Antithrombotic and anticoagulant effects of the direct thrombin inhibitor dabigatran, and its oral prodrug, dabigatran etexilate, in a rabbit model of venous thrombosis.

BACKGROUND: Oral anticoagulant therapies targeted at thrombin are being developed to overcome limitations associated with current standard therapies. OBJECTIVES: This study was undertaken to assess and compare the antithrombotic and anticoagulant effects of the novel, selective and reversible, direct thrombin inhibitor (DTI), dabigatran, and its oral prodrug dabigatran etexilate, to that of unfractionated heparin (UFH), hirudin and melagatran using a rabbit model of venous thrombosis. METHODS: A rabbit model of venous thrombosis consisting of endothelial damage with blood flow reduction was used with minor modifications. RESULTS: All compounds demonstrated a dose-dependent reduction in thrombus formation following i.v. administration with complete or almost complete inhibition at the highest doses. Dabigatran (in the dose range 0.03-0.5 mg kg(-1)) had a 50% effective dose of 0.066 mg kg(-1). By comparison, UFH (5-50 U kg(-1)), hirudin (0.01-0.05 mg kg(-1)) and melagatran (0.01-0.3 mg kg(-1)) had a 50% effective dose of 9.8 U kg(-1), 0.016 mg kg(-1) and 0.058 mg kg(-1), respectively. Similarly, oral dabigatran etexilate (1-20 mg kg(-1)) inhibited thrombus formation in a dose-dependent manner. Maximum inhibition was achieved within 1 h of administration, suggesting a rapid onset of action. For both routes of administration, inhibition of thrombus formation directly correlated with prolongation of the activated partial thromboplastin time. CONCLUSIONS: These findings demonstrate the potent anticoagulant and antithrombotic activity of dabigatran as a selective thrombin inhibitor in a rabbit model of venous thrombosis. Notably, dose-dependent and long-lasting antithrombotic efficacy was observed after application of its oral form dabigatran etexilate, which is currently undergoing phase III clinical development.

Effects of telmisartan on renal excretory function in conscious dogs.

The present study investigated the effects of telmisartan, a selective AT1 receptor antagonist, on renal function in dogs. Conscious female dogs were treated with (i) vehicle (controls) and three doses of telmisartan (0.03 mg/kg, 0.1 mg/kg and 0.3 mg/kg) administered intravenously; (ii) vehicle and three doses of telmisartan (0.3 mg/kg, 1.0 mg/kg and 3.0 mg/kg) administered orally; or (iii) 1.0 mg/kg per day telmisartan orally for 12 days. Eight dogs were used for each experiment. Each of the four treatments in (i) and (ii) was administered 7 days apart. During the 6 h after intravenous administration, urine volume was significantly higher in dogs treated with telmisartan 0.1 mg/kg (8.5 +/- 1.6 ml/kg) and 0.3 mg/kg (7.0 +/- 0.9 ml/kg) than controls (2.7 +/- 0.3 ml/kg; P < 0.05), and renal sodium excretion was increased significantly with telmisartan 0.03 mg/kg (803 +/- 124 micromol/kg), 0.1 mg/kg (1039 +/- 213 micromol/kg) and 0.3 mg/kg (966 +/- 161 micromol/kg) versus controls (159 +/- 21 micromol/kg; P < 0.05). Oral telmisartan at doses of 1.0 mg/kg and 3.0 mg/kg also produced significant increases in urine volume (7.2 +/- 1.1 ml/kg and 6.6 +/- 1.2 ml/kg, respectively) and renal sodium excretion (599 +/- 146 micromol/kg and 555 +/- 131 micromol/kg, respectively) compared with controls (2.8 +/- 0.5 ml/kg and 80 +/- 33 mciromol/kg; P < 0.05) over the 6-h post-dose period. Telmisartan at all intravenous doses and at 3.0 mg/kg orally increased the urinary excretion of chloride significantly over the 6-h post-dose period compared with vehicle alone. The excretion of potassium and creatinine were unchanged by any treatment. Telmisartan 1.0 mg/kg administered orally for 12 days produced similar results. In conclusion, acute intravenous or oral as well as subchronic oral administration of telmisartan to conscious dogs promotes diuresis and natriuresis without affecting potassium or creatinine excretion.

AT1 receptor antagonist telmisartan administered peripherally inhibits central responses to angiotensin II in conscious rats.

The effects of systemic treatment with the AT1 receptor antagonist telmisartan on central effects of angiotensin II (Ang II), namely, increase in blood pressure, vasopressin release into the circulation, and drinking response, were investigated in conscious, normotensive rats. The central responses to i.c.v. Ang II (30 ng/kg) were measured at 0.5, 2, 4, and 24 h following acute i.v. or acute and chronic oral telmisartan application. At a dose of 10 mg/kg i.v., the drinking response to i.c.v. Ang II was completely blocked over 4 h, while the pressor response and the release of vasopressin in response to i.c.v. Ang II were blocked by 60 to 80%. The inhibition of the centrally mediated pressor and drinking response to Ang II was sustained over 24 h. The lower doses of telmisartan (0.3 and 1 mg/kg) significantly inhibited the Ang II-induced actions over 4 h. A consistent 24-h inhibition of the central responses to i.c.v. Ang II was obtained after acute and chronic oral treatment with 30 mg/kg telmisartan. Oral treatment with 1 and 3 mg/kg telmisartan produced a slight but inconsistent inhibition of the central actions of Ang II. Telmisartan concentrations measured in the cerebrospinal fluid following 8 days of consecutive daily oral treatment (1-30 mg/kg) ranged from 0.87 +/- 0.27 ng/ml (1 mg/kg/day) to 46.5 +/- 11.6 ng/ml (30 mg/kg/day). Our results demonstrate that, following peripheral administration, the AT1 receptor antagonist telmisartan can penetrate the blood-brain barrier in a dose- and time-dependent manner to inhibit centrally mediated effects of Ang II.

Structural basis for inhibition promiscuity of dual specific thrombin and factor Xa blood coagulation inhibitors.

BACKGROUND: A major current focus of pharmaceutical research is the development of selective inhibitors of the blood coagulation enzymes thrombin or factor Xa to be used as orally bioavailable anticoagulant drugs in thromboembolic disorders and in the prevention of venous and arterial thrombosis. Simultaneous direct inhibition of thrombin and factor Xa by synthetic proteinase inhibitors as a novel approach to antithrombotic therapy could result in potent anticoagulants with improved pharmacological properties. RESULTS: The binding mode of such dual specific inhibitors of thrombin and factor Xa was determined for the first time by comparative crystallography using human alpha-thrombin, human des-Gla (1--44) factor Xa and bovine trypsin as the ligand receptors. The benzamidine-based inhibitors utilize two different conformations for the interaction with thrombin and factor Xa/trypsin, which are evoked by the steric requirements of the topologically different S2 subsites of the enzymes. Compared to the unliganded forms of the proteinases, ligand binding induces conformational adjustments of thrombin and factor Xa active site residues indicative of a pronounced induced fit mechanism. CONCLUSION: The structural data reveal the molecular basis for a desired unselective inhibition of the two key components of the blood coagulation cascade. The 4-(1-methyl-benzimidazole-2-yl)-methylamino-benzamidine moieties of the inhibitors are able to fill both the small solvent accessible as well as the larger hydrophobic S2 pockets of factor Xa and thrombin, respectively. Distal fragments of the inhibitors are identified which fit into both the cation hole/aromatic box of factor Xa and the hydrophobic aryl binding site of thrombin. Thus, binding constants in the medium-to-low nanomolar range are obtained against both enzymes.

Comparative antihypertensive and renoprotective effects of telmisartan and lisinopril after long-term treatment in hypertensive diabetic rats.

This study compared the cardiovascular and renal effects of long-term telmisartan (3 and 10 mg/kg/day)and lisinopril (10 mg/kg/day) in an animal model combining hypertension and diabetes mellitus. It was a parallel-group study of diabetic, spontaneously hypertensive rats (SHR), treated with control or active treatment for eight months. A non-diabetic SHR control group was run in parallel. Diabetes was induced by streptozotocin (45 mg/kg i.v.) in SHRs aged 9-10 weeks. Animals were treated with telmisartan (3 or 10mg/kg/day), lisinopril (10 mg/kg/day) or vehicle. Plasma glucose levels, blood pressure (BP), and urinary protein and albumin excretion were measured monthly. Telmisartan treatment significantly reduced BP of diabetic SHRs in a dose-dependent manner (p<0.05, low-dose, n= 18; p<0.01, high-dose, n=15). The BP reduction in the lisinopril group was similar to that in the telmisartan 10 mg/kg/day group. Compared with non-diabetic SHRs, untreated diabetic SHRs developed severe proteinuria and albuminuria over the experimental period (p<0.01). In diabetic SHRs, proteinuria and albuminuria were dose-dependently and significantly attenuated by treatment with telmisartan (p<0.01 with the higher dose) and lisinopril (p<0.01). Compared with the untreated diabetic SHRs, cardiac hypertrophy was significantly reduced after treatment with both doses of telmisartan and with lisinopril. Telmisartan, 10 mg/kg/day, but not lisinopril, significantly attenuated the diabetes-induced increase in glomerular volume. In conclusion, telmisartan, 10 mg/kg/day, is at least as beneficial as lisinopril, 10 mg/kg/day, in lowering BP, reducing cardiac hypertrophy and attenuating renal excretion of protein and albumin in this model.

Effects of telmisartan, hydrochlorothiazide and their combination on blood pressure and renal excretory parameters in spontaneously hypertensive rats.

The effects of telmisartan and hydrochlorothiazide (HCTZ) alone and in combination on blood pressure (BP) and renal excretory function were investigated in male spontaneously hypertensive rats (SHR) after oral administration for five consecutive days. Four treatments were studied: vehicle (0.5% Natrosol), telmisartan 3 mg/kg, HCTZ 10 mg/kg, and telmisartan 3 mg/kg+HCTZ 10 mg/kg. The effects on BP and heart rate were studied in 40 SHRs (10 animals per group) using an implanted telemetry device. Renal excretory function was assessed in 76 SHRs (18 animals per group, of which nine were used for urine sampling and nine for blood sampling). The telmisartan/HCTZ combination produced the greatest reductions in trough DBP (-44+/-1.5 mmHg), SBP (-60+/-1.9 mmHg) and mean BP (mBP; -53+/-1.7 mmHg) after five days of therapy (p<0.05 vs. vehicle and vs. telmisartan). Telmisartan monotherapy also decreased DBP, SBP and mBP significantly (p<0.05), but only minor BP fluctuations occurred in SHRs receiving HCTZ or vehicle. Telmisartan/HCTZ elevated heart rate by approximately 12 beats per minute (p<0.05 vs.control). Significant increases in urine volume and Na+, Cl, K+, creatinine and glucose excretion were observed with HCTZ treatment (p<0.01). Telmisartan/HCTZ also promoted renal water and electrolyte excretion (p<0.01); the diuretic effect appeared to be greater with the combination than with HCTZ alone, and there was some attenuation of urinary K+ loss. Elevated blood urea nitrogen levels were observed only in HCTZ-treated SHRs. These results indicate that the antihypertensive efficacy of telmisartan in SHRs is augmented by co-administration with HCTZ. The combination did not affect renal excretory function, with the exception of an increase in blood urea nitrogen and a possible amelioration of HCTZ-related K+ depletion.

Effect of angiotensin II and telmisartan, an angiotensin1 receptor antagonist, on rat gastric mucosal blood flow.

BACKGROUND: Angiotensin II (ATII) has been suggested to contribute to shock-induced dysfunction of the gastric circulation. AIM: To substantiate this conjecture, the effects on gastric mucosal haemodynamics and the hyperaemic response to acid back-diffusion of ATII and the angiotensin AT1 receptor antagonist, telmisartan, were examined in normal rats and in animals subjected to haemorrhage. METHODS: Gastric mucosal blood flow in phenobarbital-anaesthetized rats was recorded with the hydrogen clearance technique, and acid back-diffusion was induced by perfusing the stomach with ethanol (25%) in HCl (0.05 M). RESULTS: Intravenous infusion of ATII (0.3-10 nmol/min/kg) led to dose-dependent hypertension and a reduction of blood flow and vascular conductance in the gastric mucosa. The gastric hyperaemia caused by acid back-diffusion was attenuated by ATII (1 nmol/min/kg). These effects of ATII were antagonized by intravenous injection of telmisartan (1-10 mg/kg) which per se caused hypotension and dilated the gastric mucosal vasculature, but did not modify the gastric mucosal hyperaemia evoked by acid back-diffusion. Hypotension induced by haemorrhage (1.3 mL blood per 100 g body weight) failed to alter the hyperaemia due to acid back-diffusion, but caused gastric mucosal vasoconstriction, an effect that was left unaffected by telmisartan. CONCLUSIONS: ATII constricts the rat gastric microvasculature via an action involving AT1 receptors. The effects of telmisartan indicate that endogenous ATII contributes to the homeostatic regulation of gastric vascular tone but does not compromise the ability of the gastric microvasculature to react to influxing acid. These results negate the concept that ATII contributes to the gastric vascular perturbances in haemorrhagic shock.

Effects of AT1 receptor blockade on blood pressure and the renin-angiotensin system in spontaneously hypertensive rats of the stroke prone strain.

The aim of the study was to assess the effects of chronic angiotensin I receptor blockade on blood pressure, the renin-angiotensin system in plasma and kidney and the extent of renal damage in spontaneously hypertensive rats of the stroke prone strain (SHRsp). Four months old male SHRsp rats were orally treated with a high (10 mg/kg b.w. per day) or a low dose (1 mg/kg b.w. per day) of the AT1 receptor antagonist Telmisartan and compared to Losartan- (20 mg/kg b.w. per day), Captopril-treated (50 mg/kg b.w. per day) or untreated control groups for 38 days. Despite a similar extent of blood pressure reduction in all groups (except low dose Telmisartan), high dose Telmisartan but not Losartan or Captopril significantly reduced left ventricular weight by 24% compared to controls (p<0.05). Renal damage as assessed by urinary albumin or glomerulosclerosis index was significantly reduced in all treatment groups (p<0.02). Plasma renin concentration was significantly elevated (p<0.02) and plasma angiotensinogen significantly lowered (p<0.05) in all pharmacologically treated group compared to controls. In the kidney, renin-mRNA as well as AT1 receptor gene expression were elevated in all treatment groups, but no significant changes were found for renal angiotensinogen-mRNA. Chronic oral treatment of genetically hypertensive rats by the AT1 receptor antagonist Telmisartan reveals a blood pressure lowering and reno-protective effect of this drug comparable to other AT1 receptor antagonists or converting enzyme inhibitors, and demonstrates a marked reduction of cardiac hypertrophy by Telmisartan in this model.

Effects of an angiotensin II antagonist on organ perfusion during the post-resuscitation phase in pigs.

BACKGROUND: The aim of this study was to compare pre-arrest and post-resuscitation organ perfusion values and to investigate whether, during the post-resuscitation phase, administration of the angiotensin II antagonist telmisartan (TELM) 10 min after restoration of spontaneous circulation (ROSC) could improve organ flow in comparison to placebo. RESULTS: Five minutes after ROSC in the TELM group, blood flow in the cortex and myocardium increased to 583% (P < 0.05) and 137% (not significant), respectively, whereas blood flow of the colon, stomach and pancreas decreased to 50% (P < 0.05), 28% (P < 0.05) and 19% (P < 0.05) of pre-arrest values, respectively. At 90 min after ROSC, pre-arrest perfusion values both in non-splanchnic and splanchnic organs were achieved. At no point in time were there significant differences between the two groups with respect to organ blood flow or speed of recovery of organ perfusion. CONCLUSIONS: During the post-resuscitation phase, organ blood flow is characterized by the coincidence of increased cerebral and myocardial blood flow and decreased intestinal blood flow. Administration of TELM 10 min after ROSC did not improve the recovery of organ perfusion.

Effects of the AT1-selective angiotensin II antagonist, telmisartan, on hemodynamics and ventricular function after cardiopulmonary resuscitation in pigs.

The purpose of this study was to investigate the effects of the angiotensin II (ANG II) antagonist, telmisartan, on hemodynamics, myocardial function and myocardial blood flow during the postresuscitation phase in a porcine model of CPR and to compare these to saline. After 4 min of ventricular fibrillation and 5 min of closed-chest CPR, defibrillation was performed in 16 domestic pigs to restore spontaneous circulation (ROSC). Ten minutes after ROSC, animals were allocated to receive either the ANG II antagonist, telmisartan, at a dose of 1 mg/kg (n = 8) or saline (n = 8). Hemodynamics, myocardial function and myocardial blood flow were measured prearrest and at 5, 30, 90 and 240 min after ROSC. Using a Swan-Ganz catheter with a fast responding-thermistor and a micromanometer tipped catheter, right ventricular end-diastolic and end-systolic volume, right ventricular ejection fraction, left ventricular contractility were 67 +/- 6 ml (mean +/- S.E.M.), 42 +/- 4 ml, 38 +/- 2%, 2036 +/- 77 mmHg/s in the telmisartan group and 82 +/- 2 ml (P < 0.05), 59 +/- 3 ml (P < 0.01), 28 +/- 2% (P < 0.01), 1596 +/- 82 mmHg/s (P < 0.01) in the control group, at 240 min after ROSC. No significant differences in mean aortic and pulmonary artery pressure, cardiac index or myocardial blood flow between the two groups were found. We conclude that the ANG II antagonist telmisartan administered during the postresuscitation phase in pigs increases myocardial contractility without changing cardiac index, systemic vascular resistance, pulmonary vascular resistance, or myocardial perfusion.

Reduction of cardiac hypertrophy in TGR(mREN2)27 by angiotensin II receptor blockade.

TGR(mREN2)27 is a transgenic rat harboring the murine Ren-2 gene and exhibit fulminant hypertension and marked heart hypertrophy. In order to study the role of angiotensin II in the increase of cardiac mass, these animals were treated with antihypertensive and non-antihypertensive doses of the angiotensin II receptor AT1 antagonist Telmisartan for 9 weeks. All doses led to significant reductions of heart hypertrophy detected by the evaluation of the diameter of cardiac muscle bundles. We conclude from this study that cardiac hypertrophy in TGR(mREN2)27 is characterized by an increased volume of cardiomyocytes and an unchanged amount of fibrous tissue and that angiotensin II plays an important role in the mechanisms leading to this phenotype.

BIBP 3226, the first selective neuropeptide Y1 receptor antagonist: a review of its pharmacological properties.

Based on the assumption that the pharmacophoric groups interacting with the Y1 receptor are located in the C-terminal part of neuropeptide Y, low molecular weight compounds with high affinity and selectivity for the Y1 receptor were designed and synthesized. The prototype BIBP 3226 possesses affinity for the Y1 receptor in the nanomolar range. In addition, this compound is selective displaying rather low affinity for Y2, Y3, Y4 and a set of 60 other receptors. Both biochemical and pharmacological studies showed that BIBP 3226 behaves as a competitive antagonist. Using BIBP 3226 it was possible to investigate the role of NPY and/or Y1 receptors in blood pressure regulation. The interesting observation was that antagonism to Y1 receptors had no major influence on the basal blood pressure but attenuated stress induced hypertension. This strongly supports the hypothesis that NPY is mainly released during stress involving intense sympathetic nervous system activation. Moreover, BIBP 3226 can be used to characterize NPY receptor subtypes. For instance, we were able to show that presynaptic NPY receptors mediating catecholamine release do not solely belong to the Y2 subtype, but that presynaptic Y1 receptors also exist. In conclusion, BIBP 3226 has been shown to be an important tool for the elucidation of the physiological role of Y1 receptors in the cardiovascular system.

Pharmacological characterization of the selective nonpeptide neuropeptide Y Y1 receptor antagonist BIBP 3226.

The present study was undertaken to investigate the in vitro and in vivo pharmacological profile of the novel, nonpeptide neuropeptide Y (NPY) Y1-selective antagonist, BIBP 3226 [(R)-N2-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]-D-arginine-am ide], and a recently described peptidic structure [Ile-Glu-Pro-Orn-Tyr-Arg-Leu-Arg-Tyr-NH2, cyclic (2,4'), (2',4)-diamide]. BIBP 3226 antagonized the NPY Y1 receptor-mediated decrease in the twitch response in the rabbit vas deferens preparation with a pKb value of 6.98 +/- 0.06 (n = 16). It showed no affinity (EC50 > 1 microM) for NPY Y2 receptors in the rat vas deferens. NPY-induced increases in perfusion pressure in the isolated perfused rat kidney and rabbit ear preparations were antagonized with IC50 values of 26.8 +/- 4.5 (n = 4) and 214 +/- 30 nM (n = 4), respectively. The NPY-mediated potentiation of the noradrenaline elicited increase in perfusion pressure in the rat mesenteric bed was antagonized with an IC50 value of 976 (542-1760) nM. The NPY-induced increase in blood pressure in the pithed rat was inhibited by BIBP 3226 dose-dependently (ED50 = 0.11 +/- 0.03 mg/kg i.v.), whereas no effect of BIBP 3226 (1 mg/kg i.v.) was observed for the noradrenaline-, angiotensin-, endothelin- or vasopressin-induced pressor response. The data presented demonstrate that BIBP 3226 is a competitive and NPY Y1-selective antagonist. The peptidic compound proved to possess high potency for NPY Y1 receptors, but showed both agonistic as well as antagonistic properties.(ABSTRACT TRUNCATED AT 250 WORDS)

Subtype selectivity and antagonistic profile of the nonpeptide Y1 receptor antagonist BIBP 3226.

In the present study, the subtype specificity and species selectivity of the nonpeptide BIBP 3226, as well as its in vitro antagonism of neuropeptide Y (NPY)-mediated second messengers have been investigated. Radiolabeled NPY is potently displaced by BIBP 3226 [(R)-N2-(diphenylacetyl)-N-[(4-hydroxyphenylmethyl]-D- arginine amide] on human Y1 receptor expressing Chinese hamster ovary-K1 cells (Ki = 0.47 +/- 0.07 nM). SK-N-MC human neuroblastoma cells (Ki = 5.1 +/- 0.5 nM) and the rat parietal cortex membranes (Ki = 6.8 +/- 0.7 nM). The interaction of BIBP 3226 with the Y1 receptor is stereoselective, because the (S)-enantiomer of the (R)-configured BIBP 3226 displays almost no affinity (Ki > 10,000 nM). In contrast, concentrations up to 10 microM BIBP 3226 do not displace [125I]NPY from the human Y2 receptor (neuroblastoma cell line SMS-KAN), the rabbit Y2 receptor (kidney) and the rat Y2 receptor (hippocampus). Functional antagonism could be shown for the human Y1 receptor: 0.1 microM BIBP 3226 antagonizes the NPY induced Ca++ mobilization (pKb = 7.5 +/- 0.17) as well as the NPY-mediated inhibition of cyclic AMP synthesis (pKb = 8.2 +/- 0.24) in SK-N-MC cells. In contrast, none of the formerly described putative antagonists PYX-2, [D-Trp32]NPY and benextramine could be characterized as high affinity Y1 receptor antagonists. The 18 amino acid NPY analog EXBP 68 Ile-Glu-Pro-Orn-Tyr-Arg-Leu-Arg-Tyr-NH2, cyclic (2,4'), (2',4')-diamide] displayed Y1-selective affinity with in vitro antagonistic properties (Ki = 0.33 +/- 0.04 nM and pKb = 8.4 +/- 0.07) in SK-N-MC cells. Therefore, BIBP 3226 is the first potent and subtype-selective nonpeptide Y1 receptor antagonist.

Angiotensin II receptor blockade in TGR(mREN2)27: effects of renin-angiotensin-system gene expression and cardiovascular functions.

OBJECTIVE: To study the effect of angiotensin II receptor AT1 blockade on blood pressure, gene expression and pathomorphology of transgenic rats harbouring the mouse Ren-2 gene [TGR(mREN2)27], that develop fulminant hypertension while exhibiting suppressed components of the circulating renin-angiotensin system. DESIGN: TGR(mREN2)27 were treated orally with the newly developed AT1-specific angiotensin receptor antagonist Telmisartan, 4'-[(1,4'-dimethyl-2'-propyl[2,6'-bi-1H-benzimidazol]-1'-yl) methyl]-[1,1'-biphenyl]-2-carboxylic acid, in three doses (0.1, 1 and 3 mg/kg body weight) for 9 weeks. METHODS: The concentrations of the renin-angiotensin system components were analysed in plasma and tissues by radioimmunoassay. Messenger RNA levels for the angiotensinogen and renin genes were quantified by RNAase protection assay in several tissues. Heart hypertrophy and kidney morphology and function were monitored at the end of the treatment. RESULTS: In contrast to 0.1 mg/kg, 1 and 3 mg/kg Telmisartan normalized tail blood pressure measured once a week. Plasma renin and angiotensin II concentration increases were dose-dependent. The renin-angiotensin system genes in various cardiovascular organs were differentially regulated by angiotensin II receptor blockade. Treatment with Telmisartan stimulated angiotensinogen gene expression in the liver, kidney and heart, whereas it remained unchanged in the hypothalamus, thymus and adrenal gland. In the kidney, the expression of the endogenous, but not of the mouse Ren-2 gene, was increased in parallel to the renin concentration. Telmisartan reduced the severe glomerulosclerosis and proteinuria as well as cardiac hypertrophy observed in untreated TGR(mREN2)27 even with the lowest dose of 0.1 mg/kg, at which the blood pressure of the rats still exceeded 225 mmHg and the plasma renin-angiotensin system parameters were unchanged. CONCLUSION: From these experiments using a specific antagonist we can conclude that high blood pressure in TGR(mREN2)27 is angiotensin II-dependent. Furthermore, the expression of the renin-angiotensin system genes seems to be regulated not only by blood pressure and the plasma renin-angiotensin system but also by other, tissue-specific mechanisms. Pathomorphological changes in the kidney and in the heart do not seem to be caused by the systemic hypertension exclusively, but are also influenced by angiotensin II directly.

Labeling of neuropeptide Y receptors in SK-N-MC cells using the novel, nonpeptide Y1 receptor-selective antagonist [3H]BIBP3226.

The binding of tritium-labelled BIBP3226, N2-(diphenylacetyl)-N-[(4-hydroxy-phenyl)methyl]-D-arginine amide, to human neuroblastoma SK-N-MC cells was investigated. [3H]BIBP3226 reversibly binds to neuropeptide Y receptors of the Y1 subtype expressed in SK-N-MC cells with a KD of 2.1 +/- 0.3 nM (mean +/- S.E.M., n = 3) and a Bmax of 58,400 +/- 1100 sites/cell. Non-specific binding did not exceed 30% of the total radioactivity bound at KD. In competition experiments [3H]BIBP3226 is concentration-dependently displaced by neuropeptide Y and its peptide analogues with an affinity pattern neuropeptide Y = [Leu31, Pro34]neuropeptide Y >> neuropeptide Y-(18-36). This rank order of potencies is consistent with the interaction of [3H]BIBP3226 with neuropeptide Y receptors of the Y1 subtype. Therefore, [3H]BIBP3226 can be used as selective ligand to study neuropeptide Y Y1 receptors.

Adrenomedullin mediates vasodilation via CGRP1 receptors.

Adrenomedullin is a recently discovered 52 amino acid polypeptide with potent hypotensive activity. The peptide possesses 21% homology with the amino acid sequence of human calcitonin gene-related peptide-alpha (hCGRP-alpha). In 125I-hCGRP-alpha receptor binding experiments using membranes from human neuroblastoma cells (SK-N-MC) adrenomedullin is a potent competitor with a Ki of 0.37 nM. In SK-N-MC cells hCGRP-alpha and adrenomedullin concentration-dependently increase cAMP levels with -logEC50 values of 9.65 and 7.75, respectively. Both responses were attenuated in the presence of 30 nM CGRP[8-37], a CGRP1 receptor antagonist. In isolated rat hearts, perfused at constant flow, bolus infusion of adrenomedullin (1 to 100 nM) resulted in a concentration-dependent, pronounced and long-lasting vasodilation with an approximate EC50 of about 3 nM. This effect was markedly attenuated in the presence of 100 nM CGRP[8-37]. In this model, bolus infusion of hCGRP-alpha (0.01 to 100 nM) evoked a comparable vasodilation with an approximate EC50 of 0.5 nM. This effect was also potently inhibited in the presence of CGRP[8-37]. These results suggest that adrenomedullin-mediated vasodilation is linked to the activation of CGRP1 receptors in the coronary vascular system.

The first highly potent and selective non-peptide neuropeptide Y Y1 receptor antagonist: BIBP3226.

The design and subsequent in vitro and in vivo biological characterisation of the first potent and selective non-peptide neuropeptide Y Y1 receptor antagonist, BIBP3226 ((R)-N2-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]-argininami de) is reported. BIBP3226 displaced 125I-labelled neuropeptide Y with high affinity (Ki = 7 nM) from the human neuropeptide Y Y1 receptor and proved to be highly selective. BIBP3226 displayed potent antagonistic properties both in in vitro and in vivo models and thus represents the first selective non-peptide neuropeptide Y Y1 receptor antagonist.

Mutations in transmembrane segment VII of the AT1 receptor differentiate between closely related insurmountable and competitive angiotensin antagonists.

Chimeric constructs between the human and the Xenopus laevis AT1 receptor have demonstrated, that the binding of non-peptide angiotensin antagonists is dependent on non-conserved residues located deep in transmembrane segment VII of the AT1 receptor. Here we have studied four pairs of closely related antagonists each consisting of a competitive and an insurmountable compound differentiated by one out of three different types of minor chemical modifications. None of the antagonists bound to the Xenopus receptor and the binding of all of the compounds to the human receptor was severely impaired by the introduction of non-conserved residues from transmembrane segment VII of the Xenopus receptor. In all four pairs of antagonists the competitive compound was affected more by these substitutions than the corresponding insurmountable one (209 vs. 22, 281 vs. 29, 290 vs. 29 and 992 vs. 325-fold increase in Ki values). A similar pattern was observed in response to substitution of a single non-conserved residue in transmembrane segment VII, Asn295 to Ser. These results indicate that a common molecular mechanism distinguishes the interaction of insurmountable and competitive antagonists with the AT1 receptor.

Enzyme release into the interstitial space of the isolated rat heart induced by changes in contractile performance.

OBJECTIVE: The aim was to investigate changes in interstitial concentration and release of creatine kinase in isolated perfused rat hearts after an experimental inotropic stimulation or after recovery from a negative inotropic intervention (low Ca2+ or high K+ buffer). METHODS: Interstitial transudate emerging at the surface of the heart and venous effluent were analysed for creatine kinase. RESULTS: The interstitial concentration of creatine kinase was always higher, by a factor of 25 to 100 (range from 10 to 580 mU.ml-1), than the concentration in the venous effluent (close to or below the limit of detection: 0.4 mU.ml-1). Continuous stimulation with a submaximal effective concentration (8 nM) of isoprenaline for 30 min resulted in an initial transient increase in the interstitial release of creatine kinase to about 160% of the control (p < 0.05). Similarly, in a second series, three repeated (5 min) periods of inotropic stimulation also caused a significant and transient increase in the interstitial release of creatine kinase to a maximum of 180%. Change to a buffer containing 2.0 mM Ca2+ after a 60 min period of low Ca2+ perfusion (0.25 mM) led to restoration of contractile function and an immediate and transient increase in the interstitial release of creatine kinase to about 900%. After a period of cardiac arrest (16 mM K+ for 60 min), perfusion with 4 mM K+ also immediately restored cardiac function, and led to an increase in creatine kinase release of 2500%. In additional experiments dilatation of the left ventricle by inflating an intraventricular balloon during cardioplegic perfusion induced a significant fivefold increase in the interstitial release of creatine kinase, which was further enhanced 3.5-fold during the subsequent recovery period. CONCLUSIONS: The coincidence of an increase in or a restoration of cardiac contractile function and an increase in the interstitial enzyme release suggests that myocardial enzyme release may occur in response to physiological stimuli during different episodes of metabolic or mechanical stress, as well as under pathophysiological conditions.