TASK‐1 channel blockade by AVE1231 increases vasocontractile responses and BP in 1‐ to 2‐week‐old but not adult rats

Background and Purpose The vasomotor role of K2P potassium channels during early postnatal development has never been investigated. We tested the hypothesis that TASK‐1 channel (K2P family member) contribution to arterial vascular tone and BP is higher in the early postnatal period than in adulthood. Experimental Approach We studied 10‐ to 15‐day‐old (“young”) and 2‐ to 3‐month‐old (“adult”) male rats performing digital PCR (dPCR) (using endothelium‐intact saphenous arteries), isometric myography, sharp microelectrode technique, quantitative PCR (qPCR) and Western blotting (using endothelium‐denuded saphenous arteries), and arterial pressure measurements under urethane anaesthesia. Key Results We found mRNA of Kcnk1–Kcnk7, Kcnk12, and Kcnk13 genes to be expressed in rat saphenous artery, and Kcnk3 (TASK‐1) and Kcnk6 (TWIK‐2) were most abundant in both age groups. The TASK‐1 channel blocker AVE1231 (1 μmol·L−1) prominently depolarized arterial smooth muscle and increased basal tone level and contractile responses to methoxamine of arteries from young rats but had almost no effect in adult rats. The level of TASK‐1 mRNA and protein expression was higher in arteries from young compared with adult rats. Importantly, intravenous administration of AVE1231 (4 mg·kg−1) had no effect on mean arterial pressure in adult rats but prominently raised it in young rats. Conclusion and Implications We showed that TASK‐1 channels are important for negative feedback regulation of vasocontraction in young but not adult rats. The influence of TASK‐1 channels most likely contributes to low BP level at perinatal age.


| INTRODUCTION
Adaptive changes in the blood supply of organs are a hallmark of their growth and maturation (Štulcová, 1977). The vascular system of the immature organism is characterized by a number of structural and functional features, including, but not limited to, low arterial pressure and greatly altered mechanisms of arterial tone regulation (D. K.  D. Gaynullina et al., 2013;Longo & Goyal, 2013;Mochalov et al., 2018;Sofronova et al., 2016). Interestingly, a few studies addressed the role of potassium channels in tone regulation of the systemic vasculature during the early postnatal period. Thus, in the immature period, BKCa channel blockade was ineffective regarding tone regulation in rat basilar arteries (Gollasch et al., 1998), and BKCa and Kv channel blockade produced only very small effects in sheep middle cerebral arteries (Teng, Nauli, Brayden, & Pearce, 2002), in contrast to later developmental stages. In rat aorta, the effects of BKCa channel blockade also increased with maturation, but the anticontractile influence of Kv channels was higher in the neonatal period (Gomez, Ghisdal, & Morel, 2000).
Recently, we showed that in rat saphenous arteries in the early postnatal period, the negative feedback regulation of vasocontraction by BKCa channels was small, but the influence of Kv1, Kir, and Kv7 channels was quite pronounced compared with adult animals (Shvetsova, Gaynullina, Tarasova, & Schubert, 2019).
Thus, these data suggest that the contribution of potassium channels to arterial tone regulation in the early postnatal period varies depending on the vascular bed and/or channel type and, therefore, cannot be predicted for channels not studied yet.
There is one potassium channel family, the K2P channels, whose role in arterial tone regulation has been studied with comparatively low intensity so far. Research over the last years has changed the image of these channels from being simply leak channels to regulatory channels having important roles in the control of cell excitability (Renigunta, Schlichthörl, & Daut, 2015). Several members of the K2P channel family have been shown to be expressed differentially in various arteries (Gurney & Manoury, 2009). Most published studies have been focused on TASK-1 channels expressed in the majority of the arteries investigated so far (Gurney & Manoury, 2009). This channel was suggested to mediate changes in arterial tone induced by pH alterations (Gardener et al., 2004) or hypoxia (Nagaraj et al., 2013;Olschewski et al., 2006) in rabbit, rat, and human arteries. Moreover, TASK-1 channels have been proposed to be involved in human pulmonary arterial hypertension (Antigny et al., 2016;Ma et al., 2013).
However, the contribution of TASK-1 channels to the regulation of vascular tone during early postnatal development has never been addressed.
We tested the hypothesis that TASK-1 channel contribution to arterial vascular tone and BP is higher in the early postnatal period than in adulthood. To evaluate the functional impact of TASK-1 channels, we used AVE1231, which was initially described as a Kv1.5 channel blocker, but then demonstrated higher affinity for TASK-1 channels (Kiper et al., 2015). To provide evidence for the specificity of the effect of AVE1231 on TASK-1 channels in relation to its possible effects on Kv1.5 channels, we used DPO-1, a specific Kv1 channel blocker (Lagrutta, Wang, Fermini, & Salata, 2006;Tsvetkov et al., 2016).

| Animals
Male Wistar rats aged 10 to 15 days ("young" in the following text) and 2 to 3 months ("adult" in the following text) were used in this study (obtained from Janvier, France, or the Institute of General Pathology and Pathophysiology, Russia). Rats were killed under CO 2 anaesthesia by decapitation. Animal studies are reported in compliance with the ARRIVE guidelines (Percie du Sert et al., 2020) and with the recommendations made by the British Journal of Pharmacology (Lilley et al., 2020). Approval for the use of laboratory animals and all procedures used in this study was granted by German and Russian institutional committees on animal welfare (I-17/17 and 93-g, respectively). Rats have been used for research on potassium channel function in early postnatal ontogenesis in many studies (Belevych, Beck, Tammaro, Poston, & Smirnov, 2002;Gollasch et al., 1998;Shvetsova et al., 2019). Since the male body is not subjected to cyclical hormonal changes, we decided to employ a commonly used approach, namely, to explore a novel physiological mechanism only in males. The animals What is already known • TASK-1 channels are expressed in arteries of the pulmonary and systemic circulation.
• TASK-1 channel blockade by AVE1231 increases contractile responses in pulmonary arteries.

What this study adds
• TASK-1 channel expression is higher in arteries of 1-to 2-week-old compared with adult rats.
• TASK-1 channels contribute to arterial vascular tone and the setting of the BP level in the early postnatal period.
What is the clinical significance • The pathogenesis of cardiovascular diseases in neonates can be associated with TASK-1 channel dysfunction.
• Potential vasopressor actions of TASK-1 channel blockers should be considered in the recently suggested TASK-1 channel-targeted treatment of cardiac arrhythmia.
were provided with food and water ad libitum and housed in a room with a controlled temperature and a 12-h light-dark cycle.

| Wire myograph experiments
The saphenous artery, a muscle-type artery of the cutaneous circulation, was chosen as the main object for the present study. The arteries were carefully cleaned from surrounding tissue, cut into 2-mm-long segments, and mounted in a wire myograph (410A or 620M, DMT A/S, Denmark) to measure isometric force. Right after mounting, the endothelium was gently denuded using a rat whisker.
The experimental protocol included two sequential concentration-response relationships to methoxamine (concentration range from 0.01 to 100 μmol·L −1 ). The first relationship was started 20 min after the end of the activation procedure. Thereafter, one arterial segment was incubated for 20 min with a potassium channel blocker (AVE1231, DPO-1, or their combination) and the other onewith an equivalent volume of the solvent (time control). Then the second concentration-response relationship was obtained (for details, see Figure 2).
To calculate active force values at each methoxamine concentration, the force value at the fully relaxed state was subtracted from all recorded values. All active force values obtained during the second concentration-response relationship (which are shown in Figure 3a-f) were expressed as the percentage of the maximum active force developed during the respective first concentration-response relationship.
Presentation of data in percentage allows to eliminate variability due to differences in the size of different vessels. To estimate the initial sensitivity of arteries to methoxamine, individual concentrationresponse relationships were fitted to a sigmoidal dose-response (variable slope) equation using GraphPad Prism 7.0 (La Jolla, CA, USA, RRID:SCR_002798) to obtain pD 2 (the negative logarithm of EC 50 ).

| Membrane potential measurements
Smooth muscle membrane potential was measured simultaneously with isometric force using the sharp microelectrode technique as described in our previous study (Shvetsova et al., 2019). Briefly, saphenous arteries were isolated, denuded of the endothelium, and Active force values were calculated as the percentage of the maximum force obtained during the concentration-response relationship to methoxamine.

| Measurement of mRNA expression levels in arterial tissue by digital PCR
Endothelium-intact saphenous arteries were quickly isolated from young and adult rats. The preparation procedure was carried out in ice-cooled solution (for composition, see Section 2.2.). After isolation, arteries were frozen in liquid nitrogen and stored at −80°C until further manipulations. Each sample included four arteries from two young rats or one artery from an adult rat.
Arteries were homogenized using the TissueLyser (Qiagen

| Measurement of mRNA expression levels in arterial tissue by qPCR
Saphenous arteries were isolated, carefully cleaned from surrounding tissue, cut into 8-mm-long segments, and quickly mounted in an icecooled analogue of a wire myograph system, to remove the endothelium using a rat whisker. Endothelium-denuded arterial segments were kept in RNA-later solution (Qiagen) at −20°C pending further procedures. Each sample included four arterial segments, each from two young rats, or two arterial segments from one adult rat. RNA was confirmed by about 10-fold drop of Nos3 mRNA content in endothelium-denuded versus endothelium-intact samples ( Figure S1).

| Measurement of protein abundance in arterial tissue by Western blotting
Four saphenous arteries from two young rats or two saphenous arteries from one adult rat were used for one sample. The endothelium was rapidly removed after isolation of the artery in the same way as described for qPCR. Then arterial samples were quickly frozen in liquid nitrogen and kept at −80°C till further analysis.
Samples were homogenized in ice-cold SDS buffer (

| BP measurement
Haemodynamic measurements were performed in urethaneanaesthetized rats (1.2 g·kg −1 , i.p.). Urethane is widely used as anaesthesia in acute experiments with a terminal point, and it provides long-term (1 h or more) and stable anaesthesia in rats, including 1-week-old pups (Mochalov et al., 2018). Other types of anaesthesia do not match these requirements and may exert undesirable side effects. For example, isoflurane was shown to activate some potassium channels, including several K2P family members (Patel et al., 1999). During the initial 10-15 min, arterial pressure and heart rate (HR) were allowed to stabilize. Then the ganglionic blocker chlorisondamine (2.5 mg·kg −1 , i.v.) was injected through a venous catheter, and a 10-to 15-min-long baseline recording was obtained.
Then AVE1231 (4 mg·kg −1 , i.v.) or its solvent DMSO (0.5 ml·kg −1 ) was injected through the second venous catheter, and the recording was continued for more than 15 min.
The BP signal was processed beat-to-beat to estimate mean arterial pressure (MAP) and HR. Then MAP and HR were averaged in 1.5-to 2-min-long intervals: (i) before chlorisondamine administration; (ii) before AVE1231 or DMSO administration; and (iii) at the peak response to AVE1231 or the corresponding time period after administration of DMSO.

| Data and statistical analysis
The data and statistical analysis comply with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology (Curtis et al., 2018). Treatment of arterial segments with certain substances within each experimental group was randomized. Blinding of the operator was not feasible because vessel responses observed by the operator to manage the experiment permitted inferences about the treatment. However, data analysis was performed semi-blinded by an independent analyst.
The number of experiments was selected during experimental design based on considerations not to employ an unnecessary amount of animals (3R principles) and to get evidence for an effect of reasonable size in both age groups. Based on the experience of previous studies, the number of animals/tissue samples in each group was at least 6, except for one sample in Figure S1 with Concentration-response relationships to methoxamine between two groups were compared using repeated measures ANOVA.
Statistical analyses of values of vessel diameter, force, membrane potential, mRNA contents, and protein contents were performed using a two-sided unpaired Student's t test or Mann-Whitney U test, depending on the type of data distribution. Differences were accepted as statistically significant if the P value was less than 0.05.

| Expression pattern of K2P channels in saphenous arteries of adult and young rats
At the first step of the study, we evaluated the abundance of different K2P family members in arterial tissue on the mRNA level using dPCR.
Among them, mRNA for Kcnk3 and Kcnk6 was the most abundant ( Figure 1a). Next, we measured the mRNA expression of K2P family members in endothelium-intact saphenous arteries (n = 7) of young rats. Similarly, mRNA for Kcnk1-Kcnk7, Kcnk12, and Kcnk13 was expressed ( Figure 1b); the highest expression level was found for Kcnk3 and Kcnk6 (Figure 1b). mRNA for Kcnk9, Kcnk10, Kcnk15, Kcnk16, and Kcnk18 was not detected in arterial samples from either adult or young animals. The functionality of the qPCR assays for these Kcnk genes was demonstrated by positive qPCR results from cDNA of rat brain for all Kcnk genes except Kcnk18. The use of seven different qPCR assays for different Kcnk18 regions confirmed the negative result and indicated a very low or absent expression of Kcnk18 in rat brain. Assuming functionality, the commercial TaqMan Kcnk18 qPCR assay was used for expression analysis in the arterial samples.

| Effects of AVE1231 and DPO-1 on arterial contractile responses in adult and young rats
The next steps in our study were focused on TASK-1 (Kcnk3) channels since this channel (i) is one of the most abundant in saphenous arteries in both age groups; (ii) is one of the most studied in the circulatory system; and (iii) is one of the few K2P channels for which a tool for functional studies is available (TASK-1 channel blocker AVE1231/A293) (Antigny et al., 2016;Kiper et al., 2015).
Importantly, the first concentration-response relationships to methoxamine for endothelium-denuded arterial segments further treated with potassium channel blockers or solvent, respectively, were not different within the same age group ( Table 1)  Note: Data are presented as mean ± SD. AVE1231 (Figures 2 and 3b). These data indicate that TASK-1 channels are important for the regulation of arterial tone in young, but not in adult animals.
Thus, its effect on vessels from young rats might be possibly related to its effect on Kv1.5 channels. In order to test this possibility, we performed additional control experiments using the In arteries from young rats, DPO-1 did not affect basal tone but increased methoxamine-induced contractions (Figure 3d). Importantly, AVE1231 in the presence of DPO-1 strongly augmented basal tone and contractions to methoxamine of arteries from young animals ( Figure 3f). These data show that the effects of AVE1231 in arteries of young rats remain after blockade of Kv1.5 channels and, therefore, are considered to be predominantly associated with its influence on TASK-1 channels.

| Effects of AVE1231 on smooth muscle cell membrane potential in adult and young rats
The resting membrane potential (Step 1 in Figure 4, combined data for AVE1231 and time-control experiments) was −65.0 ± 5.7 mV (n = 14) and −67.0 ± 3.4 mV (n = 16) in endothelium-denuded saphenous arteries of adult and young animals respectively (P = 0.26, unpaired Student's t test).
Importantly, the values of force and membrane potential at Step 1 as well as the methoxamine-induced depolarization and increase in active force (Step 2 in Figure 4; see Section 2 for detailed description) did not differ between preparations further treated with AVE1231 or solvent, respectively, within the same age group (Table 2). This setting allowed us to estimate the effects of AVE1231 by comparing the values at Steps 3-5 between AVE1231-and DMSO-treated vessels (these data are shown in Figure 5a-d).
AVE1231 did not change basal tone in arteries of adult rats  (Figures 4 and 5b) F I G U R E 2 Recordings of contractile responses of two saphenous artery segments to methoxamine in a 12-day-old rat. The first concentration-response relationships (left panels in a and b) are similar for the two arterial segments. Application of the TASK-1 channel blocker AVE1231 (1 μmol·L −1 ) causes an increase in basal tone and of the contractile responses to methoxamine (a), while incubation with an equivalent volume of the solvent (5-μl DMSO per 5-ml myograph chamber) does not affect the level of basal tone and the contractile response (b). W, washout associated with pronounced smooth muscle depolarization (Figures 4   and 5d). Importantly, the AVE1231-induced depolarization (identified as the difference between Step 4 and Step 3 in AVE1231-treated preparations; see Figure 4 and Section 2 for description) was considerably larger for young rats (19.1 [16.6-26.5] mV, n = 10) compared with adult animals (12.3 [5.7-15.4] mV, n = 7, P < 0.05, Mann-Whitney U test). Further, in young rats, AVE1231 considerably increased methoxamine-induced contraction and led to higher depolarization compared with time-control preparations (Figures 4 and 5b,d). Taken together, these data demonstrate that the stronger influence of TASK-1 channel blockade on basal tone and methoxamine-induced contractile responses in arteries of young rats compared with older animals is associated with its higher impact on membrane potential.

| Comparison of TASK-1 channel abundance in endothelium-denuded arteries of adult and young rats
In order to identify whether the observed stronger impact of TASK-1 channels on smooth muscle contractile responses and depolarization in the younger age group is associated with their higher abundance in arterial smooth muscle, we estimated mRNA and protein contents of TASK-1 in endothelium-denuded arteries of the two age groups. Indeed, the relative expression level of the Kcnk3 gene in arterial preparations was considerably higher in young compared with adult animals (Figure 6a). Further, protein abundance of TASK-1 channels was larger in smooth muscle of young compared with adult animals ( Figure 6b). Thus, our results clearly demonstrate that the larger role of TASK-1 channels at the functional level in arteries of young rats is associated with their higher expression level.

| Effects of AVE1231 on systemic cardiovascular parameters in adult and young rats
To assess the role of TASK-1 channels in the regulation of systemic haemodynamics, we evaluated the effects of AVE1231 on MAP and HR in anaesthetized rats. At the beginning of the experiment, MAP was almost two times lower in young compared with adult animals; F I G U R E 3 Effects of AVE1231, DPO-1, and their combined effects on the contractile responses to methoxamine of arteries from two age groups. (a, b) Concentration-response relationships to methoxamine in the presence of solvent (DMSO) or the TASK-1 channel blocker (AVE1231, 1 μmol·L −1 ) of arteries from adult (a; n = 6, 6) and young rats (b; n = 9, 9), respectively. (c, d) Concentrationresponse relationships to methoxamine in the presence of solvent (DMSO) or the Kv1 channel blocker (DPO-1, 1 μmol·L −1 ) of arteries from adult (c; n = 6, 6) and young (d; n = 9, 8) rats, respectively. (e, f) Concentration-response relationships to methoxamine in the presence of Kv1 channel blocker only (DPO-1, 1 μmol·L −1 ) or the Kv1 channel blocker together with TASK-1 channel blocker (DPO-1 + AVE1231, both 1 μmol·L −1 ) of arteries from adult (e; n = 6, 6) and young (f; n = 8, 8) rats, respectively. B.t., basal tone value (active force before the first concentration of methoxamine). Data are presented as mean ± SD. *P < 0.05 between AVE1231 and DMSO or DPO-1 + AVE1231 and DPO-1 application (repeated measures ANOVA). $ P < 0.05 between basal tone values of AVE1231 and DMSO or DPO-1 + AVE1231 and DPO-1 application (unpaired Student's t test) HR did not differ between the two age groups (Table S1). In order to exclude the influence of the autonomic nervous system, the effects of AVE1231 were studied after ganglionic blockade by chlorisondamine (2.5 mg·kg −1 ). After ganglionic blockade, both MAP and HR values were lower in young compared with adult rats (Table S1).
In each age group, some of the animals were further treated with AVE1231 and others with the vehicle (DMSO). Importantly, no differences in baseline values of either MAP or HR were observed between the two subgroups (further treated with AVE1231 or with DMSO) within the same age group (Table S2). In adult rats, AVE1231 induced a transient increase of MAP that was similar to the changes in the DMSO-treated subgroup (Figure 7a). In contrast, in young rats, the AVE1231-induced MAP increase was larger compared with the DMSO-treated subgroup (Figure 7b). The effect of AVE1231 on MAP plateaued after 2-3 min. Noteworthy, HR levels after administration of AVE1231 were not different compared with their levels in the DMSO-treated subgroups in both adult and young animals (Table S2).
Thus, TASK-1 blockade increased the systemic BP level only in young rats, and this effect was not associated with an altered HR.

| DISCUSSION
This study provides novel data on developmental alterations in the functioning of TASK-1 channels in arterial smooth muscle. We showed that smooth muscle mRNA and protein content of TASK-1 channels is higher in peripheral arteries from young compared with adult rats. In accordance with this, TASK-1 channels oppose vasocontraction by counteracting arterial smooth muscle depolarization only at early postnatal age but not in adulthood. Moreover, TASK-1 channel blockade affected systemic BP in young but not adult rats.

| TASK-1 channels are highly abundant in rat saphenous arteries
A number of studies have demonstrated expression of some K2P channel members in cerebral (Bryan et al., 2006) as well as pulmonary and mesenteric arteries (Gardener et al., 2004) of rats. To the best of our knowledge, for the first time, we evaluated the expression pattern of all known K2P channel family members in a rat artery, the saphenous artery, using the dPCR approach. The advantage of dPCR is that Change of force (% of maximum) 19.1 ± 14.6 14.8 ± 8.4 17.3 ± 9.9 18.2 ± 3.6 Change of membrane potential (mV) 24.0 ± 4.7 20.5 ± 6.3 20.0 ± 10.7 19.6 ± 12.7 Note: Data are presented as mean ± SD.
F I G U R E 4 Traces of an experiment with simultaneous registration of force and membrane potential of a saphenous artery from a 12-dayold-rat. Membrane potential and force were detected under five experimental conditions: (1) in the absence of any blockers or agonists-Baseline 1; (2) during methoxamine-induced contraction (about 20% of maximum force); (3) after washout of methoxamine-Baseline 2; (4) in the presence of AVE1231 (1 μmol·L −1 ); and (5) in the presence of AVE1231 (1 μmol·L −1 ) together with the same concentration of methoxamine as at Step 2 F I G U R E 6 TASK-1 channel abundance in endothelium-denuded arteries of two age groups. (a) Relative expression levels of Kcnk3 poreforming subunit mRNA in arteries from adult (n = 8) and young (n = 9) rats. Data are normalized to the geometric mean of Rn18s and Gapdh.
(b) Protein content of TASK-1 pore-forming subunit in arteries from adult (n = 7) and young (n = 6) rats. Data are normalized to loading control (determined by Ponceau S staining). The median value of TASK-1 mRNA or protein content in the adult group is taken as 100%. Data are presented as the median and interquartile range. # P < 0.05 between adult and young animals (Mann-Whitney U test) 4.3 | TASK-1 channels are important for the regulation of membrane potential and arterial tone in arteries of young rats The ability of K2P channels to conduct outward potassium currents in a wide potential range suggests their important role in the regulation of membrane potential and, therefore, vascular tone (Gurney & Manoury, 2009). Indeed, a number of studies have demonstrated a pronounced anticontractile influence of these channels in pulmonary arteries of human and rat (Antigny et al., 2016;Gardener et al., 2004).
However, a functional role of TASK-1 channels in pulmonary arteries of mice was not observed (Manoury, Lamalle, Oliveira, Reid, & Gurney, 2011;Murtaza et al., 2017). We also did not observe consid-   (Mulvany, Nilsson, & Flatman, 1982;Neild & Kotecha, 1987). Therefore, during non-receptor stimulation, arterial smooth muscle starts to contract only from a certain threshold level of membrane potential. Probably, 12-mV smooth muscle depolarization induced by AVE1231 in arteries from adult rats was subthreshold for the development of a contractile response.
In contrast, in young rats, blockade of TASK-1 channels with AVE1231 caused a strong smooth muscle depolarization, which was sufficient for the development of contraction. Previously, we demonstrated a higher contribution of Kir and especially Kv7 channels in the regulation of membrane potential and vascular tone in arteries from young compared with adult rats (Shvetsova et al., 2019). Our new data suggest that TASK-1 channels together with Kir and Kv7 channels play an important role in the negative feedback regulation of vasocontraction in the period of early postnatal ontogenesis, but not in adulthood.
To our knowledge, in this study, AVE1231 was used to study the role of TASK-1 channels in rat systemic arteries for the first time.
Previously, AVE1231 (A293) at the same concentration (1 μmol·L −1 ) was shown to increase contractile responses of isolated rat pulmonary arteries (Antigny et al., 2016). Of note, the IC 50 of AVE1231 for human TASK-1 channels in Xenopus oocytes is 0.2 μmol·L −1 , while its IC 50 against Kv1.5 is 43 times higher (Kiper et al., 2015). Thus, at the concentration used, AVE1231 would block primarily and considerably TASK-1 channels but not Kv1.5 channels. This suggestion is supported by our data showing additive effects of AVE1231 (1 μmol·L −1 ) and DPO-1 (1 μmol·L −1 ) in arteries of young rats. Since F I G U R E 7 AVE1231 increases mean arterial pressure (MAP) in young (b) but not adult (a) animals. The change of MAP was calculated as the difference between the peak MAP value after intravenous administration of vehicle (DMSO, 0.5 ml·kg −1 ) or AVE1231 (4 mg·kg −1 ) and the respective baseline value. The experiments were performed under the condition of ganglionic blockade (chlorisondamine, 2.5 mg·kg −1 ). Data are presented as the median and interquartile range. # P < 0.05 between DMSO and AVE1231 experiments (Mann-Whitney U test) the IC 50 for DPO-1 against Kv1.5 channels is less than 0.2 μmol·L −1 (Lagrutta et al., 2006), 1 μmol·L −1 of DPO-1 should block Kv1.5 channels to a large degree (in a specific manner) (Tsvetkov et al., 2016).
Thus, the large additional effect of 1 μmol·L −1 of AVE1231 is most likely due to a block of TASK-1 channels rather than a blockade of the few remaining Kv1.5 channels after DPO-1 blockade.
Therefore, TASK-1 channels counteract arterial smooth muscle depolarization and, thereby, reduce contractile responses of arterial smooth muscle in young but not adult rats, in line with the greater expression of TASK-1 channels in smooth muscle of saphenous arteries at the early postnatal period. Of note, TASK-1 channels limiting smooth muscle depolarization are most likely located primarily in the smooth muscle cells of the vessel wall since the effects of AVE1231 on basal tone and on contractile responses to methoxamine were comparable in endothelium-intact and endothelium-denuded arteries from young rats ( Figure S2).

| TASK-1 channels are important for arterial BP control in young rats
To get further insight into the physiological role of TASK-1 channels, we compared the effects of AVE1231 on MAP in the two age groups of rats. Of note, blood flow to the skin comprises up to 20% of the cardiac output (Štulcová, 1977) and, thereby, is functionally relevant.
Therefore, functional alterations of skin feed (saphenous) arteries could affect the BP level in young rats.
In our experiments, AVE1231 was dissolved in DMSO, which is the most often used vehicle for hydrophobic substances. However, DMSO may exert its own biological effects, which could confound the effects of AVE1231 (Kelava, Cavar, & Culo, 2011;Sawada & Sato, 1975). To distinguish between the effects of the blocker and the vehicle, we compared the change of MAP at the same time points in animal groups treated with AVE1231 and DMSO respectively.
In a previous study, Wirth et al. (2007) evaluated the in vivo effects of AVE1231 in pigs and goats using a dose of 3 mg·kg −1 . In our experiments, we started from a dose of 1 mg·kg −1 and then increased it in a stepwise manner to get an evident effect on BP at least in one of the experimental groups. The minimum dose of AVE1231, which affected BP, was 4 mg·kg −1 . This is close to the dose in the study by Wirth et al. (2007), especially taking into account the differences in body surface area and metabolic rate between pigs/goats and rats (Nair & Jacob, 2016;Reagan-Shaw, Nihal, & Ahmad, 2008).
The MAP level was almost two times lower in young rats compared with adult animals, which is consistent with previously published data (Mochalov et al., 2018). The intravenous administration of AVE1231 (4 mg·kg −1 ) increased MAP in young rats, but not adult animals. Similarly, loss-of-function mutations in TASK-1 channels did not change systemic arterial pressure in adult rats (Lambert et al., 2019). Therefore, TASK-1 channels have a depressor effect at the systemic level only in the period of early postnatal development.
It is important to note that our experiments were carried out under conditions of ganglionic blockade. The use of ganglionic blockade allowed us to exclude the influence of AVE1231 on the activity of the autonomic nervous system and, therefore, to evaluate its effects exclusively in the periphery. TASK-1 channels are abundant in rat brain, including the ventrolateral medulla neurons, which are involved in the autonomic control of the circulation (Kindler, Pietruck, Yost, Sampson, & Gray, 2000;Washburn, Bayliss, & Guyenet, 2003). Of note, the expression of TASK-1 channels in the rat ventrolateral medulla is developmentally regulated (Kanjhan, Anselme, Noakes, & Bellingham, 2004).
Therefore, in the absence of ganglionic blockade, AVE1231 would affect the nervous control of the circulation, which is different in adult and young rats because of the immaturity of the vasomotor innervation in the younger group (Puzdrova et al., 2014).
We did not find differences in HR between AVE1231-and DMSO-treated groups of rats at either age. Previously, no chronotropic effect of AVE1231 in a similar dose (3 mg·kg −1 ) was observed in anaesthetized pigs as well (Wirth et al., 2007). Thus, we suggest that in our experimental conditions, AVE1231 had no prominent effect on heart function.
Taken together, our in vivo observations support the in vitro data demonstrating a more prominent influence of TASK-1 channels in arteries from young compared with older rats. Based on our results, we suggest that TASK-1 channels are important regulators of vascular tone and arterial pressure in the early postnatal period. Our BP measurements point to the presence of a TASK-1 channel-dependent control of vascular resistance not only in cutaneous but also in other vascular regions.

| CONCLUSION
In conclusion, our novel findings demonstrate a pronounced negative feedback regulation of vasocontraction by TASK-1 channels in the early postnatal period in rats. Taking into account the growing incidence of primary hypertension in childhood (Flynn, 2018), it is extremely important to identify all particular players regulating vascular tone and BP in the immature organism. We suppose that a large contribution of TASK-1 channels together with Kir and Kv7 channels (Shvetsova et al., 2019) is important to reduce peripheral vascular resistance and the BP level in the newborn organism to protect the immature arteries from high transmural pressure and the immature heart from high afterload. In addition, a pronounced anticontractile role of TASK-1 channels may contribute to an increase in the blood supply to growing organs during early postnatal development. It is known that the mechanisms regulating arterial smooth muscle contraction shift from calcium independent to calcium dependent during postnatal maturation (Akopov, Zhang, & Pearce, 1998a, 1998bMochalov et al., 2018;Puzdrova et al., 2014). Similarly, the role of potassium channels regulated by calcium to a lesser extent (TASK-1, Kv7, and Kir) decreases, while the role of calcium-dependent BKCa channels increases during postnatal development (Shvetsova et al., 2019), which could be a hallmark of maturation of peripheral arteries. The mechanism providing the postnatal decline in the contribution of TASK-1 channels to the regulation of vascular tone remains elusive and could serve as a topic for future studies.