Philippe Ghisdal

Endothelial beta3-adrenoreceptors mediate nitric oxide-dependent vasorelaxation of coronary microvessels in response to the third-generation beta-blocker nebivolol.

Background—The therapeutic effects of nonspecific β-blockers are limited by vasoconstriction, thus justifying the interest in molecules with ancillary vasodilating properties. Nebivolol is a selective β1-adrenoreceptor antagonist that releases nitric oxide (NO) through incompletely characterized mechanisms. We identified endothelial β3-adrenoreceptors in human coronary microarteries that mediate endothelium- and NO-dependent relaxation and hypothesized that nebivolol activates these β3-adrenoreceptors. Methods and Results—Nebivolol dose-dependently relaxed rodent coronary resistance microarteries studied by videomicroscopy (10 &mgr;mol/L, −86±6% of prostaglandin F2α contraction); this was sensitive to NO synthase (NOS) inhibition, unaffected by the β1-2-blocker nadolol, and prevented by the β1-2-3-blocker bupranolol (P<0.05; n=3 to 8). Importantly, nebivolol failed to relax microarteries from β3-adrenoreceptor–deficient mice. Nebivolol (10 &mgr;mol/L) also relaxed human coronary microvessels (−71±5% of KCl contraction); this was dependent on a functional endothelium and NO synthase but insensitive to β1-2-blockade (all P<0.05). In a mouse aortic ring assay of neoangiogenesis, nebivolol induced neocapillary tube formation in rings from wild-type but not β3-adrenoreceptor– or endothelial NOS–deficient mice. In cultured endothelial cells, 10 &mgr;mol/L nebivolol increased NO release by 200% as measured by electron paramagnetic spin trapping, which was also reversed by NOS inhibition. In parallel, endothelial NOS was dephosphorylated on threonine495, and fura-2 calcium fluorescence increased by 91.8±23.7%; this effect was unaffected by β1-2-blockade but abrogated by β1-2-3-blockade (all P<0.05). Conclusions—Nebivolol dilates human and rodent coronary resistance microarteries through an agonist effect on endothelial β3-adrenoreceptors to release NO and promote neoangiogenesis. These properties may prove particularly beneficial for the treatment of ischemic and cardiac failure diseases through preservation of coronary reserve.

Role of Caveolar Compartmentation in Endothelium-Derived Hyperpolarizing Factor–Mediated Relaxation Ca2+ Signals and Gap Junction Function Are Regulated by Caveolin in Endothelial Cells

Background— In endothelial cells, caveolin-1, the structural protein of caveolae, acts as a scaffolding protein to cluster lipids and signaling molecules within caveolae and, in some instances, regulates the activity of proteins targeted to caveolae. Specifically, different putative mediators of the endothelium-derived hyperpolarizing factor (EDHF)–mediated relaxation are located in caveolae and/or regulated by the structural protein caveolin-1, such as potassium channels, calcium regulatory proteins, and connexin 43, a molecular component of gap junctions. Methods and Results— Comparing relaxation in vessels from caveolin-1 knockout mice and their wild-type littermates, we observed a complete absence of EDHF-mediated vasodilation in isolated mesenteric arteries from caveolin-1 knockout mice. The absence of caveolin-1 is associated with an impairment of calcium homeostasis in endothelial cells, notably, a decreased activity of Ca2+-permeable TRPV4 cation channels that participate in nitric oxide– and EDHF-mediated relaxation. Moreover, morphological characterization of caveolin-1 knockout and wild-type arteries showed fewer gap junctions in vessels from knockout animals associated with a lower expression of connexins 37, 40, and 43 and altered myoendothelial communication. Finally, we showed that TRPV4 channels and connexins colocalize with caveolin-1 in the caveolar compartment of the plasma membrane. Conclusions— We demonstrated that expression of caveolin-1 is required for EDHF-related relaxation by modulating membrane location and activity of TRPV4 channels and connexins, which are both implicated at different steps in the EDHF-signaling pathway.

Endothelial beta3-adrenoceptors mediate vasorelaxation of human coronary microarteries through nitric oxide and endothelium-dependent hyperpolarization.

Background—Coronary vessel tone is modulated in part by &bgr;-adrenergic relaxation. However, the implication of specific &bgr;-adrenoceptor subtypes and their downstream vasorelaxing mechanism(s) in human coronary resistance arteries is poorly defined. &bgr;3-Adrenoceptors were recently shown to vasodilate animal vessels and are expressed in human hearts. Methods and Results—We examined the expression and functional role of &bgr;3-adrenoceptors in human coronary microarteries and their coupling to vasodilating nitric oxide (NO) and/or hyperpolarization mechanisms. The expression of &bgr;3-adrenoceptor mRNA and protein was demonstrated in extracts of human coronary microarteries. Immunohistochemical analysis revealed their exclusive localization in the endothelium, with no staining of vascular smooth muscle. In contractility experiments in which videomicroscopy was used, the nonspecific &bgr;-agonist isoproterenol and the &bgr;3-preferential agonist BRL37344 evoked an ≈50% relaxation of endothelin-1–preconstricted human coronary microarteries. Relaxations were blocked by the &bgr;1/&bgr;2/&bgr;3-adrenoceptor antagonist bupranolol but were insensitive to the &bgr;1/&bgr;2-adrenoceptor antagonist nadolol, confirming a &bgr;3-adrenoceptor–mediated pathway. Relaxation in response to BRL37344 was absent in human coronary microarteries devoid of functional endothelium. When human coronary microarteries were precontracted with KCl (thereby preventing vessel hyperpolarization), the relaxation to BRL37344 was reduced to 15.5% and totally abrogated by the NO synthase inhibitor l-&ohgr;-nitroarginine, confirming the participation of a NO synthase–mediated relaxation. The NO synthase–independent relaxation was completely inhibited by the Ca2+-activated K+ channel inhibitors apamin and charybdotoxin, consistent with an additional endothelium-derived hyperpolarizing factor–like response. Accordingly, membrane potential recordings demonstrated vessel hyperpolarization in response to &bgr;3-adrenoceptor stimulation. Conclusions—&bgr;3-adrenoceptors are expressed in the endothelium of human coronary resistance arteries and mediate adrenergic vasodilatation through both NO and vessel hyperpolarization.

Rho-dependent kinase is involved in agonist-activated calcium entry in rat arteries

The present study was aimed at investigating whether, besides its pivotal role in Ca2+‐independent contraction of smooth muscle, Rho‐kinase is involved in the mechanisms underlying the Ca2+ signal activated by noradrenaline in arteries. In rat aorta and mesenteric artery, the Rho‐kinase inhibitor Y‐27632 (10 μM) completely relaxed the contraction evoked by noradrenaline (1 μM) and simultaneously inhibited the Ca2+ signal by 54 ± 1 % (mesenteric artery) and 71 ± 15 % (aorta), and the cell membrane depolarisation by 56 ± 11 % (mesenteric artery). A similar effect was observed in arteries contracted by AlF4−, while in KCl‐contracted arteries, Y‐27632 decreased tension without changing cytosolic Ca2+. The same effects were observed with another inhibitor of Rho‐kinase (HA1077) but not with an inhibitor of protein kinase C (Ro‐31‐8220). Effects of Y‐27632 were not prevented by incubating the artery in 25 mM KCl, with K+ channel blockers or with the Ca2+ channel blocker nimodipine. Y‐27632 did not affect either the increase in the production of inositol phosphates activated by noradrenaline, or the release of Ca2+ from non‐mitochondrial stores evoked by InsP3 in permeabilised aortic cells, or the Ca2+ signals evoked by thapsigargin or caffeine. The capacitative Ca2+ entry activated by thapsigargin was not impaired by Y‐27632, but the entry of Ba2+ activated by noradrenaline in the presence of nimodipine was blocked by 10 μM Y‐27632. These results indicate that Rho‐kinase is involved in noradrenaline activation of a Ca2+ entry distinct from voltage‐ or store‐operated channels in rat arteries.

Cellular target of voltage and calcium-dependent K(+) channel blockers involved in EDHF-mediated responses in rat superior mesenteric artery.

We have investigated the cellular target of K+ channel blockers responsible for the inhibition of the EDHF‐mediated relaxation in the rat mesenteric artery by studying their effects on tension, smooth muscle cell (SMC) membrane potential and endothelial cell Ca2+ signal ([Ca2+]endo). In arteries contracted with prostaglandin F2α (2.5 – 10 μM), relaxation evoked by ACh (0.01 – 3 μM) was abolished by a combination of charybdotoxin (ChTX, 0.1 μM) plus apamin (Apa, 0.1 μM) and was inhibited by 68±6% (n=6) by 4‐aminopyridine (4‐AP, 5 mM). ACh (0.001 – 3 μM) increased [Ca2+]endo and hyperpolarized SMCs with the same potency, the pD2 values were equal to 7.2±0.08 (n=4) and 7.2±0.07 (n=9), respectively. SMCs hyperpolarization to ACh (1 μM) was abolished by high K+ solution or by ChTX/Apa. It was decreased by 66±5% (n=6) by 4‐AP. The increase in [Ca2+]endo evoked by ACh (1 μM) was insensitive to ChTX/Apa but was depressed by 58±16% (n=6) and 27±4% (n=7) by raising external K+ concentration and by 4‐AP, respectively. The effect of 4‐AP on [Ca2+]endo was not affected by increasing external K+ concentration. In Ca‐free/EGTA solution, the transient increase in [Ca2+]endo evoked by ACh (1 μM) was abolished by thapsigargin (1 μM) and was decreased by 75±7% (n=5) by 4‐AP. These results show that inhibition of EDHF‐evoked responses by 4‐AP may be attributed to a decrease in the Ca2+ release activated by ACh in endothelial cells. The abolition of SMCs hyperpolarization to ACh by ChTX/Apa is not related to an interaction with the [Ca2+]endo.

Effect of nitro‐L‐arginine on electrical and mechanical responses to acetylcholine in the superior mesenteric artery from stroke‐prone hypertensive rat

High salt diet is known to aggravate the vascular pathology in spontaneously hypertensive stroke‐prone rats (SHR‐SP). The aim of the present study was to assess the involvement of endothelial dysfunction in this effect. Contractile tension and membrane potential were simultaneously recorded in superior mesenteric artery rings of untreated and NaCl‐loaded (1% NaCl in the drinking water) SHR‐SP and normotensive Wistar Kyoto rats (WKY). In unstimulated artery, hyperpolarization evoked by acetylcholine was not different in WKY and in NaCl‐loaded WKY; it was reduced in SHR‐SP and further reduced in NaCl‐loaded SHR‐SP. Hyperpolarization was unaffected by Nω‐nitro‐L‐arginine (L‐NA) but was abolished in high‐KCl solution. In noradrenaline‐stimulated artery, ACh‐evoked hyperpolarization and relaxation were not different in WKY and in SHR‐SP. NaCl‐treatment did not affect the responses to ACh in WKY but decreased maximum relaxation in SHR‐SP from 93±2% to 72±7% of the contraction. In WKY, in NaCl‐loaded WKY and in SHR‐SP, L‐NA similarly shifted the concentration‐relaxation curve to ACh to the right and depressed its maximum but L‐NA did not affect the hyperpolarization to ACh. In NaCl‐loaded SHR‐SP, L‐NA blunted the effects of ACh on membrane potential and on contraction. The NO donor SNAP abolished the depolarization and the contraction evoked by noradrenaline with the same potency in WKY and in untreated SHR‐SP but was more potent in NaCl‐loaded SHR‐SP. In KCl‐contracted arteries the relaxations to ACh were not different in WKY and SHR‐SP but NaCl‐loaded SHR‐SP were more sensitive to ACh. The results showed that NaCl‐rich diet markedly reduced the L‐NA‐resistant responses to ACh and increased the sensitivity to NO in SHR‐SP.

Action of a NO donor on the excitation–contraction pathway activated by noradrenaline in rat superior mesenteric artery

The aim of the present study was to investigate the actions of NO donors in ratsuperior mesenteric artery stimulated with noradrenaline by studying their effects on isometric tension, membrane potential (Vm), cytosolic calcium concentration ([Ca2+]cyt) and accumulation of inositol phosphates. In unstimulated arteries, SNAP (S‐nitroso‐N‐acetylpenicillamine, 10 μm) hyperpolarised Vm by 3.0 ± 0.5 mV (n= 9). In KCl‐stimulated arteries, SNAP relaxed contraction without changing Vm and [Ca2+]cyt. In noradrenaline‐stimulated arteries, SNAP relaxed tension, repolarised Vm and decreased [Ca2+]cyt with the same potency. Responses to SNAP were unaffected by the following K+ channel blockers: glibenclamide, 4‐aminopyridine, apamin and charybdotoxin, and by increasing the KCl concentration to 25 mM. In SNAP‐pretreated arteries, the production of inositol phosphates and the contraction stimulated by noradrenaline were inhibited similarly. The guanylate cyclase inhibitor ODQ abolished the increase in cyclic GMP content evoked by SNAP and inhibited the effects of SNAP on contraction, Vm and accumulation of inositol phosphates in noradrenaline‐stimulated artery. These results indicate that, in rat superior mesenteric arteries activated by noradrenaline, inhibition of production of inositol phosphates is responsible for the effects of the NO donor SNAP on membrane potential, [Ca2+]cyt and contraction through a cyclic GMP‐dependent mechanism.