Inhibition of inwardly rectifying K+ channels by cGMP in pulmonary vascular endothelial cells

Larissa A. Shimoda, Laura E. Welsh, and David B. Pearse

Department of Medicine, Division of Pulmonary and Critical Care Medicine, Johns Hopkins School of Medicine, Baltimore, Maryland 21224


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Endothelial barrier dysfunction is typically triggered by increased intracellular Ca2+ concentration. Membrane-permeable analogs of guanosine 3',5'-cyclic monophosphate (cGMP) prevent disruption of endothelial cell integrity. Because membrane potential (Em), which influences the electrochemical gradient for Ca2+ influx, is regulated by K+ channels, we investigated the effect of 8-bromo-cGMP on Em and inwardly rectifying K+ (KIR) currents in bovine pulmonary artery and microvascular endothelial cells (BPAEC and BMVEC), using whole cell patch-clamp techniques. Both cell types exhibited inward currents at potentials negative to -50 mV that were abolished by application of 10 µM Ba2+, consistent with KIR current. Ba2+ also depolarized both cell types. 8-Bromo-cGMP (10-3 M) depolarized BPAEC and BMVEC and inhibited KIR current. Pretreatment with Rp-8-cPCT-cGMPS or KT-5823, protein kinase G (PKG) antagonists, did not prevent current inhibition by 8-bromo-cGMP. These data suggest that 8-bromo-cGMP induces depolarization in BPAEC and BMVEC due, in part, to PKG-independent inhibition of KIR current. The depolarization could be a protective mechanism that prevents endothelial cell barrier dysfunction by reducing the driving force for Ca2+ entry.

protein kinase G; membrane potential; ion channels


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE VASCULAR ENDOTHELIUM ACTS as a physical barrier between blood and extravascular tissue. Endothelial cell barrier dysfunction occurs in response to stimuli such as ischemia/reperfusion injury and oxidant stress, leading to increased vascular permeability and serious physiological consequences. For example, in the lung, leakage of fluids and proteins into the alveoli and interstitial spaces after disruption of endothelial cell barrier function results in pulmonary edema and compromised gas exchange.

Nitric oxide (NO) has been shown to modulate oxidant-induced endothelial cell injury in a variety of settings (3, 8, 18, 19). NO influences production of intracellular guanosine 3',5'-cyclic monophosphate (cGMP) via activation of soluble guanylate cyclase, and this signaling pathway provides a main mechanism for the physiological actions of NO (13, 24). A role for cGMP in NO-mediated enhancement of endothelial cell barrier function is supported by data demonstrating that membrane-permeable analogs of cGMP can prevent oxidant-mediated disruption of the endothelial cell barrier (22, 29). However, the mechanisms by which cGMP affords protection against oxidant injury are unknown.

During acute lung injury, the generation of reactive oxygen species is thought to contribute to endothelial cell barrier dysfunction, in part, by increasing intracellular Ca2+ concentration ([Ca2+]i) (7, 34, 35). For example, hydrogen peroxide (H2O2) increases [Ca2+]i and reduces barrier function (11, 32). Recently, we found that pretreatment of endothelial cells with 8p-8-(4-chlorophenylthio)-cGMP (8p-CPT-cGMP), a membrane-permeable analog of cGMP, prevented the increase in Ca2+ observed during H2O2 challenge (D. B. Pearse, L. A. Shimoda, A. D. Verin, N. Bogatcheva, C. Moon, G. V. Ronnett, L. E. Welsh, and P. B. Becker; unpublished results), suggesting that cGMP may alter Ca2+ signaling in response to oxidants. The electrochemical gradient, or driving force, for Ca2+ entry into endothelial cells is influenced by the membrane potential (Em) (1, 23), such that depolarization decreases, while hyperpolarization increases, Ca2+ entry. Resting Em in endothelial cells is thought to be controlled primarily by K+ channels, in particular, inwardly rectifying K+ channels (KIR) (1, 26). Thus a possible mechanism by which cGMP could control [Ca2+]i is through regulation of K+ channels and Em. Consistent with this possibility, cGMP has been shown to modulate ion channel function in a variety of cell types. For example, cyclic nucleotide-gated cation channels in neurons (12) and endothelial cells (37), KIR channels in kidney proximal tubule cells (20), and Ca2+-activated K+ (KCa) and voltage-gated K+ (KV) channels in vascular smooth muscle (2, 28, 31, 39) are activated by cGMP. In contrast, the effect of cGMP on L-type Ca2+ channels in cardiac myocytes (9), KIR in brain (16), KV channels in central nervous system neurons (25), and K+ channels in kidney epithelial cells (14) is inhibitory. The fact that cGMP was shown to be both excitatory and inhibitory suggests that regulation of ion channel activity by cGMP may be cell and/or channel specific. To date, the effect of cGMP on Em and K+ channels has not been studied in endothelial cells.

Many of the physiological effects of intracellular cGMP are produced via activation of protein kinase G (PKG), also known as cGMP-dependent protein kinase (21, 24). In general, the effects of cGMP on ion channel activity have been shown to occur through phosphorylation of channels secondary to activation of PKG (2, 9, 20, 25, 28), although PKG-independent modulation of ion channels has also been reported (14, 16, 39).

On the basis of these data, in this study, we used whole cell patch-clamp techniques to 1) verify the existence of KIR, 2) evaluate the contribution of KIR to maintenance of resting Em, 3) determine the effect of 8-bromo-cGMP on KIR and Em, and 4) evaluate the role of PKG in 8-bromo-cGMP-induced regulation of KIR channels in bovine pulmonary artery (BPAEC) and pulmonary microvascular endothelial cells (BMVEC).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Culture

BPAEC and BMVEC at the fourth passage were obtained from Cell Systems Certified (Kirkland, WA). Cells were seeded onto 25-mm glass coverslips and were sustained in DMEM (GIBCO-BRL) supplemented with 20% fetal bovine serum (Sigma, St. Louis, MO), 17 µg/ml endothelial growth factor supplement (Upstate Biotechnology, Lake Placid, NY), and 100 U/ml penicillin/streptomycin (GIBCO-BRL) at 37°C in a humidified atmosphere of 5% CO2 and 95% room air. Cells were used at the seventh passage for all experiments.

Electrophysiological Measurements

Em and membrane currents were measured by the whole cell patch-clamp technique. Em was recorded in current-clamp mode (I = 0). Membrane currents were recorded under voltage-clamp mode at a holding potential of -60 mV with step changes to various test potentials (-150 to +60 mV) to activate membrane currents. Cells were superfused with HEPES-buffered salt solution containing (in mM): 130 NaCl, 5 KCl, 1.2 MgCl2, 2 CaCl2, 10 HEPES, and 10 glucose, with pH adjusted to 7.4 with 5 M NaOH. Patch pipettes (tip resistance 3-5 MOmega ) were pulled from glass capillary tubes, fire polished, and filled with an internal solution containing (in mM): 35 KCl, 90 K-gluconic acid, 10 NaCl, and 10 HEPES, with the pH adjusted to 7.2 with KOH. The pipette solution also contained 5 mM ATP to inhibit ATP-sensitive K+ (KATP) channels and prevent current rundown. Whole cell currents were recorded on an Axopatch 200A amplifier (Axon Instrument). Pipette potential and capacitance and access resistance were electronically compensated. Voltage-clamp protocols were applied with pClamp software (Axon Instrument). Data were filtered at 5 kHz, digitized with a Digidata 1200 analog-to-digital converter (Axon Instrument), and analyzed with pClamp software (Axon Instrument). Cells with unstable Em or leak currents >5 pA were discarded. Leak subtraction, if necessary, was performed during data analysis. Cell capacitance was calculated from the area under the capacitive current elicited by a 10-mV hyperpolarizing pulse from a holding potential of -70 mV. Whole cell current was normalized to cell capacitance and expressed as pA/pF. External solutions were changed via a rapid exchange system with a multibarrel pipette connected to a common orifice positioned 100-200 µm from the endothelial cell studied. Complete solution exchange was achieved in <1 s. All experiments were conducted at room temperature (22-25°C).

Experimental Protocols

Identification of K+ currents in pulmonary vascular endothelial cells. Membrane currents were elicited by applying a 400-ms pulse from a holding potential of -60 mV to test potentials ranging from -150 to +70 mV in 20-mV increments. Peak current elicited at each test potential was normalized to cell capacitance and plotted, generating a current-voltage (I-V) curve. The measurements were made under control conditions and 3-4 min after applying Ba2+ (10 µM), a KIR channel inhibitor.

Effect of Ba2+ and 8-bromo-cGMP on Em. Em was measured in current-clamp mode with I = 0. The effects of KIR channel inhibition and cGMP on Em were evaluated by measuring Em for 1 min before, 2 min during, and 2 min after exposure to either Ba2+ (10 µM) or the membrane-permeant cGMP analog 8-bromo-cGMP (1 mM).

Effect of 8-bromo-cGMP on KIR current. We determined the effect of 8-bromo-cGMP on KIR current by measuring peak KIR current activated at 2-s intervals by a 400-ms hyperpolarization to -150 mV from a holding potential of -60 mV before and during application of 8-bromo-cGMP (1 mM). Measurements continued until stable currents (<1% change in magnitude) were attained. We further evaluated the effect of 8-bromo-cGMP on peak KIR current by comparing the I-V curves generated by step changes from -150 to -50 mV (holding potential = -60 mV, 20-mV increments) before and 3-4 min after exposure to 8-bromo-cGMP.

Role of PKG in the effect of 8-bromo-cGMP on KIR current. The involvement of PKG activation was examined by comparing the effects of 8-bromo-cGMP (1 mM) on KIR currents generated in BPAEC and BMVEC in the absence and presence of Rp-8-beta -phenyl-1, N2-etheno-cGMPS (Rp-8-PET-cGMPS, 1 nM), Rp-8-cPCT-cGMPS (50 µM), or KT-5823 (50 µM), specific inhibitors of PKG. Cells were exposed to the PKG inhibitors for 5 min (or until current was stable) before exposure to 8-bromo-cGMP. The effect of PKG inhibition on 8-bromo-cGMP-induced inhibition of KIR currents was determined by comparing the effect of 8-bromo-cGMP on I-V curves in control cells with I-V curves obtained from cells pretreated with PKG inhibitors.

Effect of extracellular cGMP on KIR currents. To determine whether 8-bromo-cGMP was inhibiting KIR channels from outside the cells or required crossing the cell membrane, we exposed cells to cGMP, which is largely cell impermeant. KIR current was activated at 2-s intervals by a 400-ms step hyperpolarization to -150 mV from a holding potential of -60 mV before and during application of cGMP (1 mM). Measurements continued until stable currents (<1% change in magnitude) were attained. We further evaluated the effect of cGMP on peak KIR current by comparing the I-V curves generated before and 3-4 min after exposure to cGMP.

Drugs and Chemicals

8-Bromo-cGMP (sodium salt) was obtained from Tocris Cookson (Baldwin, MO). Rp-8-bromo-PET-cGMPS and Rp-8p-CPT-cGMPS were obtained from Biolog Life Science Institute (La Jolla, Ca). KT-5283 was obtained from Calbiochem (San Diego, Ca). All other chemicals were obtained from Sigma. Stock solutions of Rp-8-bromo-PET-cGMPS and Rp-8p-CPT-cGMPS (5 mM) were made up in DMSO and stored at 4°C. On the day of experiment, stock solutions were diluted as needed with physiological salt solution (PSS) to appropriate concentrations. A 1-mM stock solution of KT-5283 was made daily in DMSO and diluted to a final concentration of 50 µM in PSS. 8-Bromo-cGMP and cGMP were made fresh daily in PSS.

Data Analysis

Statistical significance was determined by Student's t-test (paired or unpaired, as applicable) and two-way ANOVA with repeated measures with a Student-Newman-Keuls post hoc test. A value P < 0.05 was accepted as statistically significant. Data are expressed as means ± SE; n refers to the number of cells tested.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Identification of KIR in Pulmonary Endothelial Cells

Stepping test potentials from -150 to -70 mV elicited inward current in both BPAEC and BMVEC (Fig. 1). This inward current was observed at all test potentials negative to the resting Em (approximately -65 to -70 mV) and was voltage dependent, increasing in amplitude at more negative potentials. The activation and inactivation kinetics of this current were also voltage dependent, with current reaching peak magnitude faster (Table 1) and exhibiting faster and more pronounced inactivation as test potentials became more negative.


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Fig. 1.   Representative tracings illustrating the effect of Ba2+ (10 µM) on inwardly rectifying K+ (KIR) current in bovine pulmonary arterial endothelial cells (BPAEC) and bovine microvascular endothelial cells (BMVEC). We measured current with whole cell patch clamp in voltage-clamp mode. Current was elicited by a 400-ms step changes from a holding potential of -60 mV to test potentials of -150 to 70 mV. Graphs represent mean current-voltage (I-V) relationship before and after exposure to Ba2+ in BPAEC and BMVEC (n = 8 for BPAEC, n = 3 for BMVEC).


                              
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Table 1.   Time required to reach peak KIR current magnitude at different test potentials in BPAEC and BMVEC

The inward current was inhibited in the presence of 10 µ M Ba2+ by 77.8 ± 6.9% in BPAEC (n = 8) and by 85.4 ± 7.3% in BMVEC (n = 3) at -150 mV, consistent with the known Ba2+ sensitivity of KIR channels. Ba2+ had no significant effect on the residual outward current observed in these cells. The inward current was not affected by application of 1 mM 4-aminopyridine, an inhibitor of KV channels, or 100 nM ChTX, an inhibitor of KCa channels (data not shown).

Contribution of KIR to Resting Em in Pulmonary Endothelial Cells

Resting Em was monitored via current clamp (I = 0). Resting Em was measured for 2 min and averaged -61.7 ± 1.9 mV (ranging from -49 to -76 mV) in BPAEC and -67.5 ± 3.5 mV (ranging from -51 to -86 mV) in BMVEC. Cells with unstable resting Em or leak currents >5 pA were discarded. Application of Ba2+ (10 µM) significantly depolarized both BPAEC (from -66.0 ± 4.9 to -25.2 ± 5.6 mV) and BMVEC (from -71.4 ± 3.8 to -44.8 ± 5.9 mV) (n = 7 each, Fig. 2).


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Fig. 2.   Bar graph representing mean resting membrane potential (Em) and depolarization in response to Ba2+ in BPAEC and BMVEC (n = 7 cells each). *Significant difference from control value (P < 0.05).

Effect of 8-Bromo-cGMP on KIR and Em

Application of 8-bromo-cGMP (1 mM) caused significant inhibition of KIR current in pulmonary endothelial cells. The effect of 8-bromo-cGMP was rapid, reversible, and reproducible (Fig. 3). Inhibition of current began within seconds of application, reaching maximum effect within 4 min, and recovered to ~92% of the original magnitude upon washout of the drug. At -150 mV, 8-bromo-cGMP inhibited KIR by 26.4 ± 7.2% and 38.6 ± 7.3% in BPAEC (n = 10) and BMVEC (n = 8), respectively (Fig. 4). In both cell types, 8-bromo-cGMP did not alter the time required to reach peak current magnitude at any of the test potentials measured.


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Fig. 3.   Top: representative time course of change in peak KIR current at -150 mV in response to 8-bromo-guanosine 3',5'-cyclic monophosphate (cGMP); a: baseline; b: stable 8-bromo-cGMP response; c: recovery. Bottom: KIR current traces (at -150 mV) corresponding to baseline (a), stable 8-bromo-cGMP response (b), and recovery (c).



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Fig. 4.   Representative tracings illustrating the effect of 8-bromo-cGMP (1 mM) on KIR current in BPAEC and BMVEC. Bar graphs represent mean peak I-V relationship and average time required to reach peak current before and after exposure to Ba2+ in BPAEC (n = 10) and BMVEC (n = 8). For both BPAEC and BMVEC, the difference between the I-V curves in the presence and absence of 8-bromo-cGMP is significantly different (P < 0.01).

Consistent with inhibition of KIR current, application of 8-bromo-cGMP caused a significant depolarization of ~25-30 mV from the resting Em in both BPAEC (from -63.8 ± 5.4 to -35 ± 8.9 mV, n = 4) and BMVEC (from -70.0 ± 4.9 to -42.3 ± 9.7 mV, n = 7; Fig. 5). Similar to the inhibitory effect of 8-bromo-cGMP on KIR current, depolarization in response to 8-bromo-cGMP was reversible upon washout of the drug.


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Fig. 5.   Bar graph illustrating effect of 8-bromo-cGMP on Em in BPAEC (n = 4) and BMVEC (n = 7). *Significant difference from control value (P < 0.05).

Role of PKG in cGMP-Induced Inhibition of KIR

To evaluate whether activation of PKG played a role in the cGMP-induced inhibition of KIR channels observed in pulmonary endothelial cells, we pretreated BPAEC and BMVEC with Rp-8-PET-cGMPS (50 µM), an inhibitor of PKG. However, application of Rp-8-PET-cGMPS caused a significant inhibition of KIR channel activity, reducing peak KIR current by 52.1 ± 3.1% in BPAEC and by 51.9 ± 16.9% in BMVEC. Application of vehicle alone had no effect on KIR current (data not shown). Given the marked reduction in KIR current in response to this antagonist, another related compound was used to examine the possibility of nonspecific effects. Rp-8p-CPT-cGMPS (50 µM), a structurally related inhibitor of PKG, was used. In BPAEC, pretreatment with Rp-8-cPCT-cGMPS caused a slight inhibition of current (16.2 ± 5.5%) and did not prevent inhibition by subsequent exposure to 8-bromo-cGMP, which still inhibited peak KIR current by 28.9 ± 4.1% at -150 mV (n = 5, Fig. 6). In BMVEC, Rp-8-cPCT-cGMPS caused a greater decrease in KIR current, inhibiting peak current by 22.9 ± 10.3%, although this difference did not quite reach statistical significance (n = 3, P = 0.07). Subsequent application of 8-bromo-cGMP still resulted in a 23.5 ± 5.5% inhibition of peak KIR current.


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Fig. 6.   Graphs representing mean I-V relationship under control conditions and after exposure to Rp-8p-8-(4-chlorophenylthio)-cGMPS (Rp-8p-CPT-CGMPS, 50 µM) and subsequent exposure to 8-bromo-cGMP in BPAEC (n = 5) and BMVEC (n = 3). In cells exposed to Rp-8p-CPT-cGMPS, I-V curves in the absence and presence of 8-bromo-cGMP were statistically different (P < 0.05). Bar graphs represent %inhibition of peak KIR current elicited at a test potential of -150 mV in response to 8-bromo-cGMP under control conditions and in the presence of Rp-8p-CPT-cGMPS.

Both Rp-8-PET-cGMPS and Rp-8p-CPT-cGMPS are structurally related to 8-bromo-cGMP. Because these compounds had an inhibitory effect on KIR current, we also repeated the protocol with a structurally unrelated PKG inhibitor, KT-5823 (1 µM). Application of KT-5823 caused a small, statistically insignificant decrease in peak KIR current in both BPAEC (11.3 ± 6.1%, n = 7) and BMVEC (5.3 ± 2.5%, n = 6). Subsequent application of 8-bromo-cGMP caused 22.1 ± 4.1% and 21.9 ± 3.6% inhibitions of peak KIR current in BPAEC and BMVEC, respectively (Fig. 7). The cGMP-induced inhibition of KIR current in the presence of KT-5823 was slightly less than that observed in control cells, but the values were not statistically different.


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Fig. 7.   Graphs representing mean I-V relationship under control conditions and after exposure to KT-5823 (1 µM) and subsequent exposure to 8-bromo-cGMP in BPAEC (n = 9) and BMVEC (n = 6). In cells exposed to KT-5823, I-V curves in the absence and presence of 8-bromo-cGMP were statistically different (P < 0.05). Bar graphs represent %inhibition of peak KIR current elicited at a test potential of -150 mV in response to 8-bromo-cGMP under control conditions and in the presence of KT-5823.

Effect of Extracellular cGMP on KIR in Pulmonary Endothelial Cells

In some studies, cGMP has been shown to act as an extracellular regulator of ion channel function (14). Because the effect of 8-bromo-cGMP on KIR channels occurred rapidly and did not appear to involve activation of PKG, we next tested the possibility that 8- bromo-cGMP was inhibiting channels from outside, rather than inside, the cell. Cells were exposed to cGMP, which does not easily cross the cell membrane. In BPAEC, cGMP did not significantly alter peak KIR current (n = 5), whereas in BMVEC, peak KIR current was decreased by 15.0 ± 3.1% (n = 4, Fig. 8).


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Fig. 8.   Graphs representing mean I-V relationship before and after application of extracellular cGMP in BPAEC (n = 5) and BMVEC (n = 4). I-V curves in the absence and presence of cGMP were statistically different in BMVEC but not BPAEC (P < 0.05). Bar graph represents average %inhibition of peak KIR current elicited at a test potential of -150 mV in response to cGMP.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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In this study, we found that increasing cGMP levels in pulmonary vascular endothelial cells caused inhibition of KIR channels and depolarization. The reduction of KIR current in response to cGMP was not prevented by inhibitors of PKG and was not mimicked by external application of cGMP in BPAEC, although a slight inhibitory effect of external cGMP was observed in BMVEC. These results indicate that the inhibitory effects of cGMP on KIR current and Em are not dependent on activation of PKG and do not occur, for the most part, via extracellular interaction with the channel.

In our BPAEC and BMVEC, the measured resting Em was close to the reversal potential of K+, suggesting that the resting potential is controlled mainly by the K+ conductance. Endothelial cells have been shown to possess several types of K+ channels, including KCa, KV, KATP, and KIR (1, 26). Of these channel subtypes, KIR has been suggested to be the main contributor to regulation of Em (1, 26). We observed a KIR current in BPAEC and BMVEC that was qualitatively similar to that previously observed in other types of endothelial cells (4, 17, 36), in that the KIR current was activated in a voltage-dependent manner at test potentials negative to the resting Em and exhibited increased current magnitude and faster inactivation kinetics at more negative potentials. The inhibition of the inward current by Ba2+ verified the identity of the channel and the accompanying depolarization observed in the presence of Ba2+ confirmed that this channel contributes significantly to regulation of resting Em in this cell type.

Endothelial cell populations with a dominant chloride conductance and much more positive resting Em have been described (26). It is not clear at this point what influences the ion channel composition of endothelial cells, but a few possibilities exist to explain why we observed only KIR-dominated cells. First, differences may be explained based on location in the pulmonary vascular tree from which the cells were derived, the species, or culture conditions. In particular, bovine pulmonary endothelial cells with depolarized Em and dominant chloride conductance have been described primarily in cells from calves, whereas our studies, as well as others showing predominate K+-regulated Em in bovine pulmonary arterial endothelium were performed in cells from adult animals (5, 6, 15). Thus developmental differences could influence ion channel distribution in bovine pulmonary endothelial cells, as has been shown to occur in other pulmonary vascular cell types (10). Another possibility is that because our cells exhibit low resting [Ca2+]i (27) and were dialyzed with Ca2+-free intracellular solution, the resting [Ca2+]i level could be low enough to prevent activation of either Ca2+-activated or volume-sensitive chloride channels (26).

In pulmonary endothelial cells, we found that application of 8-bromo-cGMP resulted in a significant inhibition of KIR current magnitude. Inhibition of KIR by 8-bromo-cGMP was reversible and reproducible and thus was not due to rundown of the current. The inhibitory effect of 8-bromo-cGMP appears to be limited to current magnitude, as comparison of currents in the absence and presence of cGMP indicates that the activation and inactivation kinetics of KIR channels were not altered. cGMP has been shown to modulate ion channel activity in a number of cell types, although both activation and inactivation have been reported. Although KCa and KV channels in vascular smooth muscle (2, 28, 31, 39) and KIR in proximal tubule (20) and nonselective cation channels in olfactory receptors (12) and endothelium (37, 38) have been shown to be activated by cGMP, L-type Ca2+ channels in cardiac myocytes (9), KIR in brain (16), KV3 from neurons (25), KCa in mesangial cells (33), and K+ channels in immortalized human kidney epithelial cells (14) were inhibited by cGMP. Thus, although cGMP can regulate the function of many channels, whether the effect of cGMP is excitatory or inhibitory may be cell or channel subtype specific.

Consistent with KIR playing a role in regulation of Em, the inhibition of KIR current observed in response to 8-bromo-cGMP was accompanied by marked depolarization in both BPAEC and BMVEC. The fact that 8-bromo-cGMP caused depolarization provides evidence that the effect of cGMP on KIR channel activity has physiological implications. Em regulates Ca2+ entry in endothelial cells mainly by enhancing the electrochemical Ca2+ gradient, such that hyperpolarization increases while depolarization decreases [Ca2+]i (23). Thus through control of [Ca2+]i, depolarization in response to 8-bromo-cGMP may contribute to regulation of numerous cell processes.

In many cases, downstream effects of cGMP are signaled via activation of PKG. In endothelial cells, the inhibitory effect of 8-bromo-cGMP was not prevented by pretreatment of PKG inhibitors, indicating that the mechanisms by which cGMP regulates KIR function in this cell type do not require phosphorylation of the channel by the second messenger PKG. This contrasts with results from numerous studies indicating that both inhibition and activation of channels by cGMP involve signaling through PKG (2, 9, 20, 25, 28, 31, 33, 38). There are a few studies, however, in which PKG-independent effects of cGMP on ion channel regulation have been reported (14, 16, 39). Our results indicate that regulation of KIR channel activity by cGMP in endothelial cells is one of these instances. Although a simple explanation for the variability in PKG dependence may be that not all ion channels contain a PKG phosphorylation site, this is contradicted by evidence that regulation of KCa channels by cGMP has been demonstrated to be both PKG dependent (2, 28, 31) and independent (14). The difference in the role of PKG in cGMP signaling may instead reflect variations in cell type and intracellular milieu.

Interestingly, the Rp compounds used to inhibit PKG had an inhibitory effect on the KIR channel. Indeed, the inhibition caused by Rp-8-PET-cGMPS was greater than that observed in response to 8-bromo-cGMP. One reason for this phenomenon may be that inhibition of KIR by Rp-8-PET-cGMPS and Rp-8p-CPT-cGMPS reflected alleviation of PKG-dependent activation of KIR channels under basal conditions, as basal phosphorylation by PKG has been demonstrated to play a role in regulation of ion channel activity in proximal tubule epithelial cells (14). A second possibility is that an aspect of the structural composition of the Rp compounds allowed them to function in a manner similar to 8-bromo-cGMP and mimic the effect on KIR current. To distinguish between these two possibilities, we used KT-5823, a PKG antagonist structurally unrelated to 8-bromo-cGMP. Application of KT-5823 did not alter baseline function of the channels, indicating that the effect of Rp-8-PET-cGMPS and Rp-8p-CPT-cGMPS was not related to inhibition of basal PKG activation. Because the effect of 8-bromo-cGMP on KIR channel function was independent of PKG activation, and Rp-8-PET-cGMPS and Rp-8p-CPT-cGMPS appeared to be acting as partial agonists and mimicked the effect of 8-bromo-cGMP, we hypothesize that the structural composition of these drugs may allow direct interaction with, and regulation of, the channel. This possibility suggests an area for future study.

Our data indicate that the effect of 8-bromo-cGMP on KIR current occurred rapidly and appeared to be independent of PKG activation. This raised the possibility that 8-bromo-cGMP did not require entry into the cell but, rather, was inhibiting KIR channels via an external interaction with the channel. Extracellular regulation of ion channels by cGMP has been previously reported in kidney epithelial cells (14) and cerebellar neurons (30). This possibility was tested in our cells by challenging BPAEC and BMVEC with extracellular cGMP. Extracellular cGMP had no effect on KIR current in BPAEC. Only a minimal inhibitory effect was observed in BMVEC, much smaller than the response to 8-bromo-cGMP. The results from this experiment suggest that although it is possible for cGMP to act externally to regulate KIR channel activity in some situations, the inhibitory effect of 8-bromo-cGMP on KIR that we observed was due primarily to intracellular signaling mechanisms. In addition, the difference in response to external cGMP between BMVEC and BPAEC reinforces recent interest in the possibility that pulmonary endothelial cells can respond to physiological stimuli differently based on location in the pulmonary vascular tree.

In summary, the results of this study indicate that cGMP can inhibit KIR channels and cause membrane depolarization in pulmonary endothelial cells via an intracellular mechanism that does not require activation of PKG. Increasing intracellular cGMP levels, with NO or membrane-permeable analogs of cGMP, has been shown to protect against lung injury due to ischemia/reperfusion and oxidant stress. Because the permeability changes induced by ischemia/reperfusion and oxidants involve gap formation between endothelial cells secondarily to increased [Ca2+]i, the results from this study suggest that inhibition of KIR and depolarization of endothelial cells in response to cGMP may modulate [Ca2+]i and protect against oxidant-induced endothelial cell injury by reducing the driving force for Ca2+ entry into the cell.


    ACKNOWLEDGEMENTS

This work was supported by American Heart Association Scientist Development Grant AHA9930255N (awarded to L. A. Shimoda) and Established Investigator Award (to D. B. Pearse).


    FOOTNOTES

Address for reprint requests and other correspondence: L. A. Shimoda, Div. of Pulmonary and Critical Care Medicine, Johns Hopkins Univ., 5501 Hopkins Bayview Cir., JHAAC 4A.52, Baltimore, MD 21224 (E-mail: shimodal{at}welch.jhu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

March 22, 2002;10.1152/ajplung.00469.2001

Received 11 December 2001; accepted in final form 18 March 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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