Inhibition of neurogenic contractions in renal arteries and of cholinergic contractions in coronary arteries by the presumed inhibitor of ADP-ribosylation factor 6, NAV2729

Abstract

NAV2729 is a presumed inhibitor of the monomeric GTPase ADP ribosylation factor 6 (ARF6) and inhibits smooth muscle contraction outside the cardiovascular system. Its effects on vascular smooth muscle contraction or a possible role of ARF6 in vasocontraction have not yet been examined. Here, we report effects of NAV2729 on neurogenic and agonist-induced contractions in renal interlobar and coronary arteries. Contractions of pig interlobar and coronary arteries were induced in an organ bath by agonists or by electric field stimulation (EFS). Owing to divergent characteristics of both vessel types, EFS-induced contractions were only examined in interlobar arteries, and contractions by agonists acting on muscarinic receptors only in coronary arteries. NAV2729 inhibited frequency-dependent EFS-induced contractions of interlobar arteries. The degree of inhibition was similar using 5 µM and 10 µM NAV2729. Inhibition of EFS-induced contractions was resistant to a nitric oxide synthase inhibitor and to diclofenac. The neurogenic and adrenergic character of EFS-induced contractions was confirmed by inhibition by tetrodotoxin and prazosin. In coronary arteries, NAV2729 (5 µM) inhibited concentration-dependent contractions induced by carbachol and methacholine. Contractions induced by α₁-adrenergic agonists, endothelin-1, the thromboxane receptor agonist U46619, or serotonin remained unchanged by NAV2729 in both vessel types. NAV2729 inhibits neurogenic contractions in interlobar arteries and contractions induced by cholinergic agonists in coronary arteries. In both vessel types, NAV2729 does not inhibit contractions induced by receptor agonists other than those acting on muscarinic receptors. Addressing effects in other vessels and in other smooth muscle-rich organs merits further attention.

Keywords: ARF6; NAV2729; Vascular smooth muscle; Vasocontraction.

Fig. 1

High-molar KCl-induced contractions in renal interlobar and coronary arteries. Contractions by high-molar KCl were induced by the addition of a 2 M KCl solution to normal Krebs–Henseleit solution in organ bath chambers, before construction of frequency and concentration response curves (followed by washout before construction of curves and before the addition of DMSO or NAV2729) (A). These KCl-induced contractions were later used for reference of EFS- and agonist-induced contractions, assessed in the same tissues. DMSO was used as solvent for NAV2729 and applied to controls, in amounts required for corresponding NAV2729 in the same experiments. To estimate possible contributions from elevated chloride concentrations due to the hyperosmolar addition of KCl (B), vessels were first exposed to additional 80 mM NaCl (by addition of a 2 M solution to normal Krebs–Henseleit solution in organ bath chambers), followed by washout and assessment of KCl-induced contractions as described above. To assess effects of DMSO and NAV2729 on KCl-induced contractions (C), contractions by KCl were induced before and after application of DMSO or NAV2729 (5 µM), and the second KCl-induced contraction was expressed as the percentage of the first KCl-induced contraction. Shown are values from each single channel in mN (A, B), and single values (means from double determinations; paired values obtained from the same tissue and in the same experiment are marked by shared symbols) from each experiment (C)

Fig. 2

Effects of NAV2729 on EFS-induced contractions in renal interlobar arteries. Contractions were induced by different frequencies of EFS, 30 min following administration of NAV2729 at final concentrations of 5 µM (A), 10 µM (B), 5 µM in the presence of L-NAME (200 µM, in controls and in NAV2729 groups), and diclofenac (3 µM, in controls and in NAV2729 groups) (C), or solvent (controls). Shown are means ± SD in frequency response curves (together with p values for whole groups from two-way ANOVA in inserts), and all single E_max values and frequencies inducing 50% of the maximum EFS-induced contraction (Ef₅₀) from all experiments (calculated by curve fitting) in scatter plots (p value from paired Student’s t test), from experiments using tissues from n = 5 animals in A and C and from n = 7 animals in B. For each single experiment, tissue from one animal was allocated to the control and the NAV2729 group, which were examined in the same experiment. In scatter plots, paired values obtained from the same tissue and in the same experiment are marked by shared symbols

Fig. 3

Effects of tetrodotoxin and prazosin on EFS- and noradrenaline-induced contractions in renal interlobar arteries. Contractions were induced 30 min following administration of tetrodotoxin (1 µM) (A) or prazosin (100 nM) (B) by different frequencies of EFS, or of prazosin (100 nM) by cumulative concentrations of noradrenaline (C) in interlobar arteries, or of solvent (controls). Shown are means ± SD in frequency and concentration response curves (together with p values for whole groups from two-way ANOVA in inserts), and all single E_max values and frequencies inducing 50% of the maximum EFS-induced contraction (Ef₅₀) or pEC₅₀ values for noradrenaline from all experiments (calculated by curve fitting) in scatter plots (p value from paired Student’s t test), from experiments using tissues from n = 5 animals in each series. For each single experiment, tissue from one animal was allocated to the control and the drug group, which were examined in the same experiment. In scatter plots, paired values obtained from the same tissue and in the same experiment are marked by shared symbols

Fig. 4

Effects of NAV2729 on agonist-induced contractions in renal interlobar arteries. Contractions were induced by cumulative concentrations of noradrenaline (A, B), phenylephrine (C), endothelin-1 (D), U46619 (E), or serotonin (F), 30 min following administration of NAV2729 (5 µM or 20 µM before noradrenaline, 5 µM before all other agonists) or solvent (controls). Shown are means ± SD in concentration response curves, from experiments using tissues from n = 6 animals for noradrenaline and 5 µM NAV2729, n = 5 animals for noradrenaline and 20 µM NAV2729, n = 6 animals for phenylephrine, and n = 5 animals in each series in D–F. For each single experiment, tissue from one animal was allocated to the control and the NAV2729 group, which were examined in the same experiment

Fig. 5

Effects of NAV2729 on cholinergic contractions in coronary arteries. Contractions were induced by cumulative concentrations of carbachol (A) or methacholine (B), 30 min following administration of NAV2729 (5 µM) or solvent (controls). Shown are means ± SD in concentration response curves (together with p values for whole groups from two-way ANOVA in inserts), and all single E_max values and pEC₅₀ values from all experiments (calculated by curve fitting) in scatter plots, from experiments using tissues from n = 5 animals for each series. For each single experiment, tissue from one animal was allocated to the control and the NAV2729 group, which were examined in the same experiment. In scatter plots, paired values obtained from the same tissue and in the same experiment are marked by shared symbols

Fig. 6

Effects of NAV2729 on agonist-induced contractions in coronary arteries. Contractions were induced by cumulative concentrations of endothelin-1 (A), serotonin (B), U46619 (C), phenylephrine (D), or methoxamine (E), 30 min following administration of NAV2729 (5 µM) or solvent (controls). Shown are means ± SD in concentration response curves, from experiments using tissues from n = 6 animals for U46619 and phenylephrine, n = 11 animals for serotonin, and n = 5 animals for each other series. For each single experiment, tissue from one animal was allocated to the control and the NAV2729 group, which were examined in the same experiment