Hydrogen Sulfide-Induced Vasodilation: The Involvement of Vascular Potassium Channels

Hydrogen sulfide (H2S) has been highlighted as an important gasotransmitter in mammals. A growing number of studies have indicated that H2S plays a key role in the pathophysiology of vascular diseases and physiological vascular homeostasis. Alteration in H2S biogenesis has been reported in a variety of vascular diseases and H2S supplementation exerts effects of vasodilation. Accumulating evidence has shown vascular potassium channels activation is involved in H2S-induced vasodilation. This review aimed to summarize and discuss the role of H2S in the regulation of vascular tone, especially by interaction with different vascular potassium channels and the underlying mechanisms.


INTRODUCTION
Hydrogen sulfide (H 2 S) has been recognized as the third gasotransmitter following nitric oxide and carbon monoxide. Endogenous H 2 S is mainly produced by enzyme pathways in vivo, including cystathionine γ-lyase (CES), cystathionine β-synthase (CBS), cysteine aminotransferase and 3mercaptopyruvate sulfurtransferase (3-MST) (Olas, 2015). In the vascular system, H 2 S is principally synthesized by CES (Szabo, 2012). Biogenesis of H 2 S or downregulation of H 2 S pathway has been found in a variety of vascular diseases, such as hypertension, atherosclerosis, diabetes and pulmonary hypertension (Barton and Meyer, 2019;Dillon et al., 2021;Dorofeyeva et al., 2021;Roubenne et al., 2021). H 2 S plays an important role in regulation vascular homeostasis, such as angiogenesis, proliferation of vascular smooth muscle cells, leukocyte adhesion, oxidative stress and vascular dilation . The mechanisms underlying H 2 S induced vascular dilation are not completely understood.
Potassium channels are important determinant of vascular tone (Lorigo et al., 2020;Burkhoff et al., 2021). Activation of potassium channels in vascular cells leads to membrane hyperpolarization and subsequent vasodilatation. Five types of potassium channels have been recognized in vascular cells, including voltage-dependent potassium channels (K V ), Ca 2+ -activated potassium channels (K Ca ), ATP-sensitive potassium channels (K ATP ), inward rectifier potassium channels (Kir), and tandem two-pore potassium channels (K 2P ) (Dogan et al., 2019). Accumulating evidence has shown vascular potassium channels activation is involved in H 2 S-induced vasodilation Marinko et al., 2021;Wen et al., 2021). However, the interaction of H 2 S with different vascular potassium channels and the underlying mechanisms have not been reviewed systematically. This review aimed to summarize and discuss the effects of H 2 S in the regulation of vascular tone, especially by interaction with different vascular potassium channels and the underlying mechanisms.

ROLE OF HYDROGEN SULFIDE IN VASCULAR DISEASES AND VASODILATION
Biogenesis of H 2 S or downregulation of H 2 S has been found in a variety of vascular diseases. Plasma H 2 S levels were significantly lower in ever-treated hypertensive patients compared with normal subjects, and were higher in patients with wellcontrolled blood pressure than in patients with grade 2 and 3 hypertension, suggesting that the reduction of endogenous H 2 S plays a role in the pathophysiology of hypertension (Sun et al., 2007). The H 2 S generating enzymes, CSE and 3-MPST were significantly reduced in cutaneous tissue of hypertension adults (Greaney et al., 2017); CSE and CBS expression in lymphocytes were reduced of hypertensive patients (Cui et al., 2020). In patients with portal hypertension, plasma H 2 S was significantly lower than that in normal subjects, and the level of H 2 S was negatively correlated with the severity of the disease . In patients with pulmonary hypertension, plasma level of H 2 S was decreased (Sun et al., 2014) and negatively correlated with pulmonary artery diameter (Liao et al., 2021). The increase of H 2 S predicted lower pulmonary artery diameter (Liao et al., 2021). Thus, H 2 S may be involved in these vascular diseases, providing a new method for the diagnosis and treatment of these vascular diseases.
Accumulating evidence indicated endogenous H 2 S or H 2 S supplementation may prevent the development of the vascular diseases. 8 weeks administration of NaHS, a H 2 S donor, lowered tail artery pressure in spontaneously hypertensive rats (SHR) (Sun et al., 2015). Tain et al. (2018) also reported early short-term NaHS treatment prevented the development of hypertension in SHRs. Exogenous supply of H 2 S reduced pulmonary hypertension (Chunyu et al., 2003). The intravenous injections of H 2 S donors, Na 2 S and NaHS, decreased systemic arterial pressure in the anesthetized rats (Yoo et al., 2015). The predominantly effect of H 2 S or the H 2 S donors on regulation of vascular tone is vasodilation, although H 2 S has vascular constrictive effects in certain conditions (Orlov et al., 2017). NaHS produced vasorelaxation in different vessel beds, including aorta (Köhn et al., 2012), coronary artery (Hedegaard et al., 2014), carotid artery (Centeno et al., 2019), and mesenteric small artery (White et al., 2013). GYY4137, a slow-releasing H 2 S donor, induced concentration-dependent relaxation in rat mesenteric arteries (Abramavicius et al., 2021) and bovine posterior ciliary artery (Chitnis et al., 2013). N-phenylthiourea (PTU) and N, N′-diphenylthiourea (DPTU) compounds, showing long-lasting H 2 S donation, promoted vasodilation in rat aortic rings (Citi et al., 2020). Novel H 2 S donors, AP67 and AP72, also elicited a dose-dependent relaxation in phenylephrine precontracted-bovine posterior ciliary arteries vessels (Kulkarni- Chitnis et al., 2015). L-cysteine, the substrate for CSE, caused a vasodilation in mouse mesenteric artery (Hart, 2020). However, the mechanisms of H 2 S induced-vasodilation are not fully understood. It may be closely related to vascular potassium channels.

HYDROGEN SULFIDE-INDUCED VASODILATION VIA ACTIVATION OF POTASSIUM CHANNELS
Potassium channels play important roles in regulating of membrane potential and vascular tone. There are many studies having been carried out on H 2 S-induced vasodilation via activation of potassium channels in different vascular beds. Modulation of potassium channel activity by H 2 S donors may be a novel treatment for vascular diseases. This review presents the effects and mechanisms of H 2 S-induced vasodilation via activation of potassium channels, including ATP-sensitive potassium channels (K ATP ), calcium activated potassiumchannels (K Ca ) and voltage dependent potassium channels (K V ).

ATP-Sensitive Potassium Channels
The active K ATP channel structurally is a hetero-octamer of four Kir6 subunits at the center and four sulfonylurea receptor (SUR) subunits surrounded (Li et al., 2017). Kir6 forms the channel pore and SURs are regulatory subunits (Alquisiras-Burgos et al., 2022). Activation of K ATP channels can induce membrane hyperpolarization of the vascular smooth muscle cell, and then lead to the vasodilation.
H 2 S acted as a K ATP channel regulator. Glibenclamide, a K ATP channel blocker, significantly inhibited vasodilation-induced by NaHS in carotid arteries of diabetic rabbits (Centeno et al., 2019). H 2 S was involved in the anticontractile effect of perivascular adiposetissue on aorta of hypertensive pregnant rat and the anticontractile effect was eliminated by K ATP blocker (Souza-Paula et al., 2020). NaHS decreases the myogenic response of cerebral arterioles and this effect was partially mediated by K ATP channels (Liu et al., 2012). Na 2 S-induced relaxation of human umbilical vein was also impaired by glibenclamide (Mohammed et al., 2017). Maximum vadilation of constricted mouse aorta elicited by NaHS was unaffected by removal of the endothelium but significantly attenuated by glibenclamide (Al-Magableh and Hart, 2011). In addition to Na 2 S and NaHS, other H 2 S donors, such as DPTU, caused vasodilation in rat aorta and was abolished by K ATP blocker (Citi et al., 2020). ZYZ-803, a novel H 2 S and NO conjugated donor, relaxed the aortic contraction induced by PE and the effect was suppressed by glibenclamide (Wu et al., 2016). Glibenclamide significantly attenuated the dilation effect induced by AP67 and AP72 (H 2 S donors) (Kulkarni- Chitnis et al., 2015) and GYY4137 (Chitnis et al., 2013) respectively on bovine ciliary arteries. H 2 S plays a role in the vasodilation in response to the reagents, such as moringa oleifera leaves (Aekthammarat et al., 2020) and sulforaphane (Parfenova et al., 2020), and their vasodilation effects was also reduced by glibenclamide. These data suggested an important role of K ATP in H 2 S-induced vasodilation and the possible mechanisms are discussed as below.
First, H 2 S directly activated K ATP channel. H 2 S increased the whole-cell currents and the single channel open probability of K ATP channels. Inhibition of endogenous H 2 S production reduced whole-cell K ATP currents in mesenteric artery smooth muscle cells (Tang et al., 2005). Second, K ATP channel can be sulfhydrated by H 2 S. H 2 S-mediated sulfhydration and hyperpolarization are absent in cells overexpressing C43S mutant Kir 6.1. Mutating the site of sulfhydration (Kir 6.1 C43S) inhibited H 2 S-induced hyperpolarization. Sulfhydration augments K ATP activity by enhancing Kir 6.1-PIP2 binding and reducing Kir 6.1-ATP binding (Mustafa et al., 2011). Third, H 2 S-induced vasodilation by activating K ATP is mediated by its subunits. Liang et al. (2011) reported glibenclamide partially reversed H 2 S induced potassium currents in piglet cerebral arterial smooth muscle cells and H 2 S-induced vasodilation was much smaller in cerebral arteries of SUR2 subunit knockout mice than in wild type mice. Additionaly, H 2 S promoted vasodilation in SHR, partly through upregulating the expression of Kir6.1 and SUR2B subunits of K ATP . H 2 S inhibited phosphorylation of forkhead transcription factors FOXO1 and FOXO3a, stimulated their nuclear translocation, and enhanced their binding with Kir6.1 and SUR2B promoters (Sun et al., 2015). However, several studies indicated K ATP channel had no effect in hydrogen sulfide mediated vasodilation, as K ATP blocker did not attenuate H 2 S-induced vasodilation (Streeter et al., 2012;White et al., 2013;Kutz et al., 2015), suggesting other potassium channels are involved in H 2 S-induced vascular regulation.
The activation of BK channel by H 2 S is mainly mediated by the following two mechanisms. First, H 2 S activates BK channels by increasing channel currents. BK current density was 91 ± 42 pA/pF under baseline condition while 201 ± 31 pA/pF after exposure to Na 2 S at testing potential 150 mV in mouse coronary arterial smooth muscle cells (Chai et al., 2015). Second, BK channel was activated by Ca 2+ influx increase induced by H 2 S. Naik et al. (2016) reported Na 2 S increased sulfhydration of transient receptor potential vanilloid type 4 channels in aortic endothelium cells. Activation of transient receptor potential vanilloid type 4 channels by H 2 S caused Ca 2+ influx, resulting in BK channels activation in endothelial cells and subsequent endothelial hyperpolarization, and vascular dilation. Furthermore, the activation of endothelial BK channels by H₂S can also increase Ca 2+ sparks in mesenteric artery smooth muscle cells, thus resulted in vasodilation (Jackson-Weaver et al., 2013). IK and SK channels are also distributed in vascular cells (Liu et al., 2018). H 2 S-induced vasodilation was partly inhibited by selective IK and SK channel inhibitor charybdotoxin and SK channel inhibitor apamin. H 2 S stimulated IK and SK in endothelial cells, leading to hyperpolarization and vasodilation (Mustafa et al., 2011). Cheng et al. (2004) also reported charybdotoxin/apamin-sensitive K + channels in endothelium cells had an important role in H 2 S effect on rat mesenteric artery. The expression of SK2.3 was decrease in CSE knockout mice or by CSE inhibitor, and was upregulated by H₂S (Tang et al., 2013). Expression of IK was enhanced in carotid arteries from diabetic rabbits, and the relaxant action of NaHS was significantly inhibited by charybdotoxin and 4-amynopiridine in diabetic arteries (Centeno et al., 2019). Thus, H₂S-induced vasodilation was partly through IK and SK.

Voltage Dependent Potassium Channels
Kv channels is a diverse family of outwardly rectifying potassium channels (including K V1 -K V12 ) (Hua et al., 2022). Each K V channel comprises a α-subunit and an ancillary β-subunit. αsubunits form the ion conducting pore and β-subunits modulate the activity of α-subunits. K V channels are major determinants of membrane potential and play a key role in regulating vascular dilation (Rhee and Rusch, 2018). And the distribution of K V1 , K V7 , and K V9.3 are reported in vascular smooth muscle cells of different vessel beds (Nieves-Cintrón et al., 2018).
NaHS relaxed carotid arteries precontracted by phenylephrine of diabetic rabbits, while 4-amynopiridine, a K V channel blocker, significantly inhibited the relaxant effect of NaHS, suggesting a role of K V in H 2 S induce vasodilation in diabetes (Centeno et al., 2019). Vasodilation to NaHS was also inhibited by blocking K V channels in mouse mesenteric arteries (Hart, 2020), but only a significant shift to the right of the concentration-response curve of NaHS without affecting maximum dilation in the aorta (Al-Magableh and Hart, 2011). K V7 channel, encoded by KCNQ gene, is thought as a powerful mechanism to H 2 S induced vasorelaxation. NaHS produced concentration-dependent vasorelaxation, which was blocked by XE991, a K V7 channel inhibitor. Perivascular adipose tissue released H 2 S, which play a major role in control of vascular tone via K V7 channel (Schleifenbaum et al., 2010). XE991 also inhibited H₂S-induced vasodilatation of porcine coronary arteries (Hedegaard et al., 2014) and murine aortas (Köhn et al., 2012). GYY4137induced relaxation was also inhibited by K V7 blocker (Abramavicius et al., 2021). The mechanism may be related to persulfidation of K V7 channels by CBS-derived H 2 S .

CONCLUSION
Taken together, H 2 S is involved in vascular diseases, such as hypertension, diabetes, pulmonary hypertension and so on. H 2 S causes vasodilation in numerous vascular beds and regulates vascular tone by interaction with different vascular potassium channels, including K ATP , K Ca , and K V channels via the following different mechanisms. H 2 S can directly activated vascular K ATP and BK channels and increased sulfhydration of K ATP and K V7 channels. H 2 S promoted vasodilation in partly through upregulating the expression of Kir6.1 and SUR2B subunits of K ATP , or BK channel activation by increased Ca 2+ influx. Thus, H 2 S may present a potential therapeutic target on vascular diseases. However, the vasorelaxant effect of H 2 S in different vessel beds is complex. More studies are needed to focus on the activation mechanisms of H 2 S on potassium channels, and novel, low-releasing H 2 S donors are required for treatment in the future.

AUTHOR CONTRIBUTIONS
X-YL wrote the manuscript. R-XW and L-LQ edited the manuscript. All authors read and approved the final manuscript.

FUNDING
This study was supported in part by grants from the National Natural Science Foundation of China (Nos. 82000317 to X-YL and 81770331 to R-XW), Top Talent Support Program for Young and Middle-aged People of Wuxi Health Committee (BJ2020018 to L-LQ) and Research Foundation from Wuxi Health Commission for the Youth (Q202034 to L-LQ).

ACKNOWLEDGMENTS
Thanks to all the peer reviewers for their opinions and suggestions.