Exenatide induces aortic vasodilation increasing hydrogen sulphide, carbon monoxide and nitric oxide production

We performed our experiments with the permission of the Hungarian Local Animal Experiment Committee in accordance with the ‘Principles of laboratory animal care’ (NIH publication no. 85–23, revised 1985). Adult, 10–12 week old (280–340 g) male CFY Sprague-Dawly rats were kept on a standard chow. Animals were originally purchased from Charles River Laboratories GmbH (Sulzfeld, Germany). On the day of the experiment, after anesthetization with ether, rats were killed by decapitation using guillotine.

The thoracic aorta was gently excised from rats and was placed in oxygenated (95% O₂/5% CO₂), ice-cold Krebs solution (119 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂PO₄, 25 mM NaHCO₃, 1.2 mM Mg₂SO₄, 11.1 mM glucose, 1.6 mM CaCl₂*2H₂O, pH 7.4). As described earlier, the perivascular fat and connective tissue were gently removed [12, 14], and 2 mm long segments of the vessel were mounted on two stainless steel wires (40 μm in diameter), and placed in 5 ml organ baths of a wire myograph (Danish Multimyograph Model 610 M, DMT- USA Inc., Atlanta, GA, USA). Vessel rings were kept in Krebs solution at 37 ºC, pH 7.4 and were continuously oxygenated with a gas containing 95% O₂ and 5% CO₂. The rings were placed under a tension of 1 g [3]. Isometric tension was continuously recorded. After a 30 minutes resting period, vessel rings were preconstricted with 100 nM epinephrine as described earlier, which in our previously performed experiments had shown 60% contraction force of the 60 mM KCl contraction [15]. After all vessel segments had reached a stable contraction plateau, increasing doses of exenatide (Byetta® injection, Bristol-Myers Squibb–AstraZeneca, Budapest, Hungary) were administered to the organ baths, and relaxant responses were assessed. The dose of exenatide that was applied to relax the aorta correlated with the dose of epinephrine we used to preconstrict the vessels. Plasma epinephrine level is approximately 30 pM at rest, while in our experiments we used 100 nM, which is a 3000 times higher concentration [16]. The plasma exenatide level was found to be 70 pM, while in our experiments we used a 4500 times higher concentration [17].

In order to identify the extracellular and intracellular mediators of the vasodilator effect of exenatide we performed a series of experiments. Prior to contracting the vessels with epinephrine we preincubated the vessels (n = 5 of each experiment) with different materials. To determine whether the vasodilation due to exenatide evoked via the GLP-1R, we preincubated vessels with GLP-1R antagonist exendin(9–39) (32 μM, 30 min). Because the affinity of exendin(9–39) to bind GLP-1R is smaller than that of exenatide, we applied a ten times higher concentration of the receptor antagonist than the highest dose of exenatide [18]. In one set of experiments we mechanically removed the endothelium of the vessels by gently rubbing a hair through it. The effect of denudation was verified by the loss of response to 3 μM acetylcholine. We incubated one group of vessels with the eNOS inhibitor L-NAME (300 μM, 30 min). Other vessels were incubated with the potent heme oxygenase inhibitor Tin-protoporphyrin IX dichloride (10 μM, 30 min), others with DL-Propargylglycine, inhibitor of cystathionine-γ-lyase (10 mmol/l, 30 min), or with the relatively selective COX-1 inhibitor indomethacin (3 μM, 30 min). We tested the effects of free radical scavengers superoxide dismutase (SOD; 200 U/ml, 30 min) and catalase (1000 U/ml,30 min). H89 hydrochloride (5 μM, 30 min) was used to block protein kinase A (PKA), and 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ, 3 μM, 30 min) was used to inhibit the effect of soluble guanylyl cyclase (sGC). To block the large-conductance calcium-activated potassium channels (BK_Ca channels) some vessels were incubated with tetraethylammonium (TEA, 2 mM) for 30 minutes [13]. To block the ATP-sensitive potassium (K_ATP) channels we used glibenclamide (10 μM, 30 min) [13]. KCNQ-type voltage-dependent potassium channels were blocked by incubation with XE991 (30 μM, 15 min) [14]. The Na⁺/Ca²⁺-exchanger was blocked by incubation with its specific inhibitor SEA0400 (4 μM, 30 min) [19].

To exclude the effect of spontaneous vessel relaxation we performed untreated time-control experiments; however, the spontaneous vessel relaxation of untreated aortic rings was not significant. To test the effect of the specific inhibitors on the permanence of the epinephrine-induced plateau, we performed a row of control experiments, and found that most of the chemicals had a slight vasodilatory effect which could not have a significant influence on the results.

Chemicals were purchased from Sigma-Aldrich, St. Louis, MO, USA, except for Tin-protoporphyrin IX dichloride, which was purchased from Santa Cruz Biotechnology (Dallas, Texas, USA); XE991 was purchased from Ascent Scientific Ltd. (Avonmouth, Bristol, UK), and epinephrine was purchased from Richter-Gedeon Hungary (Budapest, Hungary). SEA0400 was synthesized in the Institute of Pharmaceutical Chemistry, University of Szeged, Szeged, Hungary by Professor Ferenc Fülöp.

Myodaq 2.01 M610+ software was used for data acquisition and display. We expressed the rate of relaxation caused by exenatide as the percentage of the contraction evoked by epinephrine.

After precontracting the vessels with epinephrine, time-control experiments showed that spontaneous vessel relaxation was not significant (Figure 1A). Following the epinephrine-induced contraction, in an other set of experiments we administered increasing doses of exenatide to the organ baths to assess the vasoactive effect of this GLP-1R agonist. We found a dose-dependent relaxation of the rat thoracic aorta due to exenatide (Figure 1B).

In our experiments exenatide induced vasodilation in a GLP-1R dependent manner, since preincubation with the specfic GLP-1R antagonist exendin(9–39) almost entirely blocked the vasodilation when the maximal dose of exenatide was applied, and totaly inhibited relaxation when smaller concentrations of the GLP-1 agonist were administered to the chambers (Figure 2A).

When the endothelium of the thoracic aorta was mechanically removed, the relaxation due to exenatide was significantly decreased (Figure 2B).

Incubation of vessels with the eNOS inhibitor L-NAME led to a mild but significant decrease in the relaxation of the rat thoracic aorta (Figure 3A). To determine further mediators of the vasodilator effect of exenatide, we examined the role of CO and H₂S. When we preincubated vessels with Tin-protoporphyrin, a potent heme oxygenase inhibitor, the vasorelaxation to exenatide was significantly reduced (Figure 3B). The inhibition of NO-synthesis and the inhibiton CO-production only partially decreased the rate of vasodilation: we therefore wished to prove that the third gasotransmitter, H₂S also plays a part in the vasoactive effect of exenatide. The inhibition of cystathionine-γ-lyase by preincubating vessels with PPG resulted in a significant decrease in the rate of relaxation (Figure 3C). Comparing the effects of these three gasotransmitters leading to vasodilation in response to exenatide, H₂S seemed to have the most remarkable effect.

Incubation of vessels with the COX inhibitor indomethacin for 30 minutes significantly decreased vasodilation to exenatide (Figure 3D).

To determine the role of ROS in the vasorelaxation caused by exenatide, we preincubated vessels with superoxide dismutase or with catalase. The rate of relaxation was significantly decreased in both experiments; however, SOD proved to be a more potent inhibitor of vasodilation to exenatide (Figure 4).

In order to determine the second messenger of the dilatation caused by exenatide, we incubated vessels with H89, an inhibitor of PKA. This caused only a mild decrease in the vasorelaxation at a low concentration (Figure 5A). In turn, inhibition of soluble guanylyl cyclase by ODQ significantly inhibited the vasorelaxation at higher concentrations as well (Figure 5B).

Preincubating vessels with three different potassium channel blockers before adding increasing concentrations of exenatide to the myograph chambers resulted in a signifcant decrease in the relaxation of all cases. Incubating one group of vessels with TEA, an inhibitor of the BK_Ca channels, demonstrated inhibition of vasodilation (Figure 6A). Relaxation was also inhibited by a blockade of the K_ATP channels by preincubation with glibenclamide (Figure 6B). In the group of vessels preincubated with XE991, a KCNQ (a type of K_v channels) channel inhibitor, most of the vasodilation was abolished (Figure 6C). However, there was a less pronounced decrease of vasodilation with the inhibition of BK_Ca and K_ATP channels compared to the inhibition of K_v channels.

We found that SEA0400, an inhibitor of the Na⁺/Ca²⁺-exchanger markedly inhibited vasorelaxation. Preincubation of vessels with SEA0400 almost completely abolished the whole of the vasodilation caused by exenatide (Figure 6D).