Hydrogen Sulfide Relaxes Human Uterine Artery via Activating Smooth Muscle BKCa Channels

Opening of large conductance calcium-activated and voltage-dependent potassium (BKCa) channels hyperpolarizes plasma membranes of smooth muscle (SM) to cause vasodilation, underling a key mechanism for mediating uterine artery (UA) dilation in pregnancy. Hydrogen sulfide (H2S) has been recently identified as a new UA vasodilator, yet the mechanism underlying H2S-induced UA dilation is unknown. Here, we tested whether H2S activated BKCa channels in human UA smooth muscle cells (hUASMC) to mediate UA relaxation. Multiple BKCa subunits were found in human UA in vitro and hUASMC in vitro, and high β1 and γ1 proteins were localized in SM cells in human UA. Baseline outward currents, recorded by whole-cell and single-channel patch clamps, were significantly inhibited by specific BKCa blockers iberiotoxin (IBTX) or tetraethylammonium, showing specific BKCa activity in hUASMC. H2S dose (NaHS, 1–1000 µM)-dependently potentiated BKCa currents and open probability. Co-incubation with a Ca2+ blocker nifedipine (5 µM) or a chelator (ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 5 mM) did not alter H2S-potentiated BKCa currents and open probability. NaHS also dose-dependently relaxed phenylephrine pre-constricted freshly prepared human UA rings, which was inhibited by IBTX. Thus, H2S stimulated human UA relaxation at least partially via activating SM BKCa channels independent of extracellular Ca2+.


Introduction
Normal pregnancy is associated with dramatically increased uterine perfusion, reflected by as high as 20-80-fold rises in uterine blood flow in the third trimester in a singleton pregnant woman [1]. Pregnancy-associated uterine vasodilation is rate-limiting for pregnancy health since rise in uterine blood flow delivers nutrients and O 2 from the mother to fetus and exhausting CO 2 and metabolic wastes from the fetus to mother, mandatory to support fetal development and survival. Constrained uterine blood flow has been implicated in preeclampsia, intrauterine growth restriction, and other pregnancy diseases [2,3], not only raising the morbidity and mortality of the fetus and the mother during pregnancy, but also predisposing them more susceptible to cardiovascular and other metabolic disorders later in life [4,5].
The mechanisms underlying pregnancy-associated uterine vasodilation are complex and incompletely understood; however, compelling evidence has pinpointed down a key role of locally produced vasodilators in relaxing the uterine artery (UA) smooth muscle (SM). Many vasodilators have been identified to play a role in mediating uterine vasodilation, with prostacyclin and nitric oxide as the most studied forms [6][7][8]. However, systemic inhibition of prostaglandin synthesis by indomethacin does not result in concurrent systemic or uteroplacental vasoconstriction, suggesting that uterine blood flow is not directly dependent on maintained prostaglandin synthesis [9]. Local UA NO within 1 h after hysterectomy and placed in chilled culture medium and transported to the laboratory. Portions of each UA was allocated to be fixed in 4% paraformaldehyde or snap-frozen in liquid N 2 , and the rest was used for organ bath studies.

Isolation and Culture of Primary UA Smooth Muscle Cells (hUASMC)
Fresh UA was washed at least 3 times with cold sterilized PBS. Connective tissues around the vessels were carefully removed and the lumen was flushed with ice-cold DMEM. After removal of EC by filling the lumen with 0.1% collagenase (type II) in phosphate-buffered saline (PBS) for 15 min at 37 • C, we cut the EC-denuded artery into ≈1 cm long rings and then soaked them in 0.05% collagenase for 20 min. The smooth muscle was then mechanically separated under a 50× stereo microscopy. The isolated smooth muscle was minced and then digested with collagenase for 30-45 min at 37 • C. Fetal bovine serum (FBS, final concentration = 10%) was added to terminate digestion. Single SM cells were collected and plated in 10 cm dishes and cultured in DMEM containing 10% FBS and 1% penicillin/streptomycin. After 7-day culture, hUASMC colonies were marked. Each colony was then picked up by using a cloning disc presoaked with 1% trypsin/EDTA as previously described [17]. Each colony was transferred into a well of a 12-well plate and cultured until ≈90% density. The cells were then stored in liquid N 2 for experimental use within 3 passages.

Immunofluorescence Microscopy
Sections (6 µm) of paraffin-embedded UA rings were dehydrated and treated with proteinase K for antigen retrieval for 10 min at 37 • C, followed by rinsing 3 times with PBS. After incubation with 1% bovine serum albumin (BSA) in PBS to block nonspecific binding for 30 min at room temperature, the sections were incubated with anti-human BK Ca β1 (1:50) or γ1 (1:50) subunit at 4 • C overnight. IgG was used as negative control. All antibody incubations were performed in 0.5% BSA/PBS. The sections were washed 3 × 10 min with PBS, and then incubated with Alexa 568 mouse immunoglobulin (IgG, 1:1000) for 1 h at room temperature. After 3 × 10 min washing with PBS, the sections were blocked with 1% BSA/PBS for 30 min at room temperature. The sections were incubated with anti-human CD31 (1:200) at 4 • C overnight, washed, and then incubated with Alexa 488 anti-mouse IgG (1:1000) for 1 h at room temperature. The sections were washed and then mounted with anti-fade mounting medium containing DAPI. Sections were examined under a confocal laser scanning microscope (Olympus SV3000) and images were acquired for quantifying levels of BK Ca subunits (mean red fluorescence intensity) in SMC and EC as previously described [12].

RNA Extraction and Reverse Transcription Polymerase Chain Reaction (RT-PCR)
Total RNAs were extracted from the main UA tissue (≈100 mg) or cultured hUASMC (≈2 × 10 5 cells) using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and quantified by OD 260/280. Complementary DNA was synthesized by reverse transcription with random primers and AMV Reverse Transcriptase (Promega, Madison, WI, USA) and then used for detecting mRNAs of BK Ca subunits by PCR with gene-specific primers as listed in Table 1. PCR was run as follows: 95 • C for 5 min, followed by 38 cycles of 95 • C for 30 s, 62 • C for 30 s, and 72 • C for 30 s, and then 72 • C for 5 min and 4 • C. The amplicons were confirmed by sequencing.

Western Blot
UA and cultured hUASMC proteins were extracted using a lysis buffer as previously described [32]. Equal amounts of total protein extracts (20 µg/lane) were separated on 10-15% SDS-PAGE and transferred to polyvinylidene difluoride membrane. Proteins were determined by immunoblotting with antibodies against anti-human BK Ca β1 (1:100) or γ1 (1:200) subunits in Tris-buffered saline (TBS) containing 5% BSA as described previously [12]. β-actin was determined as a control for sample loading.

Electrophysiology
Electrophysiological experiments were performed as described previously [33,34]. Briefly, cultured primary hUASMC were used for whole-cell, inside-out, and outside-out recordings with an Axonpatch-200B connected to a Digidata 1322A using pClamp10 software (Molecular Devices, CA, USA). The patch pipettes were fabricated from borosilicate glass (Havard Apparatus) and had electrode resistances from 2-4 MΩ with an access resistance from 3-10 MΩ. Cells with current leakage less than 100 pA in the whole-cell mode were selected for analysis. Sampling frequencies for whole-cell current and single-channel recordings were 1 kHz and 5 kHz, respectively. Data were filtered with a low-pass 4-pole Bessel filter set at 1 kHz, which results in a 10-90% rise time of 350 µs. For whole-cell and outside-out single-channel recordings, the bath solution contained (mM) 144 NaCl, 5 KCl, 2 CaCl 2 , 0.5 MgCl 2 , 10 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), and 10 glucose, at pH 7.4 adjusted with 10N NaOH. The recording pipette solution contained (mM) 140 KCl, 1 MgCl 2 , 5 Na 2 ATP, 5 EGTA, and 2.5 CaCl 2 , at pH 7.2. The final free Ca 2+ concentration was calculated by the Webmaxc extended calculator (http://www.stanford.edu/~cpatton/webmaxcE.htm) and estimated to be 10 µM in the control pipette solution, which was adjusted for indicated free Ca 2+ concentration in the text by changing CaCl 2 concentration or by adding EGTA.
BK Ca channels keep open with intracellular free Ca 2+ higher than 50 µM [35], making it hard to qualify the channel activity with a continuous high free Ca 2+ level. Thus, we performed all the tests with intracellular free Ca 2+ no higher than 10 µM. Ca 2+ -free recordings were performed with the same bath solution containing 5 mM EGTA. Channel blockers were added into the bath solutions unless stated otherwise. For inside-outside single-channel recordings, the pipette and the bathe solutions are the same as the pipette solutions of whole-cell recordings as described above. Test solutions were applied via a gravity-driven system controlled by VCS-66MCS (Warner Instrument, Hemden, CT, USA). For rapid solution exchange (≈300-500 ms), we held membrane patches in a stream of the experimental solution from a second pipette. Single-channel current amplitudes were calculated by fitting amplitude histograms to a Gaussian distribution. Channel open probability was expressed as P open = NPo/n, where NPo = [(to)/(to + tc)]. P open = open probability for one channel; to = sum of open times; tc = sum of closed times; N = actual number of channels in the patch; and n = maximum number of individual channels observed in the patch. Experiments were repeated at least 3 times and data were calculated as the mean ± SEM (standard error of the mean). The linear regression is shown in the single channel current-voltage (I-V) curve. P open was fit with Gaussian function. Single-channel conductance (g, pico Siemens, pS) was calculated using I/U; I = single-channel current (pA), U = membrane potential (mV).
The whole-cell patch-clamp technique was used to record K ATP channel currents as previously described [18]. The bath solution for recording whole-cell K ATP current contained (mM) 140 NaCl, 5.4 KCl, 1.2 MgCl 2 , 10 HEPES, 1 EGTA, and 10 glucose, with pH adjusted to 7.4 with NaOH. The pipette solution contained (mM) 140 KCl, 1 MgCl 2 , 10 EGTA, 10 HEPES, 5 glucose, 0.3 Na 2 ATP, and 0.5 MgGDP, with pH adjusted to 7.2 with KOH. Cells were superfused continuously with the bath solution at a rate of approximately 2 mL/min. Solution change in the recording chamber was accomplished within 30 s.
All patch clamp recordings were carried out at room temperature (20-22 • C). NaHS was used as a source of H 2 S; working solutions were prepared immediately before use as H 2 S gas evaporates 10-15% from the solution within 30 min at 37 • C [36]. Stock solution of nifedipine was dissolved in DMSO; the final DMSO concentration did not exceed 0.05%, which did not change the currents in control experiments.

Organ Bath Studies
Freshly prepared UA rings (2-5 mm in length) were placed in ice-cold Krebs-Ringer bicarbonate (KRB) bath solution containing (mM) 118.5 NaCl, 4.75 KCl, 1.2 MgSO 4 , 1.2 KH 2 PO 4 , 25 NaHCO 3 , 2.5 CaCl 2 , and 5.5 glucose, with pH 7.4 adjusted with HCl. The UA rings were mounted onto a tension transducer (JZJ01H) under a stable resting tension in organ bath chambers containing 5 mL of KRB solution at 37 • C, gassed with 95% O 2 and 5% CO 2 . The rings were allowed to equilibrate for at least 30 min, with chamber solution changed every 15 min. Endothelium integrity was determined by response to 10 µM acetylcholine as previously described [12]. Only endothelium-intact rings were used, which were preload with a tension at 1.5 g after equilibration; contraction was recorded when the tension was stable for at least 15 min. Rings were pre-contracted with 10 µM phenylephrine. Rings rapidly responding to phenylephrine in 5 min with more than 2 mN contraction were selected for recording the dose-response relaxation curves of NaHS in the presence or absence of the selective BK Ca channel blockers. Each drug was allowed at least 5 min to respond. Changes in the isometric tension were recorded and analyzed with a Multiple Channel Physiology Signal Recording System (RM-6240EC, Chengdu Instrument Factory, Chengdu, China).

Statistics
Results are expressed as means ± standard error. Significant levels were determined by using the paired Student's t-test or one-way ANOVA followed by Bonferroni test for multiple comparisons, whichever appropriate, using GraphPad Prism 8. Significant difference was accepted at p < 0.05.

Expression of BK Ca Channels in UA In Vitro and Primary UASMC In Vitro
BK Ca channels are tetramer formed by the pore-forming α subunits, along with the regulatory β1-4 and γ1-4 subunits [22,23]. By using RT-PCR and sequencing conformation, we detected α, β1, β3, β4, and γ1-3, but not β2 and γ4, mRNAs in pregnant human UA and cultured primary hUASMC ( Figure 1A). Since β1, γ1, and γ3 subunits are the most important ones for mediating UA adaptation to pregnancy [27,31,37], we further examined their proteins in human uterine arteries and cultured hUASMC. We tested two commercially available antibodies against γ1 and γ3 subunits to detect their protein levels by Western blot and immunofluorescence microscopy. The γ1 subunit was only detectable by Western blot with one antibody (PA5-38058) but not by immunofluorescence microscopy, whereas γ3 subunit was detectable by immunofluorescence microscopy with the Abcam antibody (ab121412) but not by Western blot with all other commercial antibodies. Immunoblotting detected β1 and γ1 proteins in both UA and cultured hUASMC and they did not change in three passages ( Figure 1B). Immunofluorescence microscopy analysis revealed that both VSM and EC expressed β1 and γ3 proteins; however, levels of both β1 and γ3 proteins in SM cells were significantly greater than that in the CD31 + EC. In addition, histological analysis showed that both β1 and γ3 proteins are not expressed in all ECs as β1 or γ3 proteins were only found in some regions of the CD31 + EC linings ( Figure 1C).

Functional BK Ca Channels in Primary hUASMC In Vitro
To determine if BK Ca channels were functional in cultured hUASMC, we introduced whole-cell and single-channel patch clamp with the selective BK Ca channel blockers: iberiotoxin (IBTX, 100 nM) or low concentration of TEA (1 mM). Ion currents were elicited in response to a series of voltage pulses from −60 mV holding potential to +80 mV in steps of 10 mV. Both IBTX and TEA blocked the outward current significantly compared with the baseline holding membrane potential from +40 mV to +80 mV (p < 0.05, Figure 2A-C). In the inside-out patch, cultured hUASMC BK Ca channels showed a single-channel conductance of 201 ± 19.08 pS (n = 8) in a symmetrical high K + solution (140 mM) on both sides of the cell membrane, which was consistent with reported values [38] (Figure 2D,E). In outside-out/inside-out single-channel recording with 100 nM free Ca 2+ in the pipette solution at +40 mV holding membrane potential, the observed single-channel activities were blocked by IBTX or TEA, confirming the observed 200 pS channels to be BK Ca channels ( Figure 2F). Open probability (P open ) of the channels was decreased from 0.04 ± 0.009 (n = 10) to 0.0019 ± 0.00046 (n = 5, p < 0.05) by IBTX, and to 0.0026 ± 0.0011 (n = 5, p < 0.05) by TEA. These results indicate the presence of IBTX-and TEA-sensitive functional BK Ca channels in hUASMC in vitro. To determine if BK Ca channels were functional in cultured hUASMC, we introduced whole-cell and single-channel patch clamps with the selective BK Ca channel blockers, IBTX (100 nM) and low concentration of TEA (1 mM), separately. Ion currents were elicited in response to a series of voltage pulses from −60 mV holding potential to +80 mV in steps of 10 mV. Both IBTX and TEA significantly blocked the outward current in comparison with the baseline holding membrane potential from +40 mV to +80 mV (p < 0.05, Figure 2A-C). In the inside-out patch, cultured hUASMC BK Ca channels showed a single-channel conductance of 201 ± 19.08 pS (n = 8) in a symmetrical high K + solution (140 mM) on both sides of the cell membrane ( Figure 2D,E). With 100 nM free Ca 2+ in the pipette solution at +40 mV holding membrane potential, the single-channel BK Ca currents were blocked by IBTX or TEA ( Figure 2F). P open of BK Ca decreased significantly from 0.04 ± 0.009 (n = 10) to 0.0019 ± 0.00046 (n = 5, p < 0.05) by IBTX, and to 0.0026 ± 0.0011 (n = 5, p < 0.05) by TEA, indicating the presence of IBTX-and TEA-sensitive functional BK Ca channels in primary hUASMC in vitro.

H 2 S Increased Ca 2+ -Activated and Voltage-Dependent K + Currents in hUASMC
When sodium hydrosulfide (NaHS) was applied to the extracellular solution, it rapidly dissociated into Na + and HS − , and HS − associated with H + to produce H 2 S. However, only the H 2 S molecule, but not HS − , is able to pass the plasma membrane, as H 2 S possess approximately fivefold greater lipophilic solubility than water [39]. Addition of NaHS (100 µM) caused a significant and reversible increase of membrane outward currents, and current voltage relationships were obtained within 1-3 min after NaHS incubation. NaHS on BK Ca activity was assessed with whole-cell and single-channel recordings. NaHS significantly augmented the whole-cell outward current from 60 mv membrane potential (p < 0.05, Figure 3A-C), which was sensitive to 1 mM TEA (p < 0.05, Figure 3A-C), indicating that the augmented outward currents were BK Ca -mediated. In single-channel recordings, NaHS increased P open from baseline (0.1258 ± 0.01) to 0.3107 ± 0.02, and standard bath solution reversed the NaHS-induced P open to 0.1533 ± 0.01; most of the outward currents were sensitive to 1 mM TEA (p < 0.05, Figure 3A-C). With 10 µM free Ca 2+ in the pipette solution at +40 mV holding membrane potential, NaHS increased P open of BK Ca from 0.468 ± 0.04226 to 0.7742 ± 0.02664 (p < 0.01). The H 2 S-induced P open of BK Ca was also observed at lower holding potentials from −10 mV to + 20 mV (n = 6, p < 0.05 vs. baseline, Figure 3F). NaHS stimulated BK Ca activity in a U-shaped concentration-dependent manner; NaSH at 100 and 500 µM significantly increased P open of BK Ca channels by 166.6 ± 29% and 198.1 ± 35% (n = 10), respectively. Low (10 µM) and high (1 mM) concentrations of NaHS also increased P open by 134.9 ± 24% and 160.2 ± 62% (n = 10), but these responses did not differ statistically from the controls ( Figure 3G).

H 2 S-Induced BK Ca Activation Is Redox-Sensitive
The activity of BK Ca channels depends on the redox state of the sulfhydryl groups in the channel proteins [42][43][44], and oxidation reduces BK Ca activity [45,46]. To study if the NaHS-induced BK Ca activation is redox-dependent, we determined the effects of a reducing agent dithiothreitol (DTT, 1 mM) added into the bath solution on the NaHS-induced P open of BK Ca . Treatment with NaHS increased P open of BK Ca from baseline 0.036 ± 0.011 to 0.119 ± 0.032 (p < 0.05); co-incubation with DTT decreased the NaSH-induced P open of BK Ca to 0.072 ± 0.034 (p < 0.05) ( Figure 5A,B). Co-incubation with DDT blocked NaHS-induced BK Ca activation; however, this effect was rapidly diminished and then all channel activities were blocked ( Figure 5C). DTT alone did not alter BK Ca channel P open (Figure 5B,C).

H 2 S Relaxed Human UA via BK Ca Channel
Incubation with increasing concentrations (1, 10, 100, 500 µM) NaHS stimulated dose-dependent relaxation of freshly prepared human UA rings that were pre-constricted with 10 µM phenylephrine ( Figure 6A). Pretreatment with the selective BK Ca channel inhibitor IBTX (100 nM) blocked the NaHS-induced UA relaxation ( Figure 6B).  concentrations (1, 10, 100, and 500 µM) of NaHS was then applied sequentially to relax the preconstricted UA ring. A representative dose-response curve of H 2 S-induced UA relaxation was shown to represent similar results of three UA ring preparations from three patients. (B) Bar graph summarizing the effects of NaHS on human UA (hUA) relaxation. NaHS at 100 and 500 µM decreased the artery tension to 69.3 ± 6.6% and 57.6 ± 10.8% of the maximum contraction of PE. * p < 0.05 compared with NaHS at 0. (C) NaHS (100 µM) decreased artery tension to 64.6 ± 6.7% of the maxi contraction induced by PE, and the NaHS-induced UA relaxation was reversed by co-incubation with the BK Ca channel blocker iberiotoxin (IBTX, 100 nM). * p < 0.05.

H 2 S Did Not Activate K ATP Channels in hUASMC
Since K ATP channels are direct effectors of H 2 S [36,47,48], we determined whether H 2 S activates K ATP channels in hUASMC. Treatment with NaHS (300 µM) [18] did not alter baseline inward currents stimulated by 140 mM K + , indicative of K ATP channel activity ( Figure 7A); however, co-incubation with the K ATP channel blocker glibenclamide (10 µM) inhibited K ATP channel activity ( Figure 7A,B).

Discussion
Consistent with the well-documented vasodilatory effect of H 2 S in many systemic arteries [15,36,49,50], we were the first to report that H 2 S dilates pressurized UA in a pregnancyand vascular bed-dependent manner in rats [12]. The current study demonstrates for the first time that H 2 S activates BK Ca channels in hUASMC, as well as the fact that incubation of the specific BK Ca channel blocker IBTX completely blocks H 2 S-induced relaxation of pre-constricted human UA rings in vitro. These findings provide direct evidence for a role of smooth muscle BK Ca channels in mediating the vasodilatory effects of H 2 S in the UA, further supporting the notion that H 2 S is a novel UA vasodilator.
Endogenous H 2 S is a gaseous signaling molecule that is mainly synthesized by CBS and CSE in various human tissues, while other enzymes such as 3-mercaptopyruvate sulfurtransferase (3MST) in combination with cysteine aminotransferase (CAT) may also play a role [51]. Our recent studies have consistently shown that H 2 S production is upregulated in the UA via selectively upregulating EC and SM CBS expression, without altering the expressions of CSE, 3MST, and CAT in vivo [11,12,32] and in human UAEC in vitro [17]. In this study, NaHS was used as a source of H 2 S. In aqueous solution, NaHS dissociates to Na + and HS − , and HS − associates with H + to produce H 2 S. In neutral solution, one-third of NaHS exists as H 2 S, and the remaining two-thirds are present as HS − [52]. Thus, the solution of H 2 S is about ≈66% of the original concentration of NaHS [53]. The liberation of <1 mM Na + from NaHS is negligible since the bath solution contained 145 mM Na + . The concentrations of NaSH used in this study ranged from 1 to 1000 µM, which did not change the pH of the buffered solution. The concentration of NaSH used in most of the experiments was 100 µM, equivalent to ≈60 µM H 2 S, which is close to the physiological plasma levels (less than ≈50 µM) of H 2 S in humans [51]. Our data show that addition of 100 µM NaSH significantly activated BK Ca channels in hUASMC and dilated human UA rings in vitro, showing that H 2 S is a physiological UA dilator.
Activation of K ATP channels was the first mechanism that has been shown to mediate H 2 S-induced vasodilation in rat mesentery artery [19], which has been confirmed by many follow-up studies in other vessels [36,47,48]. However, activation of K ATP accounts for no more than half of the effect of H 2 S to relax most vessels [54]. Likewise, opening of BK Ca channels results in K + efflux, causing membrane hyperpolarization of vascular SMC as a key mechanism for vasodilation [40]. UA BK Ca activity increases in pregnant sheep [55]. Local infusion of TEA to block BK Ca channels abolishes pregnancy-induced UA dilation in vitro [27] and inhibits pregnancy-associated uterine blood flow in vivo [28][29][30], while local infusion of glibenclamide to block the K ATP channels does not significantly affect baseline pregnancy-associated uterine blood flow [20]. Consistently, we did not observe a significant effect of H 2 S on K ATP channels in hUASMC. Why H 2 S, unlike other systemic SMCs, does not activate K ATP channels in hUASMC warrants further elucidation. Nonetheless, our current data, along with data from in vivo studies using blockers of various K + channels to determining their role in pregnancy-associated rise in uterine blood flow [20,28,30], suggest that activation of SM BK Ca channels is important for mediating H 2 S-induced UA dilation.
BK Ca channels, also known as BK/MaxiK/Slo1/K Ca 1.1 channels, are K + channels of largest single-channel conductance (≈200-300 pS) [55]. The essential structure of BK Ca channels consist of the α-ubunit and can be complemented with the regulatory subunits, including the β isoforms (1-4) and γ isoforms (1)(2)(3)(4) [56,57]. The β1 subunit is essential for increasing voltage sensitivity when intracellular free Ca 2+ is beyond 1 µM [22,58]. The γ1-γ4 are auxiliary subunits that greatly modify channel activity in mammalian cells [25,26,[59][60][61]. The expression and their physiological and pathological functions of SM BK Ca channels have been well studied in other tissues in mammalians [62], but their distribution and function remains to be understudied in UA smooth muscle cells (UASMC). Previous studies have shown SM expression of α and β1 [63] and γ1 [31] subunits in UA; the α subunit is constitutively expressed and the β1 and γ1 subunits are significantly upregulated in pregnancy [31,55]. Herein, we show the expressions of α, β1, β3, β4, and γ1-3, but not β2 and γ4, mRNAs, and β1 and γ1 and γ3 proteins in hUA and cultured hUASMC. Which subunit(s) of these isoforms are responsible for the H 2 S-induced BK Ca activity in hUASMC? Our current study did not provide any data to address this important question; however, β1-containing BK Ca channels are sensitive to IBTX and low concentration of TEA [22,64]; the similar pharmacological properties with IBTX and TEA obtained in this study has implicated a functional role of β1 subunit in H 2 S-induced BK Ca activity in hUASMC, consistent with previous studies showing that β1 subunits are upregulated and are important for increasing SM BK Ca activity in the UA in response to estrogen stimulation and during pregnancy [55,65]. The γ1 subunit containing BK Ca channels are featured by the ≈120 mV leftward shift at 0 and elevated cytosolic Ca 2+ , which facilitates BK Ca channel activity [22]; the γ3 is less studied but also related to Ca 2+ sensitivity of the channel [25]. γ1 subunit is upregulated sevenfold in mouse UA in pregnancy [31]. Future studies are warranted to delineate whether they are involved in the H 2 S-induced UASMC BK Ca activity since γ1 and γ3 proteins are highly expressed in hUA and retained in hUASMC in culture.
How does H 2 S activate BK Ca channels in hUASMC? With BK Ca channels being Ca 2+ -activated and voltage-dependent ion channels, activation requires either elevation of intracellular Ca 2+ or depolarization of cell membrane [66]. The free intracellular Ca 2+ concentration under resting conditions is ≈150 nM, although it is oscillating in some cells, and can increase as high as 500 nM [67]. In addition, Ca 2+ concentrations in the vicinity of BK Ca channels after influx through Ca 2+ channels are between 4 and 30 µM [68], which are dramatically higher compared to average cytoplasmic free internal Ca 2+ concentrations. Free internal Ca 2+ concentrations used in our experiments are within this range. In resistance-sized cerebral arteries, ryanodine receptor-sensitive Ca 2+ sparks in sarcoplasmic reticulum (SR) activate BK Ca channels [69], while in the resting state of cerebral artery activation of BK Ca channels relies on Ca 2+ influx through L-type voltage-dependent calcium channels (LTCC) [70]; however, this is not the case in coronary or mesenteric arteries, indicating that different mechanisms for BK Ca channel activation varies among vessels from different vascular beds. In hUASMC, blockade of LTCC using nifedipine does not affect H 2 S-induced BK Ca activity recorded by whole-cell patch clamp, suggesting LTCC-mediated Ca 2+ influx is not involved. Similar results were also obtained with 0 free Ca 2+ bath solution containing EGTA, indicating that H 2 S-induced BK Ca activity is independent of extracellular Ca 2+ , sharing similar properties with the H 2 S-responsive BK Ca channels in rat pituitary tumor cells [71]. In ovine UASMC, recent studies have shown that ryanodine-receptor sensitive Ca 2+ sparks are important for pregnancy and estrogen stimulation of BK Ca channel activity [72]. In rat mesenteric arteries, H 2 S-induced vasodilation requires activation of endothelial BK Ca channels and smooth muscle Ca 2+ sparks [21]. Thus, future studies are needed to determine if SR Ca 2+ sparks mediate activation of the H 2 S-induced BK Ca channels in UASMC.
Apart from Ca 2+ and voltage, many other mechanisms are also involved in regulating BK Ca channel activity, including phosphorylation by protein kinases such as protein kinase A (PKA), PKG, and PKC; PKA and PKG activate BK Ca channels through modulating the channel kinetics, while PKC shows an inhibitory manner on the channels [66]. In the present study, we show that NaHS modulates BK Ca channels directly by using outside-out single-channel patch recording. In the whole-cell patch recording mode, NaHS may modulate BK Ca channel activity indirectly through protein kinase-mediated phosphorylation. However, this idea needs to be further explored. In addition, direct sulfhydrating proteins in reactive cysteines has been recently recognized to be a major mechanism for H 2 S to elicit its biological functions [73]. Direct sulfyhydration of Kir 6.1 on C43 has been shown to be a key mechanism for H 2 S-induced K ATP channel activation [74]. In this study, the H 2 S-response BK Ca channel was found to be sensitive to DTT, which completely prevents protein cysteine modifications including sulfhydration [73]. Thus, this mechanism is highly likely involved in H 2 S-induced BK Ca channel activation in hUASMC, although detailed mechanisms around sulfhydration in terms of which subunit(s) and on which specific cysteine(s) are involved are still to be determined.

Conclusions
Altogether, we have shown herein that functional BK Ca channels are present in human UASMC, which can at least partially mediate the vascular relaxation effects of H 2 S in human UA in vitro. However, it is necessary to point out that research in H 2 S in uterine hemodynamics is still at a very early stage. Future studies are warranted to address many important questions so that a physiological and pathophysiological role of H 2 S and the underlying mechanisms in uterine hemodynamic regulation can be delineated, pertaining to normal pregnancy and hypertension-related pregnancy complications such as preeclampsia. Funding: The present study was supported in part by National Institutes of Health (NIH) grants HL70562, HD097498, and HD102451 (to D.-b.C.). The content is solely the responsibility of the authors and does not necessarily reflect the official views of the NIH.