Specific Angiotensin Research Articles

A state of reversible compensated ventricular dysfunction precedes pathological remodelling in response to cardiomyocyte-specific activity of angiotensin II type-1 receptor in mice.

ABSTRACTCardiac dysfunction is commonly associated with high-blood-pressure-induced cardiomyocyte hypertrophy, in response to aberrant renin-angiotensin system (RAS) activity. Ensuing pathological remodelling promotes cardiomyocyte death and cardiac fibroblast activation, leading to cardiac fibrosis. The initiating cellular mechanisms that underlie this progressive disease are poorly understood. We previously reported a conditional mouse model in which a human angiotensin II type-I receptor transgene (HART) was expressed in differentiated cardiomyocytes after they had fully matured, but not during development. Twelve-month-old HART mice exhibited ventricular dysfunction and cardiomyocyte hypertrophy with interstitial fibrosis following full receptor stimulation, without affecting blood pressure. Here, we show that chronic HART activity in young adult mice causes ventricular dysfunction without hypertrophy, fibrosis or cardiomyocyte death. Dysfunction correlated with reduced expression of pro-hypertrophy markers and increased expression of pro-angiogenic markers in the cardiomyocytes experiencing increased receptor load. This stimulates responsive changes in closely associated non-myocyte cells, including the downregulation of pro-angiogenic genes, a dampened inflammatory response and upregulation of Tgfβ. Importantly, this state of compensated dysfunction was reversible. Furthermore, increased stimulation of the receptors on the cardiomyocytes caused a switch in the secondary response from the non-myocyte cells. Progressive cardiac remodelling was stimulated through hypertrophy and death of individual cardiomyocytes, with infiltration, proliferation and activation of fibroblast and inflammatory cells, leading to increased angiogenic and inflammatory signalling. Together, these data demonstrate that a state of pre-hypertrophic compensated dysfunction can exist in affected individuals before common markers of heart disease are detectable. The data also suggest that there is an initial response from the housekeeping cells of the heart to signals emanating from distressed neighbouring cardiomyocytes to suppress those changes most commonly associated with progressive heart disease. We suggest that the reversible nature of this state of compensated dysfunction presents an ideal window of opportunity for personalised therapeutic intervention.

GW25-e0269 Angiotensin II receptor antagonist attenuates cigarette smoke-induced pulmonary vascular remodeling in rats: Potential involvement of angiotensin-converting enzyme-2

Renin-angiotensin system(RAS) is known to function in the development of Pulmonary vascular remodeling. Losartan, a specific angiotensin II receptor antagonist, is a well-known antihypertensive drug with a potential role in regulating angiotensin - converting enzyme-2(ACE2). To determine the effect

The inhibitory mechanism analysis of human chymase specific inhibitor TJK002

Chymase is a mast cell specific serine protease that is stored within secretory granules and released together with heparin and histamine in response to allergen challenge or other stimuli. Recent study have shown that chymase possess processing activity for biological peptides and cytokines implicated in a variety of diseases. For example, the chymases of primateds and dogs have highly specific angiotensin II generating activity, and the results of animal studies suggest the chymase contributes to the pathogenesis of cardiovascular diseases via Ang II generation. Based on the results of these studies, inhibition of chymase is expected to provide therapeutic means for the treatment of these diseases. Recently, we have developed some novel benzimidazole derived in human chymase inhibitors and the crystal structure of human chymase complexed with a nobel benzimidazole inhibitor TJK002 was obtained at 2.8A. TJK002 showed potent inhibitory activity (Ki value 2.24nM) with respect to human chymase. X-ray crystallographic structure showed that THK002 forms an anon-covalent interaction with the catalytic domain of human chymase. 4-methylbenzothiophen-3-yl moiety of TJK002 occupies the S1 pocket. The carboxylic acid for hydrogen bonds with the imidazole N (e) atom of H57 and O (g) atoms of Ser195 at the catalytic site. The binding mode of TJK002 is very unique and attractive compared other cymase inhibitors. It depends on the binding mode of TJK002 and reflects to its biological function. Because by site-directed mutagenesis, human mast cell chymase are responsible for an acidic amino acid residue preference in the P2' position of substrates. Chymase inhibitors besides TJK002 have negatively charged p2' moiety. But TJK002 doesn't have such a moiety which binds to S2' site, but it still has a strong incisory activity. We clarify its strong inhibitory mechanism of TJK002 by Fragment molecular orbital (FMO) calculation. We discovered the stacking interaction between the benzimidazole ring and His57 is quite effective for the inhibitory activity of TJK002.

Angiotensin Blockade—A Double-Edged Sword in Renal Failure

Angiotensin receptor suppression in macrophages infiltrating damaged kidneys exacerbates renal fibrosis.

OP0224 Anti-AT1R and Anti-ETAR Autoantibodies from Patients with SSC and their Agonistic Effects

BackgroundAutoantibodies reactive to angiotensin II type 1 receptor (AT1R) and endothelin 1 type A receptor (ETAR), anti-AT1R and anti-ETAR autoantibodies, are found in patients with Systemic sclerosis (SSc) as a...

Losartan

Losartan

Regulation of angiotensin II type 2 receptor gene expression in the adrenal medulla by acute and repeated immobilization stress

While the renin-angiotensin system is important for adrenomedullary responses to stress, the involvement of specific angiotensin II (Ang II) receptor subtypes is unclear. We examined gene expression changes of angiotensin II type 1A (AT(1A)) and type 2 (AT(2)) receptors in rat adrenal medulla in response to immobilization stress (IMO). AT(2) receptor mRNA levels decreased immediately after a single 2-h IMO. Repeated IMO also decreased AT(2) receptor mRNA levels, but the decline was more transient. AT(1A) receptor mRNA levels were unaltered with either single or repeated IMO, although binding was increased following repeated IMO. These effects of stress on Ang II receptor expression may alter catecholamine biosynthesis, as tyrosine hydroxylase and dopamine β-hydroxylase mRNA levels in PC12 cells are decreased with Ang II treatment in the presence of ZD7155 (AT(1) receptor antagonist) or with CGP42112 (AT(2) receptor agonist) treatment. Involvement of stress-triggered activation of the hypothalamic-pituitary-adrenocortical or sympathoadrenal axis in AT(2) receptor downregulation was examined. Cultured cells treated with the synthetic glucocorticoid dexamethasone displayed a transcriptionally mediated decrease in AT(2) receptor mRNA levels. However, glucocorticoids are not required for the immediate stress-triggered decrease in AT(2) receptor gene expression, as demonstrated in corticotropin-releasing hormone knockout (Crh KO) mice and hypophysectomized rats, although they can regulate basal gene expression. cAMP and pituitary adenylate cyclase-activating polypeptide also reduced AT(2) receptor gene expression and may mediate this response. Overall, the effects of stress on adrenomedullary AT(1A) and AT(2) receptor expression may contribute to allostatic changes, such as regulation of catecholamine biosynthesis.

Intrarenal angiotensin III is the predominant agonist for proximal tubule angiotensin type 2 receptors.

In angiotensin type 1 receptor-blocked rats, renal interstitial (RI) administration of des-aspartyl(1)-angiotensin II (Ang III) but not angiotensin II induces natriuresis via activation of angiotensin type 2 receptors. In the present study, renal function was documented during systemic angiotensin type 1 receptor blockade with candesartan in Sprague-Dawley rats receiving unilateral RI infusion of Ang III. Ang III increased urine sodium excretion, fractional sodium, and lithium excretion. RI coinfusion of specific angiotensin type 2 receptor antagonist PD-123319 abolished Ang III-induced natriuresis. The natriuretic response observed with RI Ang III was not reproducible with RI angiotensin (1-7) alone or together with angiotensin-converting enzyme inhibition. Similarly, neither RI angiotensin II alone or in the presence of aminopeptidase A inhibitor increased urine sodium excretion. In the absence of systemic angiotensin type 1 receptor blockade, Ang III alone did not increase urine sodium excretion, but natriuresis was enabled by the coinfusion of aminopeptidase N inhibitor and subsequently blocked by PD-123319. In angiotensin type 1 receptor-blocked rats, RI administration of aminopeptidase N inhibitor alone also induced natriuresis that was abolished by PD-123319. Ang III-induced natriuresis was accompanied by increased RI cGMP levels and was abolished by inhibition of soluble guanylyl cyclase. RI and renal tissue Ang III levels increased in response to Ang III infusion and were augmented by aminopeptidase N inhibition. These data demonstrate that endogenous intrarenal Ang III but not angiotensin II or angiotensin (1-7) induces natriuresis via activation of angiotensin type 2 receptors in the proximal tubule via a cGMP-dependent mechanism and suggest aminopeptidase N inhibition as a potential therapeutic target in hypertension.

Valsartan, a specific angiotensin II receptor blocker, inhibits pancreatic fluid secretion via vagal afferent pathway in conscious rats

Background/aimThe renin–angiotensin system (RAS) exists in the pancreas, but the role of RAS in the regulation of pancreatic exocrine secretion under physiological conditions has been little known. The present study addressed the RAS's effect on the pancreatic secretion by using valsartan, a specific angiotensin II receptor blocker, in conscious rats. MethodMale Wistar rats prepared with pancreatic, biliary, duodenal and jugular vein cannulas were used. To examine the role of RAS in the pancreatic secretion, valsartan at 1, 5, or 25mg/kg was administered into the duodenum via cannula, and volume of pancreatic juice and protein concentration were determined. In addition, to examine the role of RAS in hormone-stimulated pancreatic hypersecretion, pancreatic secretion was examined in response to stimulation of secretin or cholecystokinin after intraduodenal infusion of valsartan at 25mg/kg. Furthermore, to examine the mechanism of action of RAS on pancreatic secretion, intravenous infusion of atropine or perivagal application of capsaicin was conducted and then the pancreatic secretion was examined following intraduodenal infusion of valsartan at 25mg/kg. ResultsVolume of pancreatic juice, but not protein output, significantly decreased after administration of valsartan. However, administration of valsartan did not exert significant effects on secretin- or cholesystokinin-stimulated pancreatic secretion. Treatment with atropine and perivagal application of capsaicin completely abolished the suppressive effect of valsartan on pancreatic juice secretion. ConclusionPresent results suggest that RAS plays a stimulatory role in pancreatic juice secretion via cholinergic afferent pathway without affecting protein secretion and hormonally stimulated pancreatic secretion under physiological conditions.

Effects of valsartan on L-type calcium current in ventricular myocytes of rabbits after myocardial infarction

Objective To determine the effects of valsartan, a specific angiotensin Ⅱ type 1 receptor blockade, on arrhythmia in rabbits after myocardial infarction and to discuss the mechanism. Method Twentyfour rabbits were randomly (random number) divided into sham operated (SO) group ( n = 8), myocardial infarction (MI) group ( n = 8) and valsartan (VAL) group ( n = 8). The rabbits of SO group were operated upon with median stemotomy without left ventricular coronary artery hgature. The rabbits of MI group and VAL group had median stemotomy with left ventricular coronary artery ligature. After MI, the rabbits of VAL group were fed with border zone of infracted left ventricular wall and the L-type calcium current was recorded by whole-cell patch clamp technique. Results Ventricular tachycardia or fibrillation (VT/VF) episodes were markedly decreased in VAL group than that in MI group [(3.2 ± 0. 6) vs. ( 11.7 ± 1.8)] after 12 weeks. The density of Ica-L current was higher in MI group than that in SO group and VAL group [( - 9.12 ± 0.73) pA/pF vs. ( - 6.29 ± 0.65) pA/pF and ( - 6.75 ± 0.64) pA/pF], ( P ＜ 0.05), however, there were no significant differences in Ica-L current between So group and VAL group ( P ＞ 0.05). Conclusions Valsartan reduces the VT/VF episodes in rabbits after MI. The effects of valsartan may be attributed to the inhibited electrical remodeling after MI. Key words: Myocardial infarction; Ventricular arrhythmias; L-type calcium current; Patch clamp; Valsartan; Rabbit

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Objective of the present study was to develop hydrophilic polymer and hydrophobic polymer based matrix Losartan potassium sustained release tablet which can release the drug up to time of 24 hrs in predetermined rate. Formulation of Losartan potassium matrix tablet was prepared by the polymer combination in order to get required theoretical release profile. Influence of hydrophilic and hydrophobic polymer on Losartan potassium was studied. Formulated tablet were also characterized by physical and chemical parameters. In vitro release profile was check for 24 hrs to evaluate the SR matrix tablet of Losartan potassium. Losartan potassium (LP) is a potent, highly specific Angiotensin II type 1 (AT1) receptor antagonist with antihypertensive activity. It is readily absorbed from the gastrointestinal tract with oral bioavailability of about 33% and a plasma elimination half-life ranging from 1.5 to 2.5 hr. Administration of LP in a sustained release dosage would be more desirable for anti- hypertensive effects by maintaining the plasma concentrations of the drug well above the therapeutic concentration. From in vitro dissolution profile, Batch B4 was prepared with blend of HPMC K4M (67.2 mg), HPMC K200M(90mg) and Eudragit RSPO(112.5 mg), where drug release was about 94-98%. Batch B4 showed highest similarity factor values (f 2 = 67.76). KEYWORDS: Losartan potassium, HPMC K4M, HPMC K200M, Eudragit RSPO, Sustained release, Matrix tablets.

Losartan attenuates chronic cigarette smoke exposure-induced pulmonary arterial hypertension in rats: Possible involvement of angiotensin-converting enzyme-2

Chronic cigarette smoking induces pulmonary arterial hypertension (PAH) by largely unknown mechanisms. Renin–angiotensin system (RAS) is known to function in the development of PAH. Losartan, a specific angiotensin II receptor antagonist, is a well-known antihypertensive drug with a potential role in regulating angiotensin-converting enzyme-2 (ACE2), a recently found regulator of RAS. To determine the effect of losartan on smoke-induced PAH and its possible mechanism, rats were daily exposed to cigarette smoke for 6 months in the absence and in the presence of losartan. Elevated right ventricular systolic pressure (RVSP), thickened wall of pulmonary arteries with apparent medial hypertrophy along with increased angiotensin II (Ang II) and decreased ACE2 levels were observed in smoke-exposed-only rats. Losartan administration ameliorated pulmonary vascular remodeling, inhibited the smoke-induced RVSP and Ang II elevation and partially reversed the ACE2 decrease in rat lungs. In cultured primary pulmonary artery smooth muscle cells (PASMCs) from 3- and 6-month smoke-exposed rats, ACE2 levels were significantly lower than in those from the control rats. Moreover, PASMCs from 6-month exposed rats proliferated more rapidly than those from 3-month exposed or control rats, and cells grew even more rapidly in the presence of DX600, an ACE2 inhibitor. Consistent with the in vivo study, in vitro losartan pretreatment also inhibited cigarette smoke extract (CSE)-induced cell proliferation and ACE2 reduction in rat PASMCs. The results suggest that losartan may be therapeutically useful in the chronic smoking-induced pulmonary vascular remodeling and PAH and ACE2 may be involved as part of its mechanism. Our study might provide insight into the development of new therapeutic interventions for PAH smokers.

Localization of ACE2 in the renal vasculature: amplification by angiotensin II type 1 receptor blockade using telmisartan

Angiotensin-converting enzyme (ACE)2 is a carboxypeptidase that degrades angiotensin II and other peptides. In the kidney, ACE2 localization within the glomerulus and tubules is cell specific. This study was aimed to investigate the localization of ACE2 within the renal vasculature. We also studied the effect of the administration of a specific angiotensin II type 1 receptor blocker, telmisartan, on ACE2 expression in the renal vasculature. ACE2 and ACE were localized in renal arterioles using confocal microscopy and specific cell markers. Quantitative measurements of ACE2 and ACE mRNA were estimated in kidney arterioles isolated by laser capture microdissection using real-time PCR. In kidney arterioles, ACE was localized in the endothelial layer, whereas ACE2 was localized in the tunica media. In mice treated with telmisartan (2 mg.kg(-1).day(-1)) for 2 wk, ACE2 expression was increased by immunostaining, whereas ACE expression was decreased. This was reflected in a decrease in the ACE/ACE2 ratio compared with vehicle-treated controls (0.53 +/- 0.14 vs. 7.59 +/- 2.72, P = 0.027, respectively). In kidney arterioles isolated by laser capture microdissection, the ACE/ACE2 mRNA ratio was also decreased compared with control mice (1.21 +/- 0.31 vs. 4.63 +/- 0.86, P = 0.044, respectively). In conclusion, in kidney arterioles ACE2 is preferentially localized in the tunica media, and its expression is increased after administration of the angiotensin II type 1 receptor blocker, telmisartan. Amplification of ACE2 in the renal vasculature may contribute to the therapeutic action of telmisartan by increasing angiotensin II degradation.

Abstract 4368: Differential Left versus Right Atrial Transcriptional Response to Angiotensin Stimulation

Introduction : Angiotensin stimulation is associated with atrial arrhythmias. Atrial fibrillation is commonly initiated in the left atrium. Cardiac over-expression of members of the angiotensin receptor signaling pathway correlates with atrial dilatation. Similarly cardiac over-expression of the inhibitory basic leucine zipper (bZIP) transcription factors JDP2 and ATF3 result in atrial dilatation. We have recently found a signaling pathway in which Angiotensin and Isoproterenol induce ATF3 experssion in the atria. Hypothesis : ATF3 expression may differ in left versus right atria. Methods and Results : mice were IP injected with Angiotensin, Isoproterenol or Saline. ATF3 expression in the left and right atria and in ventricles was analyzed using western blots and by immuno- histochemisty staining of paraffin embedded sections. Whereas Isoproterenol induced ATF3 expression in both atria and in ventricular tissue. Angiotensin induced ATF3 expression in the left atrium and in the ventricls but not in the right atrium (fig ). Analysis for other bZIP transcription factors revealed a responce to angiotensin in both atria. Conclusions and Implication : Angiotensin but not isoproterenol induces ATF3 expression differentially in the left but not right atria. To the best of our knowledge this is the first description of left atrial specific angiotensin mediated expression. This finding may be important in understanding atrial pathophysiological response to hemodynamic challenge and pathologies such as dilatation and generation of arrhythmia.

Human brain contains a novel non-AT1, non-AT2 binding site for active angiotensin peptides

Aims To determine whether the novel non-AT1, non-AT2 binding site for angiotensins recently discovered in rodent brains occurs in the human brain. Main methods Radioligand binding assays of 125I-sarcosine 1, isoleucine 8 angiotensin II binding were carried out in homogenates of the rostral pole of the temporal cortex of human brains containing 0.3 mM parachloromercuribenzoate (PCMB), 10 µM losartan to saturate AT1 receptors, 10 µM PD123319 to saturate AT2 receptors, with or without 10 µM angiotensin II to define specific binding. Competition binding assays employed a variety of angiotensin peptides, specific angiotensin receptor antagonists, several neuropeptides and an endopeptidase inhibitor to determine pharmacological specificity for this binding site. Key findings The novel non-AT1, non-AT2 binding site was present in similar amounts in female and male brains: Bmax 1.77 ± 0.16 and 1.52 ± 0.17 fmol/mg initial wet weight in female and male brains, respectively. The K D values, 1.79 ± 0.09 nM for females, and 1.53 ± 0.06 nM for males were also similar. The binding site shows pharmacological specificity similar to that in rodent brains: sarcosine 1, isoleucine 8 angiotensin II > angiotensin III > angiotensin II > angiotensin I ’ angiotensin IV > angiotensin 1–7. Shorter angiotensin fragments and non-angiotensin peptides showed low affinity for this binding site. Significance The presence in human brain of this novel non-AT1, non-AT2 binding site supports the concept that this binding site is an important component of the brain angiotensin system. The functional significance of this binding site, either as a novel angiotensin receptor or a highly specific angiotensinase remains to be determined.

Angiotensin metabolites can stimulate receptors of the Mas-related genes family

The Mas protooncogene encodes a G protein-coupled receptor, we identified, also by using the specific angiotensin-(1-7) antagonist A-779, to be associated with intracellular signaling of the angiotensin (Ang) II metabolite Ang-(1-7). Recently, Mas-related genes (Mrg) have been identified coding for the Mrg-receptor family. All family members share high sequence homology to Mas. Most of them are orphan receptors. To proof whether structure similarities of the Mrg receptors with Mas turn them into potential receptors for Ang-(1-7) or other Ang metabolites, we transfected COS or HEK293 cells with an assortment of Mrg receptors and investigated arachidonic acid (AA) release and transcriptional activation by recording serum response factor (SRF) activation after stimulation with Ang II, Ang III, Ang IV, and Ang-(1-7). None of the investigated receptors activated transcription via SRF. Ang-(1-7) stimulated AA release already in control vector-transfected COS cells, indicating the existence of an endogenous receptor (A-779 sensitive). Though less pronounced than for Mas, two of the six studied receptors (MrgD, MRG) initiated significant AA release after stimulation with Ang-(1-7). Interestingly, Mas, MrgD, and MRG mediated Ang IV-stimulated AA release that was highest for Mas. While Ang III activated Mas and MrgX2, Ang II stimulated AA release via Mas and MRG. Thus, we identified other receptors of the Mrg family to respond on Ang-(1-7) stimulation. Furthermore, we describe first an AT(1)-independent direct Ang IV signaling and show that Ang II and Ang III mediate signaling independent of their specific receptors AT(1) and AT(2), whereby the receptor specificity differs.

The Relationship Between the Plasma Concentration of Irbesartan and the Antihypertensive Response Is Disclosed by an Angiotensin II Type 1 Receptor Polymorphism: Results From the Swedish Irbesartan Left Ventricular Hypertrophy Investigation vs. Atenolol (SILVHIA) Trial

The aim of this study was to investigate the effect of the plasma concentration of irbesartan, a specific angiotensin II type 1 receptor (AT1R) antagonist, and the blood pressure response in relation to AT1R gene polymorphisms. Plasma irbesartan was analyzed in 42 patients with mild-to-moderate hypertension and left ventricular hypertrophy from the Swedish Irbesartan Left Ventricular Hypertrophy Investigation vs. Atenolol (SILVHIA) trial, who were treated with irbesartan as monotherapy for 12 weeks. Blood pressure and irbesartan concentration were measured at trough, i.e., 24 +/- 3 h after the last dose. Five AT1R gene polymorphisms were analyzed by minisequencing. Neither the plasma concentration of irbesartan, nor any of the AT1R polymorphisms were associated with the blood pressure response to irbesartan treatment. However, the interaction term between the plasma concentration of irbesartan and the AT1R C5245T polymorphism was related to the reduction in systolic blood pressure after 12 weeks of treatment (P = 0.025). Furthermore, the plasma concentration of irbesartan was related to the change in systolic blood pressure in individuals homozygous for the AT1R 5245 T allele (r = -0.56, P = 0.030), but not for other genotypes. There was an association between plasma concentrations of irbesartan and the blood pressure response for hypertensive patients with AT1R 5245 TT. Because of the small sample size, this study needs to be viewed as hypothesis generating. This is the first study, to our knowledge, indicating that the concentration-response relationship of an antihypertensive drug may be genotype dependent.

EFFECT OF RENIN-ANGIOTENSIN SYSTEM ON REGULATION OF PANCREATIC SECRETION IN CONSCIOUS RATS

Background/Aim: Although previous studies have shown the presence of renin-angiotensin system (RAS) in the pancreas, the effect of RAS on regulation of pancreatic secretion has remained controversial. In the present study, we therefore investigated the effect of RAS on pancreatic secretion by using valsartan, a specific angiotensin II receptor blocker in conscious rats. Method: Male Wistar rats prepared with pancreatic, bilialy, duodenal, and intravenous cannulas were used in the study. Valsartan at a dose of 0, 1, 5, or 25 mg/kg was administrated into the duodenum, and volume of pancreatic fluid and protein output were determined for 4 hours. In addition, to examine the mechanism of action of RAS on pancreatic secretion, perivagal application of capsaicin (1 mg/rat) or intravenous infusion of atropine (100 μg/kg/hr) was conducted and then pancreatic secretion was examined after administration of valsartan at 25 mg/kg. Furthermore, to examine the influence of RAS on CCK or secretin-stimulated pancreatic secretion, valsartan at 25mg/kg was administrated and then pancreatic secretion was examined during intravenous infusion of CCK or secretin. Results: Pancreatic fluid secretion, but not protein output significantly decreased after administration of valsartan at 5 and 25 mg/kg. Perivagal application of capsaicin and intravenous infusion of atropine completely abolished the decrease in the pancreatic fluid secretion by valsartan. Valsartan at 25 mg/kg did not exert any effects on the CCK or secretin-stimulated pancreatic secretion. Conclusion: Present results suggest that pancreatic RAS participates in the stimulation of basal pancreatic fluid secretion via cholinergic pathway and has no influence on CCK or secretin-stimulated pancreatic secretion in conscious rats.

Abstract 1088: Prevention of Connexin 43 (Cx43) Dephosphorylation is Associated with Reduced Ventricular Tachycardia in Cardiac-Specific Angiotensin Converting Enzyme (ACE) Overexpression Mice

Introduction: Connexin 43 (Cx43) is a major component of the ventricular gap junctional complex, which provides a low resistance pathway for electrical conduction between myocytes. Reduction of Cx43 amount of phosphorylation has been implicated in the pathogenesis of ventricular tachycardia (VT) and fibrillation. Cardiac-specific angiotensin converting enzyme overexpression mice (ACE 8/8) have slow conduction, show conduction blocks, are prone to VT and sudden death, and show profound reductions in Cx43 levels and phosphorylation. Therefore, we hypothesized that reversal of Cx43 changes, by preventing the renin-angiotensin system (RAS) activation, would reduce the arrhythmic risk in these animals. Methods: Prevention of RAS activation was accomplished by one of three methods: ACE 8/8 receiving an ACE inhibitor (captopril), ACE 8/8 mice treated with a specific angiotensin receptor type-1 blocker (losartan), and ACE 8/8 mice crossed with homozygote angiotensinogen null mice. Pooled results were compared to untreated ACE 8/8 littermates. Surface electrocardiography and invasive electrophysiological study with programmed ventricular stimulation were performed on ten-week old mice, and the hearts were extracted for immunoblotting. Results: VT was induced in 10 out of 12 untreated ACE 8/8 mice, but only in 3 out of 11 treated mice (p<0.02). Total Cx43 was not changed significantly in treated animals as compared to controls, but the ratio (P/NP) of phosphorylated Cx43 (bands P1 and P2 in immunoblot) to non-phosphorylated Cx43 (band P0) showed a trend toward increase in the treatment group (0.78±0.18 vs. 1.25±0.19; P=0.09). Dividing the mice into two groups, high-risk (inducible and/or spontaneous VT) versus low-risk (no VT), we observed a significant difference in P/NP ratio (0.75±0.12 in high-risk mice vs. 1.78±0.32 in low-risk, p<0.01). Discussion: Unexpectedly, disruption of RAS signaling did not increase total Cx43 levels in ACE 8/8 mice. On the other hand, the treatments increased phospho-Cx43, suggesting that RAS controls Cx43 phosphorylation but not total amount. Even in the presence of significantly depressed levels of Cx43, small changes in Cx43 phosphorylation were associated with a significant reduction in VT inducibility.

Oral Bioavailability of Candesartan Cilexetil Suspension

Background: Candesartan cilexetil is a prodrug that is converted to candesartan during absorption from the gastrointestinal tract. Candesartan is a potent, orally active, specific angiotensin II type 1 receptor antagonist that has become a well-accepted treatment for hypertension. Pediatric patients often require alternative dosage forms, as candesartan cilexetil is not yet commercially available as a liquid formulation. Objective: To determine the relative bioavailability of candesartan by comparing a candesartan cilexetil tablet (reference formulation) with an oral candesartan cilexetil suspension (test formulation). Methods: In an open-label, randomized, 2 period, 2 treatment crossover study, 24 healthy volunteers were given a 32 mg candesartan cilexetil tablet and a 32 mg candesartan cilexetil suspension with a 7 day washout period between treatments. Results: Pharmacokinetic analyses showed that the mean relative bioavailability of the candesartan cilexetil commercial 32 mg tablet compared with the suspension of 32 mg candesartan cilexetil was 93%. The 90% CIs of the plasma AUC for the tablet versus suspension (0.861 and 0.998, respectively) were well within the bioequivalence criteria of 80–125%. Administration of candesartan cilexetil suspension was associated with a 17% increase in the maximum plasma concentration (Cmax) relative to the tablet administration (643 vs 523 nM/L, respectively). The 90% CIs on the ratio of mean Cmax for the tablet and suspension were 0.738 and 0.914, respectively. Mean time to peak plasma concentrations (tmax) after suspension administration was 3.1 hours, whereas the tablet resulted in a mean tmax of 3.9 hours. No significant differences in elimination half-life were observed between the 2 formulations. Both formulations were well tolerated, and no serious adverse events were reported. Conclusions: These results demonstrate that administration of candesartan cilexetil suspension achieves an extent of absorption and tolerability that is comparable with those of orally administered candesartan cilexetil tablets.