Willem A. Bax

Angiotensin-Converting Enzyme in the Human Heart Effect of the Deletion/Insertion Polymorphism

BACKGROUND An insertion (I)/deletion (D) polymorphism of the angiotensin-converting enzyme (ACE) gene has been associated with differences in the plasma levels of ACE as well as with myocardial infarction, cardiomyopathy, left ventricular hypertrophy, and coronary artery disease. METHODS AND RESULTS We determined the cardiac ACE activity and the ACE genotype in 71 subjects who died of noncardiac disorders. Cardiac ACE activity was significantly higher (P < .01) in subjects with the ACE DD genotype (12.7 +/- 1.9 mU/g wet wt) compared with subjects with the ID (8.7 +/- 0.8 mU/g) and the II (9.1 +/- 1.0 mU/g) genotypes. This difference was independent of sex, age, and the time required for tissue collection. CONCLUSIONS Cardiac ACE activity is highest in subjects with the DD genotype. Elevated cardiac ACE activity in these subjects may result in increased cardiac angiotensin II levels, and this may be a mechanism underlying the reported association between the ACE deletion polymorphism and the increased risk for several cardiovascular disorders.

Coronary Side-Effect Potential of Current and Prospective Antimigraine Drugs

BACKGROUND The antimigraine drugs ergotamine and sumatriptan may cause angina-like symptoms, possibly resulting from coronary artery constriction. We compared the coronary vasoconstrictor potential of a number of current and prospective antimigraine drugs (ergotamine, dihydroergotamine, methysergide and its metabolite methylergometrine, sumatriptan, naratriptan, zolmitriptan, rizatriptan, avitriptan). METHODS AND RESULTS Concentration-response curves to the antimigraine drugs were constructed in human isolated coronary artery segments to obtain the maximum contractile response (Emax) and the concentration eliciting 50% of Emax (EC50). The EC50 values were related to maximum plasma concentrations (Cmax) reported in patients, obtaining Cmax/EC50 ratios as an index of coronary vasoconstriction occurring in the clinical setting. Furthermore, we studied the duration of contractile responses after washout of the acutely acting antimigraine drugs to assess their disappearance from the receptor biophase. Compared with sumatriptan, all drugs were more potent (lower EC50 values) in contracting the coronary artery but had similar efficacies (Emax <25% of K+-induced contraction). The Cmax of avitriptan was 7- to 11-fold higher than its EC50 value, whereas those of the other drugs were <40% of their respective EC50 values. The contractile responses to ergotamine and dihydroergotamine persisted even after repeated washings, but those to the other drugs declined rapidly after washing. CONCLUSIONS All current and prospective antimigraine drugs contract the human coronary artery in vitro, but in view of low efficacy, these drugs are unlikely to cause myocardial ischemia at therapeutic plasma concentrations in healthy subjects. In patients with coronary artery disease, however, these drugs must remain contraindicated. The sustained contraction by ergotamine and dihydroergotamine seems to be an important disadvantage compared with sumatriptan-like drugs.

The current endothelin receptor classification: time for reconsideration?

The possible involvement of endothelins in a variety of diseases has attracted the attention of many pharmacologists in search of a novel therapeutic approach. The rapid development of endothelin research has resulted in the molecular characterization and pharmacological recognition of ETA and ETB receptors, and in the development of compounds selective for these receptors. However, the characterization of receptors in various assays has shown that a number of effects are mediated by receptors that do not fit the present criteria for ETA or ETB receptors. In this article, Willem Bax and Pramod Saxena address endothelin receptors in general, and atypical receptors in particular.

Prorenin, Renin, Angiotensinogen, and Angiotensin-Converting Enzyme in Normal and Failing Human Hearts Evidence for Renin Binding

BACKGROUND A local renin-angiotensin system in the heart is often invoked to explain the beneficial effects of ACE inhibitors in heart failure. The heart, however, produces little or no renin under normal conditions. METHODS AND RESULTS We compared the cardiac tissue levels of renin-angiotensin system components in 10 potential heart donors who died of noncardiac disorders and 10 subjects with dilated cardiomyopathy (DCM) who underwent cardiac transplantation. Cardiac levels of renin and prorenin in DCM patients were higher than in the donors. The cardiac and plasma levels of renin in DCM were positively correlated, and extrapolation of the regression line to normal plasma levels yielded a tissue level close to that measured in the donor hearts. The cardiac tissue-to-plasma concentration (T/P) ratios for renin and prorenin were threefold the ratio for albumin, which indicates that the tissue levels were too high to be accounted for by admixture with blood and diffusion into the interstitial fluid. Cell membranes from porcine cardiac tissue bound porcine renin with high affinity. The T/P ratio for ACE, which is membrane bound, was fivefold the ratio for albumin. Cardiac angiotensinogen was lower in DCM patients than in the donors, and its T/P ratio was half that for albumin, which is compatible with substrate consumption by cardiac renin. CONCLUSIONS These data in patients with heart failure support the concept of local angiotensin production in the heart by renin that is taken up from the circulation. Membrane binding may be part of the uptake process.

5-HT receptors mediating contractions of the isolated human coronary artery

We investigated contractile responses of the isolated human coronary artery to 5-hydroxytryptamine (5-HT), washed human platelets, sumatriptan and ergotamine. 5-HT (pD2: 6.8 +/- 0.1, Emax: 47.7 +/- 6.8 mN) and platelets (effect 14.4 +/- 2.8 mN with 3.10(10) platelets/l) caused contractile responses which were attenuated by ketanserin (1 microM). In the presence of ketanserin (1 microM), both rauwolscine (1 and 10 microM) and cyanopindolol (1 and 10 microM) caused concentration-dependent additional antagonism against contractions induced by low (< or = 1 microM) concentrations of 5-HT. Sumatriptan-induced contractions (pD2: 6.2 +/- 0.1; Emax: 10.7 +/- 2.4 mN) were antagonized to a similar extent by both rauwolscine (1 microM) and cyanopindolol (1 microM) (pKB: 6.5 +/- 0.1 and 6.4 +/- 0.1, respectively) and also by metergoline (0.1 microM; pKB: 7.2 +/- 0.1). The order of potency of antagonists against sumatriptan resembles the order reported for the human saphenous vein 5-HT1D-like receptor. No significant additional antagonism by cyanopindolol (1 microM) or rauwolscine (1 microM) against platelet-induced contractile responses was observed. Ergotamine caused potent contractile responses (pD2: 8.4 +/- 0.3, Emax: 19.4 +/- 2.4 mN). It is concluded that although 5-HT2 receptors predominantly mediate 5-HT-induced contractions, the 5-HT1-like receptor seems to play a role in coronary vasospasm caused by low concentrations of 5-HT.

Augmented contraction of the human isolated coronary artery by sumatriptan: a possible role for endogenous thromboxane.

1 The antimigraine drug, sumatriptan, contracts the human coronary artery and, in some patients, elicits chest symptoms (e.g. pressure and pain), particularly after subcutaneous administration. We studied the effects of the thromboxane A2 (TxA2) analogue, U46619 and endothelin‐1 on contractile responses to sumatriptan in the human isolated coronary artery as well as the role of endogenously produced TxA2 and endothelin‐1 in contractions evoked by sumatriptan. 2 In the presence of U46619 (1 and 3 nM), mean concentration‐response curves to sumatriptan in the human coronary artery were shifted vertically due to the initial contraction by U46619, but when this initial contraction was subtracted from the response to sumatriptan, no significant augmentation was observed. However, analysis of the degree of augmentation in individual arterial segments revealed that the augmentation was variable and related inversely to the Emax of sumatriptan in the absence of U46619 (r = 0.78 and 0.81 for 1 and 3 nM, respectively; P < 0.05). 3 Treatment with the TxA2 receptor antagonist, SQ30741 (100 nM), or incubation of vessel segments with aspirin (10 μm), significantly reduced responses to sumatriptan; in aspirin‐treated vessel segments, SQ30741 failed to decrease further the contractions to sumatriptan. The decrease in Emax of sumatriptan by both SQ30741 and aspirin correlated significantly with the Emax of sumatriptan without SQ30741 (r = 0.74; P < 0.01) or aspirin (r = 0.94; P < 0.01). In aspirin‐treated vessel segments, responses to sumatriptan were significantly augmented in the presence of U46619 (3 nM; P < 0.05). 4 The specificity of SQ30741 was demonstrated by its ability to antagonize coronary artery contractions to U46619 (pA2: 7.54 ± 0.30), but not endothelin‐1. Similarly, incubation with aspirin (10 μm) did not affect contractile responses to endothelin‐1, but significantly reduced TxA2 production in coronary artery segments as judged by a decrease in thromboxane B2 (TxB2) from 4.77 ± 0.98 to 1.38 ± 0.36 ng g−1 2h−1. 5 Endothelin‐1 (1 nM) did not significantly augment contractions to sumatriptan; there was also no relationship between the degree of augmentation and the control Emax of sumatriptan in the absence of endothelin‐1. Furthermore, unlike SQ30741 or aspirin, a high concentration (100 nM) of the nonselective ETA/ETB receptor antagonist, SB 209670, failed to affect contractile responses to sumatriptan. However, SB 209670 potently antagonized coronary artery contractions induced by endothelin‐1 with a pA2 of 8.84 ± 0.32. 6 Compared to control vascular segments, endothelial denudation did not reduce TxA2 production (with endothelium = 2.56 ± 1.38 vs. without endothelium = 12.32 ± 4.94 ng TxB2 g−1 2 h−1), suggesting that the production of TxA2 is not confined to the endothelium. The sumatriptan‐induced contractions were also unaffected by endothelial denudation. 7 The results of the present study suggest that endogenously produced TxA2 enhances contractions to sumatriptan in the human isolated coronary artery. Such a mechanism may play a role in causing chest symptoms after sumatriptan by potentiating coronary vascular contraction by sumatriptan in vivo.

5-Hydroxytryptamine-induced contractions of the human isolated saphenous vein: involvement of 5-HT2 and 5-HT1D-like receptors, and a comparison with grafted veins

SummaryThe receptors mediating the contractile effect of 5-hydroxytryptamine (5-HT) on the human isolated saphenous vein, obtained from 42 patients undergoing coronary bypass surgery, have been further characterized using a number of 5-HT-related drugs. The rank order of agonist potency was 5-carboxamidotryptamine (5-CT) ≈ 5-HT > methysergide sumatriptan ≈ α-methyl-5-HT ≈ 5-methoxy-3-(1,2,3,6-tetrahydropyridin-4-yl)-1-Hindolesuccinate (RU 24969) ≈ 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI) > 2-methyl-5-HT > 8-hydroxy-2(di-n-propylamino)tetralin (8-OH-DPAT). Flesinoxan was inactive as an agonist. Ketanserin (1 μmol/l) hardly affected sumatriptan-induced contractions but it caused a rightward shift of the upper part of the concentration-response curve of 5-HT and 5-CT. The same concentration of ketanserin caused a parallel rightward shift of the concentration-response curves of α-methyl-5-HT and DOI with pKB values of 7. 1 and 7.1, respectively. The responses to sumatriptan were antagonized by methiothepin (0.1 μmol/l), metergoline (0.1 and 1 μmol/l), rauwolscine (1 μmol/l) and cyanopindolol (1 μmol/l); the calculated pKB values were 7.3, 6.9, 7.3, 6.7 and 6.5, respectively. Contractions to 5-HT were antagonized by methysergide (1 μmol/l), methiothepin (0.1 μmol/l; pKB = 7.1), ICS 205-930 (1 μmol/l; pKB = 5.9) and flesinoxan (30 μmol/l; pKB = 5.3). Remarkably, the contractions elicited by 2-methyl-5-HT were not attenuated by ICS 205-930, but were antagonized by methiothepin (0.1 μmol/l) and, more markedly, by ketanserin (1 μmol/l).There was a high correlation between the functional pD2 values of 5-HT1-like receptor agonists (5-CT, 5-HT, methysergide, sumatriptan, RU 24969 and 8-OH-DPAT) and their reported binding affinities for the 5-HT1D receptor in human or calf brain membranes. Such a correlation for the antagonism of sumatriptan-induced responses was less marked than for the agonists, but of the 5-HT1-like receptor subtypes it was the highest for the 5-HT1D receptor identified in human or calf brain membranes.In 3 patients, undergoing heart transplantation, saphenous vein which had previously functioned as a graft for 6–11 years, was dissected out from the heart. Though the contractions to potassium were significantly smaller in the grafted veins, the pD2 and Emax values (calculated as percentage of potassium-induced contractions) for 5-HT and sumatriptan were similar to those found in the veins obtained directly from the lower leg.It is concluded that contractions in the human isolated saphenous vein induced by 5-HT are mediated by 5-HT2 receptors as well as by a 5-HT1-like receptor resembling the 5-HT1D subtype found in brain membranes. It is also to be noted that 2-methyl-5-HT, considered selective for the 5-HT3 receptor, contracts the saphenous vein mainly via 5-HT2 receptors.

5-Hydroxytryptamine stimulates human isolated atrium but not ventricle

Although 5-hydroxytryptamine (5-HT) elicits positive inotropic effects in the human isolated atrium via 5-HT4 receptors, no data are available about its effects in the ventricular myocardium. We investigated the inotropic effects of 5-HT in human healthy ventricular trabeculae and compared these effects with those in right atrial trabeculae. Baseline force of contraction as well as the response to noradrenaline, used to check inotropic responsiveness of the tissue, was significantly higher in ventricular (391 +/- 10 and 719 +/- 126 mg, respectively) than in atrial tissue (189 +/- 5 and 383 +/- 79 mg, respectively). However, 5-HT increased the force of contraction up to 309 +/- 82 mg at 10(-4) M in atrial trabeculae, but failed to affect the force of contraction in ventricular tissue. We conclude that, in contrast to atrial tissue, 5-HT is ineffective as a positive inotropic agent in human ventricular trabeculae. This finding obviously rules out the development of 5-HT4 receptor agonists for the treatment of heart failure, but suggests the absence of ventricular side-effects of 5-HT4 receptor (ant)agonists should these agents be used in the treatment of gastrointestinal disorders.

Heterogeniety of endothelin/sarafotoxin receptors mediating contraction of the human isolated saphenous vein

We investigated the effect of the ETA receptor antagonist, BQ-123 (0.1 and 1 microM), on contraction of the human isolated saphenous vein induced by endothelin-1 or sarafotoxin S6b. Contraction in response to endothelin-1 was not affected by BQ-123. In contrast, BQ-123 biphasically attenuated the contractions due to sarafotoxin S6b. These data indicate that (i) endothelin-1 induces contractions of the human saphenous vein via a non-ETA receptor and (ii) contractions in response to sarafotoxin S6b are mediated in part via a receptor different from the receptor mediating contraction due to endothelin-1.

Endothelin receptors in the human coronary artery, ventricle and atrium

SummaryIn the present experiments we investigated endothelin (ET) receptors in the human coronary artery, and in ventricular and atrial muscle using quantitative receptor autoradiography. Displacement of [125I]Sf6b (Sarafotoxin S6b) (30 pM)- and [125I]ET-1 (30 pM)-labeled binding sites was studied using ET-1, the ETA receptor selective ligand BQ-123 (cyclo[D-Asp-L-Pro-D Val-L-Leu-D-Trp-]), and the ETB receptor selective ligand [Ala1,3,11,15]ET-1.Specific binding was more dense in atrium and coronary artery (relative optical density (r.o.d.): 0.14±0.01 and 0.16±0.01, respectively) than in ventricular muscle (r.o.d.: 0.10±0.01). In the coronary artery, binding was especially dense in the media. ET-1 displaced [125I]ET-1 and [125I]Sf6b monophasically in atrium, ventricle and coronary artery. [Ala1,3,11,15]ET 1 and BQ-123 displaced [125I]ET-1 and [125I]Sf6b-labeled sites biphasically in the ventricle and in the atrium. In the human coronary artery, [Ala1,3,11,15]ET-1 and BQ-123 displaced [125I]ET-1-labeled sites monophasically (pIC50): ET-1 (9.72±0.12) > BQ-123 (6.84±0.08) > [Ala1,3,11,15]ET-1 (6.40±0.12). In contrast, [Ala1,3,11,15]ET-1 and BQ-123 displaced [125I] Sf6b-labeled coronary artery sites biphasically (high affinity pIC50: BQ-123, 9.03±0.25;[Ala1,3,11,15]ET-1, 8.40±0.14; low affinity pIC50: BQ-123, 7.24±0.14; [Ala1,3,11,15]ET-1, 6.99±0.09).These data indicate that both [125I]ET-1 and [125I] Sf6b-labeled ETA and ETB binding sites in human ventricular and atrial muscle. In the human coronary artery, both radioligands labeled ETA binding sites, but [125I] Sf6b also labeled a non-ETA, non-ETB binding site with relatively high affinity for both BQ-123 and [Ala1,3,11,15] ET-1.