Human Trabecular Meshwork Cell Volume Decrease by NO-Independent Soluble Guanylate Cyclase Activators YC-1 and BAY-58-2667 Involves the BK_Ca Ion Channel

Abstract

purpose. There is a correlation between cell volume changes and changes in the rate of aqueous humor outflow; agents that decrease trabecular meshwork (TM) cell volume increase the rate of aqueous humor outflow. This study investigated the effects of the nitric oxide (NO)-independent activators of soluble guanylate cyclase (sGC), YC-1, and BAY-58-2667 on TM cell volume and the signal transduction pathways and ion channel involved.

methods. Cell volume was measured with the use of calcein AM fluorescent dye, detected by confocal microscopy. Inhibitors and activators of sGC, 3′,5′-cyclic guanosine monophosphate (cGMP), protein kinase G (PKG), and the BK_Ca channel were used to characterize their involvement in the YC-1– and BAY-58-2667–induced regulation of TM cell volume. cGMP was assayed by an enzyme immunoassay.

results. YC-1 (10 nM–200 μM) and BAY-58-2667 (10 nM–100 μM) each elicited a biphasic effect on TM cell volume. YC-1 (1 μM) increased TM cell volume, but higher concentrations decreased TM cell volume. Similarly, BAY-58-2667 (100 nM) increased TM cell volume, but higher concentrations decreased cell volume. The YC-1–induced cell volume decrease was mimicked by 8-Br-cGMP and abolished by the sGC inhibitor ODQ, the PKG inhibitor (RP)-8-Br-PET-cGMP-S, and the BK_Ca channel inhibitor IBTX. The BAY-58-2667–induced cell volume decrease was mimicked by 8-Br-cGMP and was abolished by the PKG inhibitor and the BK_Ca channel inhibitor. Unlike the YC-1 response, ODQ potentiated the BAY-58-2667–induced decreases in cell volume.

conclusions. These data suggest that the NO-independent decrease in TM cell volume is mediated by the sGC/cGMP/PKG pathway and involves K⁺ efflux.

Aqueous humor exits the eye through the trabecular meshwork (TM) and Schlemm canal. Activation of sGC by NO-dependent donors increases the rate at which aqueous humor flows through the TM and Schlemm canal. These changes in outflow facility occur concomitantly with sGC-induced decreases in TM cell volume. sGC comprises an α-subunit and a smaller heme-containing β-subunit, ^{1 2} both of which constitute the active enzyme. Heterodimers are activated by NO binding to the heme moiety, whereas homodimers exhibit little or no synthetic activity, even in the presence of the ligand. Binding of NO to sGC results in the formation of 3′,5′-cyclic guanosine monophosphate (cGMP) from guanosine 5′-triphosphate (GTP). Increased cGMP activates protein kinase G (PKG), ³ with subsequent phosphorylation of target proteins. 

NO acting through the sGC, cGMP, and PKG pathways decreased TM cell volume in a time course that correlated with the NO-induced increases in outflow facility in perfused eye anterior segments. ⁴ Although NO is a potent regulator of IOP, chronic administration of NO donor to eyes results in lack of responsiveness and development of tolerance. ⁵ Therefore, the need to identify other activators of sGC that regulate TM cell function is of vital interest. YC-1 [3-(5′-hydroxymethyl-2′furyl)-1-benzyl indazole], ⁶ a benzyl indazole derivative, and BAY-58-2667 ⁷ are NO-independent activators of sGC. As with NO activation of sGC, YC-1, and BAY-58-2667, activation of sGC also results in increases in cGMP and PKG phosphorylation events. 

Alterations of the contractile states and volume of the TM cells would regulate aqueous humor outflow. ^{8 9 10 11 12 13 14 15 16 17} Changes in cell volume are influenced by the activities of the Na-K-2Cl cotransporter, ^{13 15 18} the Na⁺/H⁺ transporter, ¹⁸ the K⁺ and Cl⁻ channels, ^{17 18} and the large-conductance calcium-activated potassium channel (BK_Ca). ⁴ Further, it is possible that both the cellular contractile mechanisms and the cell volume regulatory mechanisms are functionally linked ^{19 20 21} because the BK_Ca channels have been shown to regulate TM cell volume and contractility ^{10 17} and outflow facility. ¹⁷ In these studies we tested the hypothesis that YC-1 and BAY-58-2667 regulate TM cell function. Specifically, we tested the ability of YC-1 and BAY-58-2667 to regulate TM cell volume, and we tested the involvement of sGC, cGMP, PKG, and the BK_Ca channel in the YC-1– and BAY-58-2667–induced response. 

Materials and Methods

Cell Culture

Eyes from human donors with no history of ocular disease or surgery were obtained from Lions Eye Institute (Tampa, FL) within 24 to 30 hours of death. Primary human TM cell lines (numbers represent the ages of the donors: HTM26, HTM71, HTM36, HTM80, and HTM86) were developed. For our experimental protocols cells from early passages (passages 3–5) were used. Human TM explants were obtained from whole eyes that were stored in a moist environment at 4°C or from corneal scleral rims stored in ophthalmic solution (Optisol; Dexol; Chiron Ophthalmics, Irvine, CA) at 4°C. TM cells were isolated after collagenase digestion of TM explants. ²² Collagenase-treated cells were grown in low-glucose (1g/L) Dulbecco modified Eagle medium (DMEM; Mediatech, Herndon ,VA) in the presence of 10% fetal bovine serum (Mediatech), 100 U/mL penicillin, and 100 μg/mL streptomycin (Mediatech) and then passaged into six-well culture dishes (Nalge Nunc International, Rochester, NY) in a tissue culture incubator at 37°C in 5% CO₂. We validated human TM cells by their morphology and the presence of dexamethasone-induced myocilin expression. ²³ For experimental protocols, TM cells were grown on chambered coverglass (Laboratory-Tek II; Nalge Nunc International, Rochester, NY) in low-glucose DMEM, as described, to 100% confluence, after which they were exposed to serum-free media for 2 days before the experiments were performed. 

Measurement of Cell Volume

Cell volume measurements were performed as previously described. ⁴ Before any drug treatments, the cells were loaded with the fluorescent dye calcein AM (2 μM) in DMEM at 37°C, in a 5% CO₂ incubator for 60 minutes to ensure a stable baseline. Coverslips containing the cells were subjected to examination under a confocal microscope (Leica, Wetzlar, Germany) and were thermostated at 37°C, and images of the same cells were acquired with a 20× objective lens at 1-μm z-step intervals to a depth of 15 μm. During the experimental protocol these cells were in their native state and were not harvested; therefore, we did not experience the movement of cells from the region of study or observe the rapid contraction and relaxation phenomenon previously described. ^{18 24} Confocal microscopy was used to calculate the number of voxels, and ImageJ software (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD; available at http://rsb.info.nih.gov/ij/index.html) was used to quantify cell volume and to identify the top and bottom edges of the cell. Images were converted from 8-bit to binary values using a threshold that was determined by analysis of fluorescent latex beads (Fluoresbrite; Polyscience Inc., Warrington, PA) of known diameter and volume that were imaged under conditions identical to those used for TM cells. A region of interest was then selected around each cell, and the ImageJ software was used to calculate the number of voxels in the region of interest in the image stack. Changes in cell volume were determined by dividing the voxel count with drug treatment by the voxel count without drug treatment. Unless otherwise stated in the text, for studies involving drug treatments, images were taken without drug treatment (0 time point) and served as controls for the treatment groups. Because our preliminary data demonstrated that the maximum decrease in TM cell volume in response to drug treatment was achieved at 20 minutes, images were taken of the same cells at 20 minutes after drug treatment. For all experiments, YC-1 (10 mM) and BAY-58-2667 (10 mM) were solubilized in a dimethyl sulfoxide-ethanol mixture for a final concentration of 0.1%. 

cGMP Assay

For cGMP measurements, cells were grown in 12-well culture dishes (Nalge Nunc International) in a tissue culture incubator at 37°C in 5% CO₂, as described. Two days before experiments, the cells were exposed to serum-free media. cGMP was assayed by an enzyme immunoassay (Amersham Biosciences, Piscataway, NJ) according to the manufacturer’s protocol. 

Materials and Reagents

Routine reagents YC-1 and iberiotoxin (IBTX) were purchased from Sigma (St. Louis, MO). Others were obtained as follows: 8-bromo-cGMP sodium salt, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) was purchased from Sigma-RBI (Natick, MA), and (RP)-8-Br-PET-cGMP-S was purchased from Calbiochem (La Jolla, CA). BAY-58-2667 was obtained from Alcon Research, Ltd. (Fort Worth, TX). 

Statistical Analysis

Statistical comparisons were performed by ANOVA, followed by the Holm-Sidak method or the Fisher least significant difference (LSD) method for comparison of significant differences among different means. 

Results

YC-1 and BAY-58-2667 Effects Are Biphasic

To quantitatively measure changes in cell volume, HTM36 and HTM80 cells were exposed to varying concentrations of YC-1 (10 nM–200 μM). ²⁵ Figure 1Ademonstrates that the action of YC-1 is biphasic in HTM cells; 1 μM significantly increased TM cell volume, but higher concentrations (50–200 μM) decreased TM cell volume. We also observed that there were no changes in cell volume over time in cells incubated in calcein AM only (Fig. 1A) . Similarly, varying concentrations of BAY-58-2667 (10 nM–100 μM) were added to TM cells, and images were captured with or without drugs. Figure 1Bdemonstrates that, as with Figure 1A , the action of BAY-58-2667 on TM cell volume is biphasic; 100 nM caused increases in TM cell volume, but higher concentrations resulted in decreases in TM cell volume. 

YC-1–Induced Decrease in Cell Volume Involves Activation of Soluble Guanylate Cyclase and cGMP

To test the involvement of sGC in the YC-1–induced decreases in cell volume, primary human HTM36 and HTM80 cells were incubated with YC-1 (150 μM) in the presence or absence of ODQ (500 nM–5 μM) ²⁶ the specific sGC inhibitor, and images were taken at 20 minutes. ODQ at 500 nM and 1 μM had no effect on the YC-1- induced decreases in TM cell volume while 5 μM significantly attenuated the YC-1 effect (Fig. 2A) . Because activation of sGC results in increased cGMP levels, we examined the ability of exogenously applied cGMP to decrease TM cell volume. Figure 2Ademonstrates that the non-hydrolyzable analog of cGMP, 8-bromo-cGMP, mimics the action of YC-1 in decreasing TM cell volume. 

To further determine whether sGC is involved in the YC-1–induced decreases in HTM cell volume, the ability of YC-1 (50–150 μM) to increase cGMP levels was tested. There was a concentration-dependent increase in cGMP levels in cells treated with varying concentrations of YC-1 that was saturating at 100 μM (Fig. 2B) . Additionally, ODQ (5 μM) abolished the ability of YC-1 (150 μM) to increase cGMP levels (Fig. 2B) . 

Unlike YC-1, however, ODQ (5 and 10 μM) potentiated the BAY-58-2667–induced decreases in TM cell volume (Fig. 2C) . Although we were unable to pharmacologically determine sGC involvement, we tested downstream effects. Cyclic GMP levels were measured in high concentration-exposed BAY-58-2667 samples. Figure 2Ddemonstrates that BAY-58-2667 (10 and 100 μM) increased cGMP levels. Similarly, 8-Br-cGMP mimicked the actions of BAY-58-2667 in decreasing TM cell volume (Fig. 2C) . 

YC-1– and BAY-58-2667–Induced Increases in Cell Volume Do Not Involve cGMP

To determine whether sGC is involved in the low-concentration YC-1– and BAY-58-2667–induced increases in cell volume, HTM36 and HTM80 cells were incubated with calcein AM and exposed to YC-1 (10 nM–25 μM) and BAY-58-2667 (100 nM), and cGMP levels were then measured. The addition of 10 nM to 25 μM YC-1 to HTM cells did not result in a statistically significant increase in cGMP levels compared with control samples (Fig. 3A) . There were no alterations in cGMP levels in TM cells incubated with YC-1 in the presence of ODQ (5 μM; Fig. 3A ). Although it appears as if there was a trend for decreases in cGMP levels when ODQ (5 μM) was added in the presence of YC-1 (1 μM), when all concentrations of YC-1 were included in the data analysis of the cGMP assay, the decrease was not statistically significant. As with low concentrations of YC-1, BAY-58-2667 (100 nM) did not result in a statistically significant increase in cGMP levels (Fig. 3B) . 

PKG Involvement in the YC-1 and BAY-58-2667 Response

The pathway downstream of sGC was tested by exposure of HTM cells to varying concentrations of the PKG inhibitor (RP)-8-Br-PET-cGMP-S (25–100 μM). HTM cells were incubated with YC-1 (150 μM) in the presence of (RP)-8-Br-PET-cGMP-S (25–100 μM) and were imaged. The addition of (RP)-8-Br-PET-cGMP-S (50 and 100 μM) resulted in attenuation of the YC-1–induced decreases in TM cell volume (Fig. 4A) . TM cells were also exposed to BAY-58-2667 (10 μM) in the presence of varying PKG inhibitor concentrations (25–100 μM) that resulted in concentration-dependent attenuation of the BAY-58-2667 effect (Fig. 4B) , providing evidence for the involvement of protein phosphorylation in mediating the YC-1– and BAY-58-2667–induced decreases in TM cell volume. 

BK_Ca Channel Involvement

To test whether activation of the BK_Ca channel is obligatory for the YC-1– and BAY-58-2667–induced decreases in TM cell volume, HTM cells were preincubated with IBTX (100 nM), ^{17 27} and images were captured at the 0 time point. YC-1 (150 μM) and BAY-58-2667 (10 μM) were then added to the cells, and images were captured at 20 minutes after drug exposure. Figure 5demonstrates that IBTX attenuated the YC-1– and BAY-58-2667–induced decreases in TM cell volume. Additionally, IBTX alone had no significant effect on HTM cell volume (Fig. 5) . 

Discussion

In this study we provide evidence that YC-1 and BAY-58-2667, NO-independent sGC activators decrease human TM cell volume through the involvement of the BK_Ca channel. Specifically, YC-1 at concentrations of 50 to 200 μM and BAY-58-2667 at concentrations of 10 to 100 μM decreased TM cell volume, and these decreases were mediated by the sGC/cGMP/PKG pathway in a manner dependent on the BK_Ca channel (Fig. 6) . The actions of YC-1, however, are biphasic, with 1 μM causing increases in TM cell volume, while higher YC-1 concentrations elicited a cell volume reduction. As with YC-1, BAY-58-2667, at higher concentrations (10–100 μM), significantly decreased TM cell volume, whereas exposure to BAY-58-2667 (100 nM) resulted in a significant increase in cell volume. The data observed at the concentrations used are consistent with the known potency of YC-1 and BAY-58-2667 because BAY-58-2667 has previously been shown to have higher potency than YC-1 in activating sGC. ²⁸ The biphasic effects of YC-1 and BAY-58-2667 could be explained by the possible existence of two binding sites for these compounds on sGC that may involve heme-dependent and independent moieties of the sGC. ⁷  

Cell volume was measured in adherent cells in their native states, and each cell was able to serve as its own control. After calcein AM dye achieved a stable baseline, cell volume was measured in response to drug treatment in isotonic media. Additionally, cells that were not treated with drugs were also imaged to assess any changes in fluorescence in response to laser exposure. It has been observed that TM cell cultures contain two distinct cell populations, ²⁹ which is consistent with the identified regions of the TM, the cribriform or juxtacanalicular region and the uveal/corneoscleral region. ^{30 31} The juxtacanalicular region and, hence, the juxtacanalicular cells are regions of high resistance to aqueous humor outflow. Although the cells in the juxtacanalicular tissue contribute little to total tissue volume, the changes in cell volume in this area may have a large contribution to outflow resistance. Although we were able to visually identify the two cell populations, we were unable to determine whether the two cell populations responded similarly to low or high YC-1 or BAY-58-2667 concentrations. 

The ability of the specific sGC inhibitor ODQ to antagonize the actions of YC-1 on TM cell volume suggested that a direct consequence of YC-1 stimulation is the activation of sGC. We measured alterations in cGMP levels in response to varying concentrations of YC-1 and demonstrated a concentration-dependent increase in cGMP levels. Higher concentrations of YC-1 caused significant increases in cGMP levels that correlated with decreases in TM cell volume; however, increased cell volume in response to 1 μM YC-1 was cGMP independent. The physiological and pharmacologic significance of this observation is unclear. ODQ abolished the YC-1–induced increases in cGMP but had no effect on basal cGMP levels, suggesting that ODQ acts by inhibiting the interaction of YC-1 with sGC. Further evidence for the involvement of cGMP in the YC-1–induced response was demonstrated by the ability of 8-Br-cGMP to mimic the actions of YC-1 in decreasing TM cell volume. In our hands, the decreases in TM cell volume in response to YC-1 were similar to decreases in cell volume in response to 8-Br-cGMP, suggesting that cGMP maybe the second messenger mediating the effects of YC-1 on cell volume. 

Unlike ODQ attenuation of the YC-1–induced decreases in TM cell volume, ODQ potentiated the BAY-58-2667 effects. Although the precise mechanisms are unclear, experimental evidence suggests that removal of the heme prosthetic group or oxidation to its ferric form by ODQ causes conformational changes in sGC such that it no longer responds to NO or YC-1 but does respond to BAY-58-2667. ³² Although we did not demonstrate sGC involvement in the BAY-58-2667–induced decreases in TM cell volume, other studies have demonstrated that BAY-58-2667 binds to and activates sGC with subsequent increases in cGMP levels. ²⁸ Similarly, we demonstrated that higher concentrations of BAY-58-2667 caused increases in cGMP levels that correlated with decreases in TM cell volume. 

Additionally, the PKG inhibitor was able to inhibit the YC-1– and BAY-58-2667–induced cell volume changes, further demonstrating the role of PKG and protein phosphorylation events in regulating TM cell volume. Our studies, however, do not preclude the involvement of other second messengers, including cAMP ^{16 33} and protein kinase C. ³⁴  

IBTX inhibited the YC-1– and BAY-58-2667–induced decreases in TM cell volume, suggesting the involvement of the BK_Ca channel and the role of K⁺ efflux in regulating the YC-1– and BAY-58-2667–induced decreases in TM cell volume. Similar observations were made in studies involving TM cells treated with NO in the presence of IBTX; preincubation of TM cells with IBTX abolished the NO-induced decreases in TM cell volume, suggesting that the BK_Ca channel is obligatory for the sGC/cGMP induced decreases in TM cell volume. ⁴ Although we do not know the mechanism(s) by which the sGC/cGMP/PKG system regulates the BK_Ca channel, other studies demonstrate that PKG phosphorylation of the α subunit of the BK_Ca channel results in its activation. ^{35 36} Thus, the possibility exists that YC-1 or BAY-58-2667 activation of PKG in TM cells could result in phosphorylation of BK_Ca channels and subsequent decreases in TM cell volume. 

Although this study suggests the involvement of the BK_Ca channel and K⁺ efflux in the BAY-58-2667– and YC-1–induced decreases in TM cell volume, other studies have demonstrated that cell volume decrease is accompanied by K⁺ and Cl⁻ efflux ³⁷ induced by the activation of K⁺ and Cl⁻ channels and/or K⁺ and Cl⁻ symport. This suggests that K⁺ efflux may initiate a parallel Cl⁻ efflux in TM cells. In other studies, exposure of TM cells to 8-Br-cGMP (50 μM) resulted in inhibition of the bumetanide-sensitive K⁺ influx, demonstrating the involvement of cGMP in Na-K-2Cl cotransport regulation. ¹³ This suggests that the Na-K-2Cl cotransporter may be regulated by YC-1 and BAY-58-2667. However, its ability to decrease TM cell volume is dependent on low bicarbonate levels ^{13 18} or blockade of the Na/H exchanger, ¹⁸ experimental conditions that were not manipulated in these studies. Furthermore, increased cGMP levels resulting from sGC and membrane guanylate cyclase activation resulted in decreased cardiac cell volume through inhibition of K⁺ influx via the bumetanide-sensitive K⁺ cotransporter. ³⁸ These observations suggest that NO-dependent and NO-independent regulation of K⁺ transport and cell volume are bidirectional, facilitating K⁺ efflux by the BK_Ca channel and inhibiting K⁺ influx by the Na-K-2Cl cotransporter. 

Additionally, the NO-dependent sGC/cGMP system plays an important role in regulating aqueous humor dynamics by regulating aqueous humor production in the ciliary processes ^{39 40} and aqueous humor outflow through the TM/Schlemms canal ⁴ with subsequent decreases in IOP. ^{5 41} Therefore, these data suggest that modulation of the volume of TM cells by YC-1 and BAY-58-2667 through the sGC/cGMP/PKG system may modify aqueous humor outflow resistance and, thus, may alter IOP. 

 Supported in part by a grant from the American Health Assistance Foundation, National Glaucoma Research, and Alcon Research Laboratories, Ltd.

 Submitted for publication November 10, 2008; revised December 11, 2008, and February 1, 2009; accepted April 23, 2009.

 Disclosure: W.M. Dismuke, None; N.A. Sharif, Alcon Research Laboratories, Ltd. (E); D.Z. Ellis, Alcon Research Laboratories, Ltd. (F)

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

YC-1– and BAY-58-2667–induced regulation of TM cell volume is biphasic. (A) YC-1–induced changes in cell volume are concentration dependent. Human TM cells were exposed to varying concentrations of YC-1 (10 nM–200 μM). Images were captured at 0 and 20 minutes. Data shown represent the mean ± SEM for control (n = 52 cells), 10 nM (n = 22 cells), 100 nM (n = 35 cells), 1 μM (n = 58 cells), 25 μM (n = 47 cells), 50 μM (n = 65 cells), 75 μM (n = 11 cells), 100 μM (n = 40 cells), 150 μM (n = 51 cells), and 200 μM (n = 49 cells). Data are expressed as percentage of initial volume at 0 time point without drugs. #Significantly different from control at P < 0.05 (ANOVA and Holm-Sidak method). *Significantly different from 1 μM YC-1 at P < 0.05 (ANOVA and Holm-Sidak method). (B) Cells were exposed to varying concentrations of BAY-58-2667 (10 nM–100 μM), and images were captured at 0 and 20 minutes. Data are expressed as percentage of initial volume at 0 time point without drugs and represent the mean ± SEM for control (n = 22 cells), 10 nM (n = 26 cells), 100 nM (n = 17 cells), 1 μM (n = 17 cells), 10 μM (n = 13 cells), and 100 μM (n = 17 cells) . #Significantly different from control at P < 0.05 (ANOVA and Holm-Sidak method). *Significantly different from 100 nM BAY-58-2667 at P < 0.05 (ANOVA and Holm-Sidak method).

Figure 2.

 

Involvement of sGC/cGMP in the YC-1– and BAY-58-2667–induced decrease in TM cell volume. (A) ODQ inhibition of the YC-1–induced decrease in TM cell volume is concentration dependent. Data are expressed as percentage of initial volume at 0 time point. For YC-1 (150 μM), the treated group represents mean ± SEM, n = 24 cells; for 8-Br-cGMP (2 mM), the treated group represents mean ± SEM, n = 71

 

cells; for YC-1 + ODQ (500 nM), the treated group represents mean ± SEM, n = 35 cells; for YC-1 + ODQ (1 μM), the treated group represents mean ± SEM, n = 31 cells; for YC-1 + ODQ (5 μM), the treated group represents mean ± SEM, n = 32 cells. *Significantly different from control at P < 0.05; ANOVA and the Holm-Sidak method. #Significantly different from the YC-1 (150 μM) treated group at P < 0.05 (ANOVA and Holm-Sidak method). (B) YC-1–induced decrease in cell volume is associated with increases in cGMP. Levels of cGMP in TM cells after incubation with YC-1 (50–150 μM) and YC-1 (150 μM) plus ODQ (5 μM). Results measured are expressed as mean ± SEM fmol of cGMP conducted in triplicate. *Significantly different from control at P < 0.05 (ANOVA and Holm-Sidak method). (C) ODQ potentiates the BAY-58-2667–induced decreases in TM cell volume. Data are expressed as percentage of initial volume at 0 time point. For BAY-58-2667 (10 μM), the treated group represents mean ± SEM, n = 38 cells; for 8-Br-cGMP (2 mM), the treated group represents mean ± SEM, n = 30 cells; for BAY-58-2667 + ODQ (5 μM), the treated group represents the mean ± SEM, n = 48 cells; and for BAY-58-2667 + ODQ (10 μM), the treated group represents the mean ± SEM, n = 44 cells. *Significantly different from control at P < 0.05 (ANOVA and Holm-Sidak method). #Significantly different from BAY-58-2667 (10 μM) treated group at P < 0.05 (ANOVA and Holm-Sidak method). (D) BAY-58-2667–induced decrease in cell volume is associated with increases in cGMP. Levels of cGMP in TM cells after incubation with BAY-58-2667 (1–100 μM). Results measured are expressed as mean ± SEM fmol of cGMP conducted in quadruplicate.

Figure 3.

 

Low concentration YC-1– and BAY-58-2667–induced increases in cell volume are not associated with significant increases in cGMP. (A) Levels of cGMP in TM cells after incubation with YC-1 (10 nM–25 μM) and YC-1 (1 μM) plus ODQ (5 μM). Results measured are expressed as mean ± SEM fmol of cGMP conducted in triplicate. (B) Levels of cGMP in TM cells after incubation with BAY-58-2667 (100 nM). Results measured are expressed as mean ± SEM fmol of cGMP conducted in quadruplicate.

Figure 4.

 

PKG is involved in the YC-1– and BAY-58-2667–induced decreases in TM cell volume. (A) Cells were incubated with YC-1 (150 μM) in the presence or absence of varying concentrations of (RP)-8-Br-PET-cGMP-S, a PKG inhibitor (PKGi; 25–100 μM). Images were taken, and cell volume was measured. Data are expressed as percentage of the initial volume at the 0 time point (mean ± SEM); n = 22 cells for the YC1-treated group, n = 21 cells for the YC-1 + PKGi (25 μM)-treated group, n = 14 cells for the YC-1 + PKGi (50 μM)-treated group, and n = 14 cells for the YC-1 + PKGi (100 μM)-treated group. *Significantly different from control (P < 0.05; ANOVA and Holm-Sidak method). #Significantly different from the 150 μM YC-1–treated group (P < 0.05; ANOVA and Holm-Sidak method). (B) Cells were incubated with BAY-58-2667 (10 μM) in the presence or absence of varying concentrations of PKGi (25–100 μM). Images were taken, and cell volume was measured. Data are expressed as percentage of the initial volume at the 0 time point (mean ± SEM); n = 37 cells for the BAY-58-2667–treated group, n = 31 cells for the BAY-58-2667 + PKGi (25 μM)-treated group, n = 84 cells for the BAY-58-2667 + PKGi (50 μM)-treated group, and n = 25 cells for the BAY-58-2667 + PKGi (100 μM)-treated group. *Significantly different from control (P < 0.05). #Significantly different from the 10 μM BAY-58-2667–treated group (P < 0.05; ANOVA and Holm-Sidak method.

Figure 5.

 

YC-1– and BAY-58-2667–induced decreases in TM cell volume involve the BK_Ca channel. TM cells were incubated with YC-1 (150 μM) and BAY-58-2667 (10 μM) in the presence or absence of IBTX (100 nM). Data are expressed as percentage of initial volume at the 0 time point and represent the mean ± SEM; n = 27 cells for the control group, n = 23 cells for the YC1-treated group, n = 37 cells for the BAY-58-2667–treated group, n = 19 cells for the IBTX-treated group, n = 41 cells for the IBTX+YC1-treated group, and n = 69 cells for the IBTX+BAY-58-2667–treated group. *Significantly different from the control group. #Significantly different from the YC-1– or BAY-58-2667–treated group (P < 0.05; ANOVA and the Fisher LSD method).

Figure 6.

 

Summary diagram of the pathway of NO-dependent and NO-independent regulation of TM cell volume. Increases in [Ca²⁺]_i result in activation of nitric oxide synthase (NOS) and the subsequent formation of NO, which then binds to and activates soluble guanylate cyclase (sGC). YC-1 and BAY-58-2667, NO-independent activators of sGC, also bind to sGC and cause increases in cGMP. cGMP then activates PKG, which may, directly or indirectly through other proteins, phosphorylate the BK_Ca channels, with subsequent K⁺ efflux and decreases in cell volume.