Jean-Vivien Mombouli

Potentiation of endothelium-dependent relaxations to bradykinin by angiotensin I converting enzyme inhibitors in canine coronary artery involves both endothelium-derived relaxing and hyperpolarizing factors.

Studies were designed to investigate the mechanisms underlying the augmentation by angiotensin I converting enzyme (ACE) inhibitors of the endothelium-dependent relaxations evoked by bradykinin. Isometric tension, tissue levels of cGMP, and transmembrane potential were measured in isolated canine coronary arteries as indications of the respective contribution of nitric oxide and endothelium-derived hyperpolarizing factor. In rings of coronary artery with endothelium, relaxations to bradykinin were potentiated by the ACE inhibitors cilazaprilat and perindoprilat. NG-Nitro-L-arginine (NLA), a nitric oxide synthase inhibitor, impaired relaxations to bradykinin. But the presence of ACE inhibitors partially restored this activity. Bradykinin stimulated the production of cGMP, and this was enhanced significantly by ACE inhibitors, indicating an augmented release of nitric oxide. NLA abolished the increase induced by bradykinin irrespective of the presence of ACE inhibitors. Electrophysiological studies revealed that bradykinin elicited an endothelium-dependent hyperpolarization of vascular smooth muscle that was insensitive to NLA and potentiated by ACE inhibitors. The bradykinin-induced hyperpolarization and NLA-resistant relaxations were transient and impaired by potassium depolarization. Thus, production of endothelium-derived hyperpolarizing factor may account for the NLA-resistant relaxations of canine coronary arteries. The relaxations induced by bradykinin were unaffected by the B1 kinin receptor antagonist des-Arg9,[Leu8]-bradykinin either in the absence or in the presence of NLA but were antagonized by the B2 kinin receptor antagonist D-Arg[Hyp3,D-Phe7]-bradykinin. Molecular exclusion chromatography of 125I-labeled [Tyr8]-bradykinin and its degradation products demonstrated that the breakdown of the kinin by isolated coronary arteries was prevented in the presence of perindoprilat.(ABSTRACT TRUNCATED AT 250 WORDS)

Endothelium-dependent hyperpolarization caused by bradykinin in human coronary arteries.

The present study was designed to determine whether bradykinin induces endothelium-dependent hyperpolarization of vascular smooth muscle in human coronary arteries, and if so, to define the contribution of this hyperpolarization to endothelium-dependent relaxations. The membrane potential of arterial smooth muscle cells (measured by glass microelectrodes) and changes in isometric force were recorded in tissues from six patients undergoing heart transplantation. In the presence of indomethacin and NG-nitro-L-arginine (NLA), the membrane potential was -48.3 +/- 0.6 and -46.9 +/- 0.6 mV, in preparations with and without endothelium, respectively, and was not affected by treatment with perindoprilat, an angiotensin-converting enzyme inhibitor. In the presence of both indomethacin and NLA, bradykinin evoked transient and concentration-dependent hyperpolarizations only in tissues with endothelium, which were augmented by perindoprilat and mimicked by the calcium ionophore A23187. Glibenclamide did not inhibit membrane hyperpolarization to bradykinin. In rings contracted with prostaglandin F2 alpha, the cumulative addition of bradykinin caused a concentration-dependent relaxation during contractions evoked by prostaglandin F2 alpha, which was not abolished by NLA and indomethacin. The present findings demonstrate the occurrence of endothelium-dependent hyperpolarization, and its contribution to endothelium-dependent relaxations, in the human coronary artery.

Endothelium-derived hyperpolarizing factor(s): updating the unknown

Endothelium-dependent hyperpolarization of vascular smooth muscle is a mechanism that contributes to the vasodilator response to shear stress and chemicals acting on endothelial receptors. The phenomenon is explained by the release from endothelial cells, of an endothelium-derived hyperpolarizing factor(s) (EDHF) (s), although its (their) exact nature is still controversial. Indeed, endothelial cells produce several substances that are capable of evoking hyperpolarization in vascular smooth muscle. However, which of these factors represents EDHF under physiological conditions remains unknown. The term EDHF should be reserved for a substance(s) that differs from both NO and prostaglandins. In this review Jean-Vivien Mombouli and Paul M. Vanhoutte consider the possible candidates for EDHF and the arguments that have lead to the proposal that these substances fulfil the functions of an endothelium-derived relaxing agent. The weaknesses of the available sudies are also discussed. The identification of EDHF would allow the understanding of its physiological role alongside other known endothelial mediators such as NO and prostacyclin. This could lead to the design of new therapies aimed at correcting the impairment of EDHF-mediated dilatation in a number of cardiovascular diseases.

Vascular endothelium: Vasoactive mediators

In most blood vessels, the endothelium generates both vasodilator and growth-stabilizing mediators under normal physiological circumstances. The vasodilator influence of the endothelium modulates the vasoconstriction induced by adrenergic nerves, bloodborne substances, and local autacoids. Nitric oxide (NO) is a major endothelium-derived vasodilator, along with prostacyclin. A third substance called endothelium-derived hyperpolarizing factors (EDHF) mediates vasodilatation in certain conduit arteries and in most resistance vessels. EDHF may be a cytochrome P-450 metabolite of arachidonic acid. NO acts mostly through an elevation of cyclic guanosine monophosphate in vascular smooth muscle, whereas prostacyclin stimulates adenylate cyclase. The mode of action of EDHF involves the activation of K+ channels. The multiplicity of the factors released by the endothelium, as well as the complexity of the interactions among these factors and those with other nonendothelial mediators, determine the extent of vasomotor control exerted locally by the endothelium.

Endothelium-dependent effects of converting-enzyme inhibitors

Angiotensin-converting enzyme (ACE) inhibitors were designed to prevent the vasoconstrictor influence of the activated renin-angiotensin system. However, it has long been suspected that the vasodilator actions of these compounds are not entirely related to inhibition of the generation of angiotensin II. Bradykinin, which is rapidly degraded by ACE, stimulates the release of endothelium-derived vasodilator mediators, including nitric oxide, endothelium-derived hyperpolarizing factor, and prostacyclin. These mediators do not contribute to the vasodilator effect of bradykinin in every arterial bed. However, the prevention by ACE inhibitors of the degradation of bradykinin-induces an augmentation of the production of these substances and thus potentiates the dilatation evoked by the peptide. The existence of a local kallikrein-kinin system in the vascular wall has been demonstrated, and locally generated kinins contribute to the acute vasodilator actions of ACE inhibitors. ACE inhibitors can potentiate endothelium-dependent dilatations evoked by neurohumoral mediators that are not substrates for ACE. Thus, the vasodilator properties of ACE inhibitors not only reflect inhibition of the renin-angiotensin system but also depend on the enhanced production of endothelium-derived mediators.

P2u receptor-mediated release of endothelium-derived relaxing factor/nitric oxide and endothelium-derived hyperpolarizing factor from cerebrovascular endothelium in rats.

BACKGROUND AND PURPOSE Stimulation of P2u purinoceptors by UTP on endothelium dilates the rat middle cerebral artery (MCA) through the release of endothelium-derived relaxing factor/nitric oxide (EDRF/NO) and an unknown relaxing factor. The purpose of this study was to determine whether this unknown relaxing factor is endothelium-derived hyperpolarizing factor (EDHF). METHODS Rat MCAs were isolated, cannulated, pressurized, and luminally perfused. UTP was added to the luminal perfusate to elicit dilations. RESULTS Resting outside diameter of the MCAs in one study was 209+/-7 micrometer (n=10). The MCAs showed concentration-dependent dilations with UTP administration. Inhibition of NO synthase with NG-nitro-L-arginine methyl ester (L-NAME) (1 micromol/L to 1 mmol/L) did not diminish the maximum response to UTP but did shift the concentration-response curve to the right. Scavenging NO with hemoglobin (1 or 10 micromol/L) or inhibition of guanylate cyclase with ODQ (1 or 10 micromol/L) had effects on the UTP-mediated dilations similar to those of L-NAME. In the presence of L-NAME, dilations induced by 10 micromol/L UTP were accompanied by 13+/-2 mV (P<0.009) hyperpolarization of the vascular smooth muscle membrane potential (-28+/-2 to -41+/-1 mV). Iberiotoxin (100 nmol/L), blocker of the large-conductance calcium-activated K channels, sometimes blocked the dilation, but its effects were variable. Charybdotoxin (100 nmol/L), also a blocker of the large-conductance calcium-activated K channels, abolished the L-NAME-insensitive component of the dilation to UTP. CONCLUSIONS Stimulation of P2u purinoceptors on the endothelium of the rat MCA released EDHF, in addition to EDRF/NO, and dilated the rat MCA by opening an atypical calcium-activated K channel.

Endothelium‐dependent relaxation and hyperpolarization evoked by bradykinin in canine coronary arteries: enhancement by exercise‐training

1 Kinins, which are produced locally in arterial walls, stimulate the release of endothelium‐derived vasodilator substances. Therefore, they may participate in the metabolic adaptation to chronic exercise that occurs in the coronary circulation. Experiments were designed to compare the reactivity to bradykinin in coronary arteries isolated from sedentary and exercised‐trained dogs (for 8–10 weeks). 2 The organ chambers used in this study were designed for measurement of isometric tension and cell membrane potential with glass microelectrodes. Rings of canine isolated coronary arteries with endothelium were suspended in the organ chambers filled with modified Krebs‐Ringer bicarbonate solution (37°C, gassed with 5% CO2 in 95 O2), and were all treated with indomethacin to prevent interference from prostaglandins. 3 Bradykinin evoked concentration‐dependent relaxations of the coronary arteries. However, the kinin was significantly less potent in relaxing coronary arteries from the sedentary dogs than those from the trained ones. 4 In the presence of NG‐nitro‐L‐arginine (an inhibitor of nitric oxide synthases), concentration‐relaxation curves to bradykinin were shifted to the right in both types of preparations. Nonetheless, the peptide was still significantly more potent in arteries from exercise‐trained animals. 5 In the electrophysiological experiments, concentration‐hyperpolarization curves to bradykinin obtained in arteries from sedentary dogs were also significantly to the right of those in vessels from exercise‐trained animals. Thus, in arteries from exercised animals, bradykinin more potently evoked the release of both nitric oxide (NO) and endothelium‐derived hyperpolarizing factor (EDHF). 7 The angiotensin converting enzyme (ACE)‐inhibitor, perindoprilat, shifted to the left the concentration‐relaxation curves to bradykinin obtained under control conditions and in the presence of NG‐nitro‐L‐arginine. The concentration‐hyperpolarization curves to bradykinin were also shifted to the left by perindoprilat. The shift induced by the ACE‐inhibitor in either type of preparation was not significantly different. 8 These findings demonstrate that exercise‐training augments the sensitivity of the coronary artery of the dog to the endothelium‐dependent effects of bradykinin. This sensitization to bradykinin may reflect an increased role of both NO and EDHF, and is not the consequence of differences in ACE activity in the receptor compartment.

ACE inhibition, endothelial function and coronary artery lesions. Role of kinins and nitric oxide.

SummaryIn healthy coronary arteries, the endothelium plays an important role in the regulation of vascular smooth muscle growth and contractility. Furthermore, the endothelium inhibits overt platelet aggregation and prevents the adhesion of white blood cells to, and their infiltration into, the vascular wall. Among the mediators of these functions of endothelial cells, nitric oxide (NO) plays a central role. Moreover, the presence of local kinin-generating enzymatic systems associated with endothelial cells, vascular smooth muscle, platelets, neutrophils and monocytes suggests that bradykinin stimulates endothelial cells to release NO locally. The activation of endothelial cells by bradykinin is inhibited by kininase II, best known as angiotensin converting enzyme (ACE). Hence, ACE inhibitors, in addition to reducing the levels of angiotensin II (a potent stimulus to vascular smooth muscle growth and contraction), cause an amplification of the release of NO and other endothelial mediators that is induced by bradykinin.Independent risk factors for coronary artery disease such as hypertension, diabetes and hypercholesterolaemia reduce the NO-dependent regulation of vascular smooth muscle contractility and growth in otherwise normal coronary arteries. This endothelial dysfunction probably also affects the inhibitory role of NO with regard to platelet aggregation and monocyte infiltration into the vascular wall. In atherosclerotic vessels, the role of NO is severely reduced. In animal models, as well as in patients with coronary artery disease, endothelial dysfunction is improved by treatment with ACE inhibitors. Although in humans the mechanism of the restoration of endothelial function is not known, in animals endogenous kinins and NO are involved. However, it is clear that this process is multifactorial, and thus probably involves both the prevention of the deleterious actions of angiotensin II and the potentiation of bradykinin.

Heterogeneity of endothelium-dependent vasodilator effects of angiotensin-converting enzyme inhibitors : role of bradykinin generation during ACE inhibition

Endothelium-derived mediators are released in response to shear stress and a variety of endogenous substances including bradykinin and angiotensins. They may contribute to the regulation of the renin-angiotensin system in the vascular wall and in the kidney. Bradykinin is a powerful agonist at endothelial cells, and the actions of this peptide, which is generated by components of the vascular wall, during angiotensin-converting enzyme (ACE) inhibition may determine some of the vascular effects of ACE inhibitors. In vitro studies demonstrate that the relaxations to bradykinin are mostly endothelium dependent and are mediated by nitric oxide, endothelium-derived hyperpolarizing factor, and/or vasodilator prostaglandins; however, these endothelium-derived relaxing factors do not always contribute simultaneously to the relaxations in every artery. The contribution of ACE in the termination of bradykinin action, relative to the other inactivation processes (including carboxypeptidases and internalization) also may determine the ability of ACE inhibitors to augment the effects of the kinin. Furthermore, it appears that the level of ACE activity and the potency of bradykinin, respectively, are not uniform in all preparations. In arteries in which bradykinin is very efficacious and in which ACE activity may be relatively low, ACE inhibitors may prolong but not amplify the responses to the peptide. The pharmacologic characteristics of the responses of the different vascular beds to bradykinin, together with the modulation of endothelium-de-pendent responses to other agonists (including purines), may be of importance in the heterogeneity of the vasodilator actions of ACE inhibitors.

Purinergic endothelium-dependent and -independent contractions in rat aorta.

The role of endothelium-derived contracting factor or factors in modulating relaxations and contractions to adenine nucleotides was examined in aortas from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) and Wistar rats. During contractions to phenylephrine, the relaxations to ATP were impaired significantly in SHR compared with WKY aortas with endothelium. In rings treated with NG-nitro-L-arginine (to inhibit nitric oxide synthase), the endothelium significantly augmented contractions evoked by ATP; this enhancement was greater in SHR compared with WKY aortas. Indomethacin (inhibitor of cyclooxygenase) and SQ 29,458 (antagonist of thromboxane/prostaglandin endoperoxide receptors) but not dazoxiben (inhibitor of thromboxane synthase) significantly augmented the maximal relaxation in WKY rats, abolished the impairment of the relaxation in SHR, and prevented the potentiation by the endothelium of the contractions evoked by ATP. In older animals (10 to 12 months old), the endothelium-dependent concentration-relaxation curves to ATP in SHR and WKY aortas treated with indomethacin were superimposable, as were the concentration-contraction curves (with NG-nitro-L-arginine present). Endothelium-dependent concentration-relaxation and -contraction curves to ADP obtained in these preparations overlapped also. In Wistar rats, the magnitude of the endothelium-dependent relaxations to either ATP or ADP were significantly smaller compared with the other strains, and the endothelium-dependent contractions were even smaller. Results show that adenine nucleotides stimulate the production of both endothelium-derived relaxing and contracting factors. Although there is no obvious age-related alteration in the capacity of aortas to release endothelium-derived relaxing factor, aging enhances endothelium-derived contracting factor activity in WKY rats.