischemia/reperfusion-stimulated upregulation of monocyte chemoattractant protein-1. CONCLUSIONS: ROSI reduced myocardial infarction and improved contractile dysfunction caused by ischemia/reperfusion injury. The cardioprotective effect of ROSI was most likely due to inhibition of the inflammatory response.

Hypertensive end-organ damage and premature mortality are p38 mitogen-activated protein kinase-dependent in a rat model of cardiac hypertrophy and dysfunction.

BACKGROUND: Numerous pathological mediators of cardiac hypertrophy (eg, neurohormones, cytokines, and stretch) have been shown to activate p38 MAPK. The purpose of the present study was to examine p38 MAPK activation and the effects of its long-term inhibition in a model of hypertensive cardiac hypertrophy/dysfunction and end-organ damage. METHODS AND RESULTS: In spontaneously hypertensive stroke-prone (SP) rats receiving a high-salt/high-fat diet (SFD), myocardial p38 MAPK was activated persistently during the development of cardiac hypertrophy and inactivated during decompensation. Long-term oral treatment of SFD-SP rats with a selective p38 MAPK inhibitor (SB239063) significantly enhanced survival over an 18-week period compared with the untreated group (100% versus 50%). Periodic echocardiographic analysis revealed a significant reduction in LV hypertrophy and dysfunction in the SB239063-treatment groups. Little or no difference in blood pressure was noted in the treatment or vehicle groups. Basal and stimulated (lipopolysaccharide) plasma tumor necrosis factor-alpha concentrations were reduced in the SB239063-treatment groups. In vitro vasoreactivity studies demonstrated a significant preservation of endothelium-dependent relaxation in animals treated with the p38 MAPK inhibitor without effects on contraction or NO-mediated vasorelaxation. Proteinuria and the incidence of stroke (53% versus 7%) were also reduced significantly in the SB239063-treated groups. CONCLUSIONS: These results demonstrate a crucial role for p38 MAPK in hypertensive cardiac hypertrophy and end-organ damage. Interrupting its function with a specific p38 MAPK inhibitor halts clinical deterioration.

Therapeutic modulation of transcription factor activity.

Aberrant gene expression is a fundamental cause of many disease-associated pathophysiologies. The pharmacological modulation of transcription factor activity therefore represents an attractive therapeutic approach to such disorders. With the exception of nuclear receptors, which are the direct targets of pharmaceuticals, other known classes of transcription factors are largely regulated indirectly by drugs that impact upon those signal transduction cascades that alter transcription factor phosphorylation and dephosphorylation and/or nuclear import. However, recent advances in drug discovery technologies now enable high-throughput screens that can identify molecules that act directly at the level of transcription factor complexes.

California dreamin' 'bout endothelin: emerging new therapeutics.

Progress in our understanding of endothelins has been extremely rapid since their discovery in 1988. A number of pharmaceutical companies have developed potent, orally active, nonpeptide endothelin receptor antagonists with efficacies in a wide variety of animal disease models and potential for treating human disease. However, only time will tell whether or not these compounds are developed into drugs that are perceived as worthy of a place in the therapeutic marketplace. If this is the case, endothelin will have been promoted from the status of striking scientific discovery to therapeutic target in a remarkably short period.

Apoptosis: a potential target for discovering novel therapies for cardiovascular diseases.

The realization that apoptosis is genetically programmed raises the exciting prospect that modulating apoptosis may provide novel approaches for treatment of cardiovascular diseases in which apoptosis has been demonstrated. Low molecular weight inhibitors of caspases and mitogen-activated protein kinases have been evaluated, with promising results in a variety of cardiovascular apoptotic models.

Human urotensin-II, the most potent mammalian vasoconstrictor identified to date, as a therapeutic target for the management of cardiovascular disease.

The novel cyclic undecapeptide human urotensin-II (hU-II) and its high-affinity G-protein-coupled receptor, GPR14, are both expressed within the human cardiovasculature (vascular smooth muscle, endothelium, myocardium, coronary atheroma, etc.) and may, therefore, contribute to the (patho)physiological regulation of cardiovascular homeostasis in humans. Indeed, hU-II is an efficacious, sustained spasmogen of mammalian isolated blood vessels including those from rats, rabbits, dogs, pigs, non-human primates and humans (where it is one to two orders of magnitude more potent than endothelin(ET)-1). In vivo, hU-II markedly alters systemic hemodynamics in the anesthetized primate (increase cardiac contractility [dP/dt], increase stroke volume, decrease total peripheral resistance) ultimately resulting in fatal cardiovascular collapse. As such, the development of selective hU-II receptor antagonists may be of utility in the management of cardiovascular disorders characterized by aberrant vasoconstriction, myocardial dysfunction and/or cardiac remodeling (e.g., myocardial infarction, congestive heart failure).

Selective ETA receptor antagonism with BQ-123 is insufficient to inhibit angioplasty induced neointima formation in the rat.

OBJECTIVE: The aim was to assess whether or not the endothelin ETA receptor selective antagonist BQ-123 could inhibit neointima formation in vivo following balloon angioplasty. METHODS: The effect of either acute administration of BQ-123 (0.1 mg.kg-1.min-1 intra-arterial infusion for 1 h before and 1 h after angioplasty) or chronic administration (bolus intraperitoneal injection, 2.5 mg.kg-1 twice daily; continuous intraperitoneal infusion, 0.8 and 8 mg.kg-1.d-1) on neointima formation was examined in rats which had undergone left common carotid artery balloon angioplasty. RESULTS: Neither acute intra-arterial infusion nor either mode of chronic intraperitoneal administration of BQ-123 had a significant effect on the degree of neointima formation observed following balloon angioplasty. CONCLUSIONS: Neither acute nor chronic ETA receptor blockade is sufficient to inhibit angioplasty induced neointima formation in the rat. Since it was previously shown that the ETA/B antagonist SB 209670 was effective in this model, while the ETA selective antagonist BQ-123 is now found to be ineffective, the data implicate the ETB receptor subtype in the pathogenesis of neointima formation.

Application of in vivo and ex vivo magnetic resonance imaging for evaluation of tranilast on neointima formation following balloon angioplasty of the rat carotid artery.

OBJECTIVE: Recent studies suggest that tranilast inhibits a variety of agents implicated in neointimal growth and restenosis in experimental animal models and humans. We report here a study evaluating the efficacy of tranilast in the rat carotid artery balloon angioplasty model, a model that mimics many aspects of the percutaneous transluminal angioplasty procedure in humans. Efficacy was determined based on in vivo and ex vivo magnetic resonance imaging (MRI) as well as by histomorphometry. The utility of this study, using a reverse paradigm, is to investigate if agents successful in the clinic can demonstrate efficacy in this animal model primary screen as measured by MRI and histomorphometry. METHODS: Tranilast (300 mg/kg/day, p.o.) was administered to Sprague-Dawley rats 3 days prior to balloon injury and continued for 14 days after injury. Three methods of measuring the vascular injury that occurs in this model were employed: (1) in vivo MRI, used to measure in vivo lumen volumes for the carotid artery once at baseline (pre-surgery) and again at 14 days post angioplasty; (2) ex vivo MRI (and histomorphometry), used to evaluate the total arterial wall thickness and the intima-to-media ratio; and (3) analysis of collagen density, used to evaluate the efficacy of tranilast to abrogate collagen synthesis and deposition following vascular injury. RESULTS: Tranilast provided 33% protection (P<0.05) from angioplasty-induced lumen narrowing as measured by MRI in vivo. The results of the ex vivo MR analysis of total wall thickness showed a 14% protection of angioplasty-induced narrowing (P<0.05), and the mean intima-to-media ratio showed a 39% (P<0.006) protection for the tranilast-treated rats. Histological analysis of the mean intima-to-media ratio demonstrated that tranilast provided 36% (P<0. 01) protection in the intima-to-media ratio. Further, treatment with tranilast showed a 52% reduction in collagen density of the intimal layer and a 70% reduction in collagen density of the medial layer of the injured arteries. CONCLUSION: The data obtained by in vivo MRI, ex vivo MRI, histology and collagen analysis demonstrate that tranilast provided significant beneficial effects in inhibiting neointimal formation in the rat carotid artery model. Also this study, to the best of our knowledge, is the first to harness complimentary information from various technologies, including lumen patency by in vivo MRI, neointimal formation by ex vivo MRI and conventional histomorphometry, and histological analysis for collagen density, to provide a comprehensive understanding of the pathology in this disease model.

Eprosartan reduces cardiac hypertrophy, protects heart and kidney, and prevents early mortality in severely hypertensive stroke-prone rats.

OBJECTIVE: Eprosartan is a selective angiotensin II type I receptor antagonist approved for the treatment of hypertension. In the present studies, eprosartan's ability to provide end-organ protection was evaluated in a model of cardiomyopathy and renal failure in stroke-prone rats (SP). METHODS: SP were fed a high fat (24.5% in food) and high salt (1% in water) diet (SFD). Eprosartan (60 mg/kg/day) or vehicle (saline control) (n = 25/group) was administered by intraperitoneally-implanted minipumps to these SP on the SFD for 12 weeks. Normal diet fed SP and WKY rats (n = 25/group) were also included for comparison (i.e. served as normal controls). Mortality, hemodynamics, and both renal and cardiac function and histopathology were monitored in all treatment groups. RESULTS: Eprosartan decreased the severely elevated arterial pressure (-12%; P < 0.05) produced by SFD but did not affect heart rate. Vehicle-treated SP-SFD control rats exhibited significant weight loss (-13%; P < 0.05) and marked mortality (50% by week 6 and 95% by week 9; P < 0.01). Eprosartan-treated SP-SFD rats maintained normal weight, and exhibited zero mortality at week 12 and beyond. Eprosartan prevented the increased urinary protein excretion (P < 0.05) that was observed in vehicle-treated SP-SFD rats. Echocardiographic (i.e. 2-D guided M-mode) evaluation indicated that SP-SFD vehicle control rats exhibited increased septal (+22.2%) and posterior left ventricular wall (+30.0%) thickness, and decreased left ventricular chamber diameter (-15.9%), chamber volume (-32.7%), stroke volume (-48.7%) and ejection fraction (-22.3%), and a remarkable decrease in cardiac output (-59.3%) compared to controls (all P < 0.05). These same parameters in eprosartan-treated SP-SFD rats were normal and differed markedly and consistently from vehicle-treated SP-SFD rats (i.e. treatment prevented pathology; all P < 0.05). Cardiac-gated MRI data confirmed the ability of eprosartan to prevent cardiac pathology/remodeling (P < 0.05). Histopathological analysis of hearts and kidneys indicated that eprosartan treatment significantly reduced end-organ damage (P < 0.01) and provided corroborative evidence that eprosartan reduced remodeling of these organs. Vehicle-treated SP-SFD rats exhibited a 40% increase in the plasma level of pro-atrial natiuretic factor that was reduced to normal by eprosartan (P < 0.05). CONCLUSION: These data demonstrate that eprosartan, at a clinically relevant dose, provides significant end-organ protection in the severely hypertensive stroke-prone rat. It preserves cardiac and renal structural integrity, reduces cardiac hypertrophy and indices of heart failure, maintains normal function of the heart and kidneys, and eliminates premature mortality due to hypertension-induced end-organ failure.

Identification of endothelin receptor subtypes in human renal cortex and medulla using subtype-selective ligands.

High affinity and high density endothelin (ET)-binding sites were identified in membranes prepared from human kidney cortex and medulla. Saturation binding experiments performed in membranes prepared from cortex and medulla using [125I]ET-1 and [125I]ET-3 revealed that the proportion of [125I]ET-3-binding sites was 30-35% less than that of [125I]ET-1-binding sites. The apparent dissociation constants and maximum binding for [125I]ET-1 and [125I]ET-3 to membranes from cortex were 91 +/- 5 pM and 165 +/- 10 fmol/mg protein, and 117 +/- 9 pM and 110 +/- 7 fmol/mg protein, respectively, whereas in medulla they were 139 +/- 10 pM and 360 +/- 11 fmol/mg protein, and 142 +/- 11 pM and 245 +/- 15 fmol/mg protein, respectively. In the presence of 10 nM sarafotoxin-6c, which is selective for ETB receptors, [125I]ET-1 binding was decreased by 65-70%, whereas [125I]ET-3 binding was totally abolished, suggesting that 65-70% of [125I]ET-1 binding and 100% of [125I]ET-3 binding was to ETB receptors. This was further confirmed by the use of a cyclic pentapeptide [cyclo(D-Trp,D-Asp,L-Pro, D-Val,L-Leu)] (BQ123), which is selective for ETA receptors. In the presence of 1 microM BQ123, [125I]ET-1 binding was decreased by 25-30%, whereas [125I]ET-3 binding was unaffected, confirming that 30-35% of ET receptors belong to the ETA subtypes, and that [125I]ET-1 bound to both ETA and ETB receptors with the same high affinity, but [125I]ET-3 bound only to ETB receptors with high affinity. These results suggest that human kidney cortex and medulla contain ETA and ETB receptors in a ratio of 30:70, and that sarafotoxin-6c and BQ123 are valuable tools in identifying the subtype of ET receptors in various tissues.

Cyclic guanosine monophosphate mediates vascular relaxation induced by atrial natriuretic factor.

The biochemical mechanism of action of synthetic atrial natriuretic factor (atriopeptin II) was studied in vascular smooth muscle of the rabbit thoracic aorta. Atriopeptin II caused a time-dependent and concentration-dependent increase in tissue levels of cyclic guanosine monophosphate that corresponded in these same tissues with vascular relaxation. The elevation of arterial cyclic guanosine monophosphate levels preceded the onset of vascular relaxation. Atriopeptin II did not alter vascular levels of cyclic adenosine monophosphate. The presence of a functionally intact vascular endothelium was not necessary for atriopeptin II to elicit vascular relaxation. Atriopeptin II-induced vascular relaxation and elevation of cyclic guanosine monophosphate levels were inhibited by the guanylate cyclase inhibitor methylene blue. These data suggest cyclic guanosine monophosphate mediates vascular relaxation produced by atriopeptin II.

Antihypertensive actions of the novel nonpeptide endothelin receptor antagonist SB 209670.

Indirect evidence has implicated endothelin-1 in the pathogenesis of hypertension. In the present study we examined such a role directly with SB 209670, a novel nonpeptide endothelin receptor antagonist. The antihypertensive and hemodynamic effects of SB 209670 were examined in conscious, unrestrained spontaneously hypertensive (SHR), normotensive Wistar-Kyoto (WKY), and renin-hypertensive rats. Sustained intravenous infusion of SB 209670 (10 micrograms.kg-1.min-1 for 6 hours) produced a significant, reversible reduction in mean arterial pressure in SHR but not in WKY rats. The antihypertensive response to 10 micrograms.kg-1.min-1 SB 209670 (approximately 25 mm Hg reduction in blood pressure) was associated with bradycardia (16% decrease in heart rate) but only a minimal reduction (3%) in cardiac output, because stroke volume was evaluated (by 15%). Therefore, the antihypertensive effect of SB 209670 resulted from a decrease (13%) in total peripheral resistance. A sustained antihypertensive effect could also be observed after intraduodenal administration of SB 209670 (3 mg/kg) in conscious SHR (reduction of approximately 35 mm Hg 5 hours after administration). SB 209670 (3 mg/kg intravenous bolus) did not alter the pressor response or tachycardia observed in pithed SHR after stimulation of thoracolumbar sympathetic outflow. SB 209670 was also antihypertensive in renin-hypertensive rats, lowering blood pressure to an extent similar to that observed in SHR. Thus, the data presented provide evidence to support a role for endothelin-1 in the pathophysiology of two animal models of hypertension.

Endothelin levels increase in rat focal and global ischemia.

Endothelin-1, a peptide exhibiting extremely potent cerebral vasoactive properties, is elevated in the cerebrospinal fluid after hemorrhagic stroke and implicated in cerebral vasospasm. The purpose of this study was to determine changes in endothelin in ischemic rat brain by assaying endothelin tissue and extracellular levels. Immunoreactive endothelin levels in ischemic brain tissue following permanent or transient focal ischemia produced by middle cerebral artery occlusion was determined. In addition, endothelin levels were assayed in striatal extracellular fluid collected by microdialysis before, during, and after global ischemia produced by two-vessel occlusion combined with hypotension. Twenty-four hours after the onset of permanent middle cerebral artery occlusion, the ischemic cortex level (0.58 +/- 0.27 fmol/mg protein) of immunoreactive endothelin was significantly (p < 0.05) increased, by 100%, over that in the nonischemic cortex (0.29 +/- 0.13 fmol/mg protein). Transient artery occlusion for 80 min with reperfusion for 24 h also resulted in a similar significant (p < 0.05) increase, 78%, in immunoreactive endothelin in the ischemic zone. Global forebrain ischemia significantly (p < 0.05) increased the level of immunoreactive endothelin collected in striatal microdialysis perfusate, from a basal level of 14.6 +/- 6.7 to 26.5 +/- 7.7 and 26.2 +/- 7.4 amol/microliters (i.e. 82 and 79%). These changes reflect the relative picomolar extracellular concentration increases during ischemia and following reperfusion, respectively. This is the first demonstration of elevated levels of endothelin in focal ischemic tissue and in the extracellular fluid in global ischemia and suggests a role of the peptide in ischemic and postischemic derangements of cerebral vascular function and tissue injury.

Identification, characterization, and functional role of phosphodiesterase type IV in cerebral vessels: effects of selective phosphodiesterase inhibitors.

The role of the phosphodiesterase type IV isozyme (PDE IV) in the regulation of cerebrovascular tone was investigated in the canine basilar artery in vitro and in vivo. The PDE isozymes extracted from the canine basilar artery were isolated by diethylaminoethanol (DEAE)-Sepharose affinity chromatography and identified based on sensitivity to isozyme-selective PDE inhibitors. [3H]cAMP hydrolysis was observed in one major and one minor peak of activity. The predominant peak was inhibited by the addition of cGMP (25%), siguazodan (26%), rolipram (39%), and the combination of siguazodan and rolipram (95%). Selective PDE IV inhibitors BRL 61063, rolipram, and denbufylline were equieffective inhibitors of [3H]-ccAMP hydrolysis mediated by PDE IV isolated from the canine basilar artery [concentrations producing 50% inhibition (IC50S) = 0.21 +/- 0.05 microM, 0.67 +/- 0.23 microM, and 0.73 +/- 0.16 microM, respectively]. In precontracted isolated ring segments of the canine basilar artery, selective PDE IV inhibitors produced potent and complete relaxation (IC50S < 150 nM). In contrast, zaprinast (a selective PDE V inhibitor) and siguazodan (a selective PDE III inhibitor) produced only weak relaxation of the basilar artery (IC50S = 4.5 microM and > 10 microM, respectively). Vasorelaxation produced by PDE IV inhibitors was not altered by removing the endothelium, 1-NAME, or adenosine receptor antagonism. In a canine model of acute cerebral vasospasm, all three selective PDE IV inhibitors reversed basilar artery spasm produced by autologous blood without altering mean arterial blood pressure. In contrast, prolonged treatment with BRL 61063 failed to alter the development of basilar spasm in the two hemorrhage canine models of chronic cerebral vasospasm. Denbufylline-induced relaxation in vitro was also significantly impaired in basilar arteries obtained from the model of chronic vasospasm. In conclusion, PDE IV appears to be the predominant isozyme regulating vascular tone mediated by cAMP hydrolysis in cerebral vessels. In addition, vasorelaxation modulated by PDE IV is compromised in chronic cerebral vasospasm associated with subarachnoid hemorrhage.

BQ-123, a selective endothelin subtype A-receptor antagonist, lowers blood pressure in different rat models of hypertension.

OBJECTIVE: To investigate the role of endothelin-1 in the pathogenesis of hypertension directly by using the selective endothelin subtype A-receptor antagonist BQ-123. METHODS: The antihypertensive and hemodynamic effects of sustained BQ-123 administration were examined in conscious, unrestrained spontaneously hypertensive rats (SHR), normotensive Wistar-Kyoto (WKY) rats and renin hypertensive rats. RESULTS: Sustained infusions of BQ-123 (0.16-164 nmol/kg per min, intravenously, for 6 h) produced dose-dependent reductions in mean arterial pressure in SHR, the maximal reduction being obtained with a dose of 16 nmol/kg per min. This reversible response was evident up to 14 h after the cessation of antagonist infusion. The antihypertensive response to a maximal dose of BQ-123 was associated with bradycardia, but only a minimal reduction in cardiac output (since the stroke volume was elevated) in the SHR. Therefore, the antihypertensive effect of BQ-123 resulted from a decrease in total peripheral resistance. In contrast, in WKY rats the infusion of the high dose (164 nmol/kg per min, intravenously for 6 h) produced a small but significant reduction in mean arterial pressure. BQ-123 did not alter the pressor response or tachycardia observed in pithed SHR following stimulation of the thoracolumbar sympathetic outflow. BQ-123 was also antihypertensive in renin hypertensive rats, lowering the blood pressure to an extent similar to that observed in SHR. CONCLUSION: The data presented indicate a role for endothelin in the pathophysiology of hypertension.

Inhibitors of dopamine beta-hydroxylase. 3. Some 1-(pyridylmethyl)imidazole-2-thiones.

The 1-benzylimidazole-2-thione moiety has been previously shown by Kruse et al. to be broadly associated with dopamine beta-hydroxylase (DBH) inhibitory activity both in vitro and in vivo in spontaneously hypertensive rats (SHR). An extension of structure-activity studies to 1-(pyridylmethyl)- and 1-(oxypyridylmethyl)imidazole-2-thiones is reported here in an attempt to exploit the pH differential that exists across the chromaffin vesicle membrane. We hypothesized that the weakly basic pyridyl compounds would diffuse into the acidic vesicles in their neutral forms where protonation and concentration would occur to enhance their in vivo effectiveness as inhibitors. To test this hypothesis, isomeric 2-, 3- and 4-(1-pyridylmethyl)imidazole-2-thiones were synthesized from the appropriate pyridinecarboxaldehydes by reductive alkylation of aminoacetaldehyde dialkyl acetal followed by imidazole-2-thione formation using acidic potassium thiocyanate. Related oxypyridyl compounds were synthesized by first preparing the appropriate aldehyde intermediate followed by conversion to the imidazole-2-thione by the same procedure. The unsubstituted pyridylmethyl compounds showed modest DBH inhibition in vitro but, consistent with a transport-mediated increase in observed potency, showed significant effects in vivo to increase the vascular ratio of dopamine to norepinephrine and to lower blood pressure.

Endothelin receptor antagonist improves pulmonary hemodynamics during lung ischemia/reperfusion injury.

BACKGROUND: We have previously shown that elevated release of endothelin-1 is associated with increased pulmonary vascular resistance (PVR) immediately after reperfusion of the transplanted lung. In the present study, we investigated the effect of ET receptor blockage on pulmonary hemodynamics and function in an ex vivo lung reperfusion model after 6 hr of cold ischemia. METHODS: Eighteen rabbits were divided into three groups: no ischemia followed by 3 hr of reperfusion (group I) and 6 hr of cold ischemia followed by 3 hr of reperfusion with either blood (group II) or blood + SB209670 (mixed ETA/ETB receptor antagonist) (group III). RESULTS: Shortly after reperfusion, mean pulmonary artery pressure, PVR, and pulmonary edema were increased, and pulmonary compliance and PO2 were decreased in group II compared with group I. Treatment with SB209670 resulted in a significant decrease in mean pulmonary artery pressure, PVR, and pulmonary edema, and improvement in pulmonary compliance and PO2. CONCLUSION: The data suggest an important role for ET-1 in lung ischemia/reperfusion injury and that the use of ET receptor antagonist immediately after transplantation may provide a new therapeutic tool in the management of early graft dysfunction.