Jean-Louis Bény

EPOXYEICOSATRIENOIC ACIDS ACTIVATE A HIGH-CONDUCTANCE, CA2+-DEPENDENT K+ CHANNEL ON PIG CORONARY ARTERY ENDOTHELIAL CELLS

1 Epoxyeicosatrienoic acids (EETs) have been described as endothelium‐derived hyper‐polarizing factors (EDHFs), based on their stimulatory effects on smooth muscle K+channels. In order to reveal a putative autocrine effect of EETs on endothelial channels, we have studied the effects of the four EET regioisomers (5,6‐EET, 8,9‐EET, 11,12‐EET and 14,15‐EET) on the high‐conductance, Ca2+‐dependent K+ (BKCa) channel recorded in inside‐out patches of primary cultured pig coronary artery endothelial cells. Currents were recorded in the presence of either 500 nm or 1 μm free Ca2+ on the cytosolic side of the membrane. 2 In 81% of experiments, EETs at < 156 nm, applied on the cytosolic side of the membrane, transiently increased BKCa channel open state probability (P0) without affecting its unitary conductance, thus providing evidence for direct action of EETs, without involvement of a cytosolic transduction pathway. 3 The four EET regioisomers appeared to be equally active, multiplying the BKCa channel Po by a mean factor of 4.3 ± 0.6 (n= 15), and involving an increase in the number and duration of openings. 4 The EET‐induced increase in BKCa channel activity was more pronounced with low initial Po. When the BKCa channel was activated by 500 nm Ca2+, application of EETs increased the initial Po value of below 0.1 by a factor of 5. When the channel was activated by 1 μm Ca2+, application of EETs increased the initial Po value by a factor of 3. 5 Our results show that EETs potentiate endothelial BKCa channel activation by Ca2+. The autocrine action of EETs on endothelial cells, which occurs in the same concentration range as their action on muscle cells, should therefore fully participate in the vasoactive effects of EETs, and thus be taken into account when considering their putative EDHF function.

Hydrogen peroxide: an endogenous smooth muscle cell hyperpolarizing factor.

Hydrogen peroxide can be released by different cells such as the nerves, the endothelial or phagocytotic white blood cells which can all interact with vascular smooth muscles. We show that hydrogen peroxide hyperpolarizes and relaxes pig coronary artery smooth muscle cells. The possibility that the endothelium derived hyperpolarizing factor released by the endothelium in response to bradykinin and substance P being hydrogen peroxide was tested using catalase, an enzyme which hydrolyses hydrogen peroxide. We find that this particular endothelial hyperpolarizing factor and hydrogen peroxide are two distinct molecules.

Endothelium-dependent Vasoactive Modulation in the Ophthalmic Circulation

The vascular endothelium is strategically located between the circulating blood and the vascular smooth muscle cells. Different agonists or stimuli transported by the circulating blood can trigger the endothelium to release potent relaxing (nitric oxide, prostacyclin, endothelium-derived hyperpolarizing factor) or contracting factors (endothelin, cycloxygenase products). These endothelium-derived vasoactive factors can modulate blood flow locally. Heterogeneity exists from one vascular bed to the other, or even between vessels, in the agonists able to stimulate the release of endothelium-derived vasoactive factors. In the ophthalmic circulation, nitric oxide and endothelin are strong vasoactive modulators. In many vascular diseases that are of importance in ophthalmology (hypercholesterolemia, arteriosclerosis, hypertension, diabetes, vasospastic syndrome, ischemia and reperfusion, etc) the function of the endothelium can be impaired. There exist different drugs that can modulate the vasoactive function of the vascular endothelium. In other words, it appears that the vascular endothelium plays an important role in both the physiology and pathophysiology of the regulation of blood flow. The modulation of this regulatory system by different drugs might open new therapeutical approaches to treat vascular disorders in ophthalmology.

Simultaneous oscillations in the membrane potential of pig coronary artery endothelial and smooth muscle cells.

1. The effects of tetrabutylammonium (TBA) on the mechanical tension and on the electrical behaviour of endothelial and smooth muscle cells were studied in intact porcine coronary artery strips. 2. Superfusion of strips with TBA (2‐20 mM) induced mechanical oscillations, leading to an increase in tonic isometric tension. 3. TBA‐induced mechanical oscillations were correlated with fluctuations of the membrane potential of endothelial cells, which were identified by iontophoretic injection of Lucifer Yellow. 4. The endothelial cell membrane potential fluctuations appeared as action potentials or smaller amplitude slow waves, and were synchronized with electrical membrane potential fluctuations of the underlying coronary smooth muscle cells. 5. Oscillations induced by TBA in smooth muscle cells were not affected by removal of the endothelium, and depended on the presence of calcium in the external medium. 6. To our knowledge, this is the first description of action potential‐like fluctuations in the endothelium. It is concluded that the oscillations were generated in the smooth muscle and that they propagate to the endothelium. The question of the mode of propagation of the signal is discussed.

Effect of Mechanical Stimulation, Substance P and Vasoactive Intestinal Polypeptide on the Electrical and Mechanical Activities of Circular Smooth Muscles from Pig Coronary Arteries Contracted with Acetylcholine: Role of Endothelium

Mechanical stimulation, substance P and vasoactive intestinal polypeptide (VIP) were found to relax the transversal strip of anterior descending branches of pig coronary arteries precontracted by acetylcholine. The effects of mechanical stimulation and substance P required the presence of intact endothelium, while VIP did not. The effect of VIP did not appear to be mediated by catecholamines. Simultaneous measurements of intracellular membrane potential and tension developed by coronary smooth muscle precontracted with Ach showed that the smooth muscle relaxation by substance P is accompanied by membrane hyperpolarization. In contrast VIP relaxed the same tissue without affecting the membrane potential. In a cascade experiment, the fluid perfused intraluminally in intact segments of coronary arteries was dropped over a de-endothelialized strip which relaxed in response to substance P and mechanical stimulation. This indicates that substance P and mechanical stimulation act by releasing from the endothelium a humoral factor that produces arterial smooth muscle relaxation.

An evaluation of potassium ions as endothelium-derived hyperpolarizing factor in porcine coronary arteries

In the rat hepatic artery, the endothelium‐derived hyperpolarizing factor (EDHF) was identified as potassium. Potassium hyperpolarizes the smooth muscles by gating inward rectified potassium channels and by activating the sodium‐potassium adenosine triphosphatase (Na+‐K+ATPase). Our goal was to examine whether potassium could explain the EDHF in porcine coronary arteries. On coronary strips, the inhibition of calcium‐dependent potassium channels with 100 nM apamin plus 100 μM charibdotoxin inhibited the endothelium‐dependent relaxations, produced by 10 nM substance P and 300 nM bradykinin and resistant to nitro‐L‐arginine and indomethacin. The scavenging of potassium with 2 mM Kryptofix 2.2.2 abolished the endothelium‐dependent relaxations produced by the kinins and resistant to nitro‐L‐arginine and indomethacin. Forty μM 18α glycyrrethinic acid or 50 μM palmitoleic acid, both uncoupling agents, did not inhibit these kinin relaxations. Therefore, EDHF does not result from an electrotonic spreading of an endothelial hyperpolarization. Barium (0.3 nM) did not inhibit the kinin relaxations resistant to nitro‐L‐arginine and indomethacin. Therefore, EDHF does not result from the activation of inward rectified potassium channels. Five hundred nM ouabain abolished the endothelium‐dependent relaxations resistant to nitro‐L‐arginine and indomethacin without inhibiting the endothelium‐derived NO relaxation. The perifusion of a medium supplemented with potassium depolarized and contracted a coronary strip; however, the short application of potassium hyperpolarized the smooth muscles. These results are compatible with the concept that, in porcine coronary artery, the EDHF is potassium released by the endothelial cells and that this ion hyperpolarizes and relaxes the smooth muscles by activating the Na+‐K+ATPase.

Loss of connexin40 is associated with decreased endothelium-dependent relaxations and eNOS levels in the mouse aorta

Upon agonist stimulation, endothelial cells trigger smooth muscle relaxation through the release of relaxing factors such as nitric oxide (NO). Endothelial cells of mouse aorta are interconnected by gap junctions made of connexin40 (Cx40) and connexin37 (Cx37), allowing the exchange of signaling molecules to coordinate their activity. Wild-type (Cx40(+/+)) and hypertensive Cx40-deficient mice (Cx40(-/-)), which also exhibit a marked decrease of Cx37 in the endothelium, were used to investigate the link between the expression of endothelial connexins (Cx40 and Cx37) and endothelial nitric oxide synthase (eNOS) expression and function in the mouse aorta. With the use of isometric tension measurements in aortic rings precontracted with U-46619, a stable thromboxane A(2) mimetic, we first demonstrate that ACh- and ATP-induced endothelium-dependent relaxations solely depend on NO release in both Cx40(+/+) and Cx40(-/-) mice, but are markedly weaker in Cx40(-/-) mice. Consistently, both basal and ACh- or ATP-induced NO production were decreased in the aorta of Cx40(-/-) mice. Altered relaxations and NO release from aorta of Cx40(-/-) mice were associated with lower expression levels of eNOS in the aortic endothelium of Cx40(-/-) mice. Using immunoprecipitation and in situ ligation assay, we further demonstrate that eNOS, Cx40, and Cx37 tightly interact with each other at intercellular junctions in the aortic endothelium of Cx40(+/+) mice, suggesting that the absence of Cx40 in association with altered Cx37 levels in endothelial cells from Cx40(-/-) mice participate to the decreased levels of eNOS. Altogether, our data suggest that the endothelial connexins may participate in the control of eNOS expression levels and function.

Endothelium-independent relaxation and hyperpolarization to C-type natriuretic peptide in porcine coronary arteries

Endothelial cells produce C-type natriuretic peptide (CNP), which has been proposed as an endothelium-derived hyperpolarizing factor. In porcine coronary arteries, we investigated the vasodilatory effects of CNP and compared them with endothelium-dependent relaxations and hyperpolarizations to bradykinin. Isolated epicardial porcine coronary arteries were studied in organ chambers, and concentration-response curves to CNP and bradykinin were obtained. Membrane potential was measured in endothelial cells and smooth muscle of intact porcine coronary arteries during stimulation with CNP or bradykinin. In precontracted porcine coronary arteries with or without endothelium, CNP (10[-10]-10[-6] M) evoked relaxations (maximum, 42 +/- 4%) smaller than those evoked by bradykinin (100 +/- 1%), blunted in preparations contracted by KCl instead of U46619 (9,11-dideoxy-11a,9a-epoxymethano-prostaglandin F2alpha; p < 0.05) and unaffected by inhibition of NO synthase (NS). CNP evoked hyperpolarization of vascular smooth muscle of similar magnitude in endothelium-intact (-4.4 +/- 1 mV) and endothelium-denuded (-4.6 +/- 1 mV) porcine coronary arteries. Bradykinin (10[-10]-10[-6] M) evoked concentration-dependent relaxations in preparations with endothelium only. Although atrial natriuretic peptide-receptor antagonist HS-142-1 (25 microM) slightly reduced the sensitivity to bradykinin (log shift at IC50, twofold; p < 0.05), it had no effect on the maximal response to bradykinin. Inhibition of NO synthase partially attenuated, whereas high potassium chloride (30 mM) markedly inhibited relaxations to bradykinin (p < 0.05). Hyperpolarization to bradykinin was much more pronounced than that to CNP (-17 +/- 3 mV; p < 0.05 vs. CNP) and was observed in endothelium-intact preparations only and unaffected by HS-142-1. In conclusion, in contrast to bradykinin, CNP induces endothelium-independent and weaker relaxation and hyperpolarization of coronary artery vascular smooth muscle, suggesting that CNP is an unlikely mediator of endothelium-dependent hyperpolarization of porcine coronary arteries.

Ca(2+)-dependent non-selective cation and potassium channels activated by bradykinin in pig coronary artery endothelial cells.

1. Using the cell‐attached and inside‐out modes of the patch‐clamp technique, we studied the Ca(2+)‐dependent ionic channels activated by bradykinin in cultured pig coronary artery endothelial cells to further understand electrophysiological events underlying cellular activation. 2. In the cell‐attached mode, bradykinin (94 nM) activated two types of Ca(2+)‐dependent channels: a high conductance K+ channel (285 pS in high symmetrical K+), whose open state probability was increased by depolarization, and a lower conductance inwardly rectifying non‐selective cation channel (44 pS in high symmetrical K+). 3. The 285 pS K+ channel was half‐maximally activated by cytosolic Ca2+ levels of 1.6 and 4.5 microM at +10 and ‐30 mV, respectively. Such local concentrations should be reached in the presence of bradykinin, which induces a mean maximal cytosolic Ca2+ rise of 1.3 microM. 4. The 285 pS K+ channel was inhibited by d‐tubocurarine, which acted by reducing the mean open time duration (flickering pattern), finally reducing the channel conductance. 5. Divalent cations such as Ca2+ could flow through the 44 pS non‐selective cation channel, with nearly the same permeability (P) as monovalent cations (PK: PNa: PCa = 1:1:0.7). 6. The cation channel appeared to be more sensitive to Ca2+ than the K+ channel, with a half‐maximal open probability induced by 0.7 microM Ca2+ on the intracellular side of the membrane. 7. In contrast to the K+ channel, the cation channel mean open time was clearly increased by bradykinin. This effect was delayed compared with the increase in the channel open state probability and was rapidly lost in the inside‐out configuration. Caffeine also activated the cation channel but more transiently than bradykinin and without any effect on the open duration. 8. In the absence of extracellular Ca2+, the bradykinin‐induced increase in cytosolic free Ca2+ was shortened temporally by 52% and reduced in amplitude by 88%, whereas the bradykinin‐induced hyperpolarization was not significantly reduced in amplitude but was shortened by 70%, thus illustrating the major role of Ca2+ influx in endothelial cell activation by bradykinin. 9. We conclude that bradykinin activates two types of Ca(2+)‐dependent channels in coronary endothelial cells: a high conductance K+ channel regulated by membrane potential, and an inwardly rectifying cation channel allowing Ca2+ entry, the cation channel being about 6 times more sensitive to Ca2+ than the K+ channel. The increase in cation channel open state probability involves an increase in open number, like the K+ channel, but also involves a rise in channel open duration. Ca2+ entry via cation channels could contribute to increase the cytoplasmic Ca2+ level, activate Ca(2+)‐dependent K+ channels, thus triggering membrane hyperpolarization when the endothelial cell is stimulated by a vasoactive agonist such as bradykinin.

Altered Dye Diffusion and Upregulation of Connexin37 in Mouse Aortic Endothelium Deficient in Connexin40

Connexin40 (Cx40), connexin37 (Cx37) and connexin43 (Cx43) are subunit proteins of gap junction channels in the vascular wall which are presumably involved in the propagation of vasomotor signals. In this study we have investigated in Cx40-deficient versus wild-type aortic endothelium to which extent loss of Cx40 impairs intercellular communication. We show in Cx40-deficient mice that expression of both Cx37 and Cx43 protein was increased approximately 3- and 2-fold over the level in wild-type endothelium, respectively. Furthermore, Cx37 immunosignals were distributed more homogeneously on contacting plasma membranes in Cx40-deficient versus with wild-type endothelium. Cx43 was not detected in endothelium but only in smooth muscle cells of the vessel wall. Iontophoretic injection of Lucifer Yellow or neurobiotin into aortic endothelium of Cx40-deficient mice showed extensive intercellular transfer of neurobiotin but not of Lucifer Yellow. In contrast, intercellular spreading of Lucifer Yellow was observed in endothelium of wild-type aorta. As shown by electron microscopy, gap junctions in Cx40-deficient endothelium were morphologically different from those of wild-type vessels. These results demonstrate that dye diffusibility of endothelial gap junctions is different in Cx40-deficient and wild-type mice, although Cx40-deficient mice retain the capability of intercellular communication. Apparently, Cx40-deficient endothelial cells upregulate and redistribute Cx37 as a molecular adaptation to the lack of Cx40.