Inotropic effects of prokinetic agents with 5-HT(4) receptor agonist actions on human isolated myocardial trabeculae.

AIMS: Besides acting as gastrointestinal prokinetic agents, 5-hydroxytryptamine(4) (5-HT(4)) receptor agonists can induce positive inotropism in human isolated atrium, but not in ventricles. We pharmacologically evaluated the gastroprokinetic 5-HT(4) receptor agonists tegaserod, prucalopride, R199715, cisapride, the cisapride metabolite norcisapride, and the 5-HT(3) receptor agonist MKC773 on human isolated myocardial trabeculae, and compared their effects with those induced by 5-HT and 5-methoxytryptamine (5-MeOT). MAIN METHODS: Atrial and ventricular trabeculae were paced and changes in contractile force were studied in the absence or presence of the 5-HT(4) receptor antagonist GR113808. Partial agonism was assessed using 5-HT(4) receptor agonists as antagonists against 5-HT. To test the contribution of L-type calcium channels, the inotropic responses to 5-HT and 5-MeOT were studied in the absence or presence of verapamil. KEY FINDINGS: Like 5-HT and 5-MeOT, cisapride and tegaserod, but not prucalopride, R19971 and MKC-733, induced concentration-dependent positive inotropic responses on atrial trabeculae, which were abolished by GR113808. The L-type calcium channel blocker verapamil attenuated inotropic responses to 5-HT and 5-MeOT. None of the agonists affected the contraction of left ventricular trabeculae. Concentration response curves to 5-HT were shifted to the right in the presence of prucalopride, cisapride, tegaserod and R199715, but not MKC-773. SIGNIFICANCE: We conclude that (i) inotropic responses to 5-HT and 5-MeOT seem to depend on L-type calcium channels, (ii) tegaserod and cisapride behave as partial 5-HT(4) receptor agonists, while prucalopride, norcisapride and MKC-733 cause no significant effects on human atrial trabeculae, (iii) R199715 seems to behave as a 5-HT(4) receptor antagonist.

Mast cell degranulation mediates bronchoconstriction via serotonin and not via renin release.

To verify the recently proposed concept that mast cell-derived renin facilitates angiotensin II-induced bronchoconstriction bronchial rings from male Sprague-Dawley rats were mounted in Mulvany myographs, and exposed to the mast cell degranulator compound 48/80 (300 microg/ml), angiotensin I, angiotensin II, bradykinin or serotonin (5-hydroxytryptamine, 5-HT), in the absence or presence of the renin inhibitor aliskiren (10 micromol/l), the ACE inhibitor captopril (10 micromol/l), the angiotensin II type 1 (AT1) receptor blocker irbesartan (1 micromol/l), the mast cell stabilizer cromolyn (0.3 mmol/l), the 5-HT2A/2C receptor antagonist ketanserin (0.1 micromol/l) or the alpha1-adrenoceptor antagonist phentolamine (1 micromol/l). Bath fluid was collected to verify angiotensin generation. Bronchial tissue was homogenized to determine renin, angiotensinogen and serotonin content. Compound 48/80 contracted bronchi to 24+/-4% of the KCl-induced contraction. Ketanserin fully abolished this effect, while cromolyn reduced the contraction to 16+/-5%. Aliskiren, captopril, irbesartan and phentolamine did not affect this response, and the angiotensin I and II levels in the bath fluid after 48/80 exposure were below the detection limit. Angiotensin I and II equipotently contracted bronchi. Captopril shifted the angiotensin I curve approximately 10-fold to the right, whereas irbesartan fully blocked the effect of angiotensin II. Bradykinin-induced constriction was shifted approximately 100-fold to the left with captopril. Serotonin contracted bronchi, and ketanserin fully blocked this effect. Finally, bronchial tissue contained serotonin at micromolar levels, whereas renin and angiotensinogen were undetectable in this preparation. In conclusion, mast cell degranulation results in serotonin-induced bronchoconstriction, and is unlikely to involve renin-induced angiotensin generation.

Steroidogenesis vs. steroid uptake in the heart: do corticosteroids mediate effects via cardiac mineralocorticoid receptors?

OBJECTIVE: To test whether glucocorticoids act as the endogenous agonist of cardiac mineralocorticoid receptors, we evaluated the cardiac effects of aldosterone and corticosterone and cardiac steroidogenesis vs. steroid uptake from plasma. METHODS AND RESULTS: Both corticosterone and aldosterone increased left ventricular pressure in the rat heart. Aldosterone decreased coronary flow, whereas corticosterone increased it. All corticosterone effects were blocked by the glucocorticoid receptor antagonist, RU486, and unaltered by the mineralocorticoid receptor antagonist, canrenoate, or the 11beta-hydroxysteroid dehydrogenase (HSD11B)2 inhibitor, carbenoxolone. Unlike mineralocorticoid receptor blockade, RU486 did not ameliorate postischemia infarct size and arrhythmias. Corticosterone, when added to the perfusion buffer, rapidly accumulated at cardiac tissue sites, reaching steady-state levels that were identical to those in coronary effluent, independently of the presence of aldosterone, RU486 or canrenoate. After stopping the perfusion, cardiac corticosterone fully washed away with a half-life of less than 1 min. Measurements of steroid-synthesizing enzyme gene expression levels in human ventricular and atrial tissue pieces from heart-beating organ donors, patients with end-stage heart failure and hypertrophic cardiomyopathy patients revealed that under no condition, the human heart was capable of synthesizing aldosterone or cortisol de novo. Yet, expression of HSD11B1, HSD11B2, mineralocorticoid receptors and glucocorticoid receptors was found, and HSD11B2 and mineralocorticoid receptors were upregulated in pathological conditions. Moreover, aldosterone reduced cardiac inotropy in a Na/K/2Cl cotransporter-dependent manner. CONCLUSION: Both cortisol/corticosterone and aldosterone accumulate in the cardiac interstitium. The presence of HSD11B2 and mineralocorticoid receptors/glucocorticoid receptors at cardiac tissue sites allows both steroids to exert their effects via separate mechanisms.

Structural abnormalities of the inferoseptal left ventricular wall detected by cardiac magnetic resonance imaging in carriers of hypertrophic cardiomyopathy mutations.

OBJECTIVES: The purpose of this study was to evaluate whether structural left ventricular (LV) abnormalities can be observed in hypertrophic cardiomyopathy (HCM) mutation carriers who have not yet developed echocardiographic signs of hypertrophy by using cardiac magnetic resonance imaging (CMR). BACKGROUND: Hypertrophic cardiomyopathy is caused by mutations of genes encoding for sarcomeric proteins. Myocyte disarray and interstitial fibrosis precede the development of regional hypertrophy in HCM mutation carriers (carriers). No macroscopic LV structural abnormalities have been observed in carriers without LV hypertrophy. METHODS: A CMR, echocardiogram, and electrocardiogram (ECG) were performed in 16 carriers. Delayed contrast enhancement imaging was used with CMR to detect fibrosis. In 16 age- and gender-matched control subjects, CMR and ECG were performed and an echocardiogram was made when structural abnormalities were detected with CMR. All carriers had an LV wall thickness <13 mm in the year before the study, measured by echocardiography. RESULTS: In 13 carriers (81%), crypts were discerned with CMR in the basal and mid inferoseptal LV wall, not detected by routine echocardiography and not observed in healthy volunteers. In 4 of the crypt-positive carriers, both the echocardiogram and ECG were normal. Two HCM carriers revealed regional hypertrophy of the inferoseptum not detected by echocardiography, and in both carriers, focal fibrosis was present. CONCLUSIONS: In carriers who have not yet developed frank hypertrophy, crypts can be detected with CMR in the inferoseptal LV wall, even when echocardiography and ECG are normal. The crypts might represent one of the early pathological alterations of myocardium in carriers that ultimately progress into manifest HCM.

Why are mineralocorticoid receptor antagonists cardioprotective?

Two clinical trials, the Randomized ALdosterone Evaluation Study (RALES) and the EPlerenone HEart failure and SUrvival Study (EPHESUS), have recently shown that mineralocorticoid receptor (MR) antagonists reduce mortality in patients with heart failure on top of ACE inhibition. This effect could not be attributed solely to blockade of the renal MR-mediated effects on blood pressure, and it has therefore been proposed that aldosterone, the endogenous MR agonist, also acts extrarenally, in particular in the heart. Indeed, MR are present in cardiac tissue, and possibly aldosterone synthesis occurs in the heart. This review critically addresses the following questions: (1) is aldosterone synthesized at cardiac tissue sites, (2) what agonist stimulates cardiac MR normally, and (3) what effects are mediated by aldosterone/MR in the heart that could explain the beneficial effects of MR blockade in heart failure? Conclusions are that most, if not all, of cardiac aldosterone originates in the circulation (i.e., is of adrenal origin), and that glucocorticoids, in addition to aldosterone, may serve as the endogenous agonist of cardiac MR. MR-mediated effects in the heart include effects on endothelial function, cardiac fibrosis and hypertrophy, oxidative stress, cardiac inotropy, coronary flow, and arrhythmias. Some of these effects occur via or in synergy with angiotensin II, and involve a non-MR-mediated mechanism. This raises the possibility that aldosterone synthase inhibitors might exert beneficial effects on top of MR blockade.

Cardioprotective effects of eplerenone in the rat heart: interaction with locally synthesized or blood-derived aldosterone?

Mineralocorticoid receptor antagonism with eplerenone reduces mortality in heart failure, possibly because of blockade of the deleterious effects of aldosterone. To investigate these effects, rat Langendorff hearts were exposed to aldosterone and/or eplerenone. Under normal conditions, aldosterone increased left ventricular pressure and decreased coronary flow. Eplerenone did not block these effects. Eplerenone reduced infarct size (from 68+/-2% to 53+/-4%; P<0.05) and increased left ventricular pressure recovery (from 44+/-2% to 60+/-5%; P<0.05) after 45 minutes of coronary artery occlusion and 3 hours of reperfusion, whereas aldosterone did not affect these parameters. To verify the origin of cardiac aldosterone, hearts were perfused with 3 to 30 nmol/L aldosterone and either frozen immediately or exposed to washout. Without washout, cardiac aldosterone was 1.5 times aldosterone in coronary effluent (CE), that is, too high to be explained on the basis of its presence in extracellular fluid. The cardiac levels of aldosterone correlated with its CE levels (r=0.81; P<0.01), and both were unaffected by eplerenone. During washout, tissue aldosterone disappeared monophasically (half life, 9+/-1 minutes), and CE aldosterone disappeared biphasically (half life 1+/-0 and 8+/-1 minutes, respectively). During buffer perfusion, cardiac aldosterone was at or below the detection limit. In conclusion, eplerenone improves the condition of the heart after ischemia and reperfusion. This does not relate to interference with the inotropic and vasoconstrictor effects of aldosterone. The majority of cardiac aldosterone, if not all, is derived from the circulation. The rapid, mineralocorticoid receptor-independent kinetics of aldosterone suggest that its accumulation in the heart involves cell surface binding rather than internalization.

The role of calcitonin gene-related peptide (CGRP) in ischemic preconditioning in isolated rat hearts.

Brief coronary artery occlusion can protect the heart against damage during subsequent prolonged coronary artery occlusion; ischemic preconditioning. The role of calcitonin gene-related peptide (CGRP) in ischemic preconditioning is investigated in isolated perfused rat hearts, by measuring CGRP release during ischemic preconditioning and mimicking this by exogenous CGRP infusion, either in the absence or presence of the CGRP antagonist BIBN4096BS. CGRP increased left ventricular pressure and coronary flow in a concentration dependent manner, which was effectively antagonized by BIBN4096BS. Rat hearts (n=36) were subjected to 45 min coronary artery occlusion and 180 min reperfusion, which was preceded by: (1) sham pretreatment, (2) BIBN4096BS infusion (1 microM), (3) preconditioning by 15 min coronary artery occlusion and10 min reperfusion, (4) as 3, but with BIBN4096BS, (5) 15 min CGRP infusion (5 nM) and 10 min washout, (6) as 5, but with BIBN4096BS. Cardiac protection was assessed by reactive hyperaemia, creatine kinase release, infarct size related to the area at risk (%), and left ventricular pressure recovery. Preconditioning increased CGRP release into the coronary effluent from 88+/-13 to 154+/-32 pg/min/g, and significantly protected the hearts by decreasing reactive hyperaemia (35%), reducing creatine kinase release (53%), limiting infarct size (48%), and improving left ventricular pressure recovery (36%). Exogenous CGRP induced preconditioning-like cardioprotection. BIBN completely abolished the cardioprotection induced by preconditioning as well as by exogenous CGRP. In conclusion, since cardioprotection of preconditioning-induced CGRP release can be mimicked by exogenous CGRP, and both can be blocked by a CGRP antagonist, results indicate an important role for CGRP in ischemic preconditioning.

Cardiac aldosterone in subjects with hypertrophic cardiomyopathy.

Left ventricular (LV) hypertrophy in subjects with hypertrophic cardiomyopathy (HCM) is variable, suggesting a role for modifying factors. Here, we determined whether aldosterone modulates hypertrophy in HCM. Cardiac and/or plasma aldosterone were measured in organ donors and HCM patients. The effect of the aldosterone synthase ( CYP11B2 ) C-344T polymorphism on LV mass index (LVMI) and interventricular septum thickness (IVS) was determined in 79 genetically independent subjects with HCM. Aldosterone in HCM hearts and plasma was similar to that in normal hearts and plasma. In HCM women, no associations between CYP11B2 genotype and any of the measured parameters were observed, whereas in HCM men, LVMI increased with the presence of the T allele. Similar T allele-related increases were observed for IVS. Multiple regression analysis revealed that the T allele-related effect on IVS occurred independently of renin, the ACE I/D polymorphism, the AT1-receptor A/C(1166)polymorphism and the AT2-receptor A/C(3123) polymorphism. In conclusion, circulating and cardiac aldosterone are normal in HCM, thereby arguing against selectively increased cardiac aldosterone production in HCM. Thus, the association between the CYP11B2 C-344T polymorphism and hypertrophy in HCM most likely relates to the T allele-related increases in circulating aldosterone. This finding raises the need for studies determining the benefit of aldosterone blockade in HCM.

Nongenomic effects of aldosterone in the human heart: interaction with angiotensin II.

Aldosterone exerts rapid "nongenomic" effects in various nonrenal tissues. Here, we investigated whether such effects occur in the human heart. Trabeculae and coronary arteries obtained from 57 heart valve donors (25 males; 32 females; 17 to 66 years of age) were mounted in organ baths. Aldosterone decreased contractility in atrial and ventricular trabeculae by maximally 34+/-3% and 15+/-4%, respectively, within 5 to 15 minutes after its application. The protein kinase C (PKC) inhibitor chelerythrine chloride, but not the mineralocorticoid receptor antagonists spironolactone and eplerenone, blocked this effect. Aldosterone also relaxed trabeculae that were prestimulated with angiotensin II (Ang II), and its negative inotropic effects were mimicked by hydrocortisone (at 10-fold lower potency) but not 17beta-estradiol. Aldosterone concentrations required to reduce inotropy were present in failing but not in normal human hearts. Previous exposure of coronary arteries to 1 micromol/L aldosterone or 17beta-estradiol (but not hydrocortisone) doubled the maximum contractile response (Emax) to Ang II. DeltaEmax correlated with extracellular signal-regulated kinase (ERK) 1/2 phosphorylation (P<0.01). Spironolactone and eplerenone did not block the potentiating effect of aldosterone. Studies in porcine renal arteries showed that potentiation also occurred at pmol/L aldosterone levels but not at 17beta-estradiol levels <1 micromol/L. Aldosterone did not potentiate the alpha1-adrenoceptor agonist phenylephrine. In conclusion, aldosterone induces a negative inotropic response in human trabeculae (thereby antagonizing the positive inotropic actions of Ang II) and potentiates the vasoconstrictor effect of Ang II in coronary arteries. These effects are specific and involve PKC and ERK 1/2, respectively. Furthermore, they occur in a nongenomic manner, and require pathological aldosterone concentrations.

Adrenal angiotensin: origin and site of generation.

BACKGROUND: Circulating angiotensin (Ang) II accumulates in adrenal tissue via binding to Ang II type 1 (AT1) receptors, reaching levels that are 15 to 20 times higher than in blood. Adrenal tissue contains a second renin transcript that gives rise to a truncated prorenin representing a cytosolic form of renin. Here we investigated what percentage of adrenal Ang II originates at adrenal tissue sites, and whether intracellular renin contributes to adrenal angiotensin production. METHODS: Concentrations of endogenous and iodine-125 (125I)-labeled Ang I and II were measured in adrenal tissue and blood from pigs after 125I-Ang I infusion. RESULTS: In the adrenal tissue in all animals, 125I-Ang I was undetectable. In untreated pigs, adrenal 125I-Ang II was 17 +/- 1 times arterial 125I-Ang II, and tissue Ang I and II were 5 +/- 1 and 388 +/- 40 times higher than plasma Ang I and II. The AT1 receptor antagonist eprosartan reduced adrenal 125I-Ang II accumulation by 80%, and increased plasma Ang II to a greater degree than tissue Ang II. As a consequence, eprosartan equally reduced the tissue/plasma concentration ratios of both Ang II and 125I-Ang II. Captopril did not alter 125I-Ang II accumulation, and acutely, but not chronically, reduced the adrenal Ang II/I ratio. CONCLUSIONS: More than 90% of adrenal Ang II originates at adrenal tissue sites. Local adrenal Ang II generation occurs extracellularly and is followed by internalization via AT1 receptor-mediated endocytosis. Enhanced angiotensin generation, combined with incomplete AT1 receptor blockade and the large adrenal AT1 receptor reserve, explains why eprosartan increased rather than decreased adrenal Ang II. Our data do not support angiotensin generation by truncated prorenin.

Genomic and nongenomic effects of aldosterone in the rat heart: why is spironolactone cardioprotective?

1. Mineralocorticoid receptor (MR) antagonism with spironolactone reduces mortality in heart failure on top of ACE inhibition. To investigate the underlying mechanism, we compared the actions of both aldosterone and spironolactone to those of angiotensin (Ang) II in the rat heart. 2. Hearts of male Wistar rats were perfused according to Langendorff. Ang II and aldosterone increased left ventricular pressure (LVP) by maximally 11+/-4 and 9+/-2%, and decreased coronary flow (CF) by maximally 36+/-7 and 20+/-4%, respectively. Spironolactone did not significantly affect LVP or CF. 3. In hearts that were exposed to a 45-min coronary artery occlusion and 3 h of reperfusion, a 15-min exposure to spironolactone prior to occlusion reduced infarct size (% of risk area) from 68+/-2 to 45+/-3%, similar to the reduction (34+/-2%) observed following 'preconditioning' (15 min occlusion followed by 10 min reperfusion) prior to the 45-min occlusion. Aldosterone exposure did not affect infarct size (71+/-5%). 4. In cardiomyocytes, aldosterone decreased [(3)H]thymidine incorporation maximally by 73+/-3%, whereas in cardiac fibroblasts it decreased [(3)H]proline incorporation by 33+/-7%. Spironolactone inhibited both effects. Ang II increased DNA and collagen synthesis, and these effects were reversed by aldosterone. 5. In conclusion, aldosterone induces positive inotropic and vasoconstrictor effects in a nongenomic manner, and these effects are comparable to those of Ang II. Aldosterone reduces DNA and collagen synthesis via MR activation, and counteracts the Ang II-induced increases in these parameters. MR blockade reduces infarct size and increases LVP recovery following coronary artery occlusion. The MR-related phenomena may underlie, at least in part, the beneficial actions of spironolactone in heart failure.

Is angiotensin II made inside or outside of the cell?

Angiotensin synthesis at tissue sites is well-established, and depends largely, if not completely, on kidney-derived renin. The exact tissue site of angiotensin generation (extracellular fluid, cell surface, intracellular compartment) is still being debated. In this review, we discuss the various possibilities, taking into consideration the intracellular occurrence/absence of prorenin, renin, angiotensinogen, angiotensin-converting enzyme, and angiotensin receptors; the local activation of prorenin to renin; the differences between in vivo and in vitro studies; and the methodologic difficulties related to angiotensin measurements. It is eventually concluded that angiotensin generation at tissue sites occurs extracellularly, most likely on the cell surface.