cAMP activates BKCa channels in pulmonary arterial smooth muscle via cGMP-dependent protein kinase

Scott A. Barman, Shu Zhu, Guichan Han, and Richard E. White

Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, Georgia 30912


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The signal transduction mechanisms defining the role of cyclic nucleotides in the regulation of pulmonary vascular tone is currently an area of great interest. Normally, signaling mechanisms that elevate cAMP and guanosine-3',5'-cyclic monophosphate (cGMP) maintain the pulmonary vasculature in a relaxed state. Modulation of the large-conductance, calcium- and voltage-activated potassium (BKCa) channel is important in the regulation of pulmonary arterial pressure, and inhibition (closing) of the BKCa channel has been implicated in the development of pulmonary hypertension. Accordingly, studies were done to determine the effect of cAMP-elevating agents on BKCa channel activity using patch-clamp studies in pulmonary arterial smooth muscle cells (PASMC) of the fawn-hooded rat (FHR), a recognized animal model of pulmonary hypertension. Forskolin (10 µM), a stimulator of adenylate cyclase and an activator of cAMP-dependent protein kinase (PKA), and 8-4-chlorophenylthio (CPT)-cAMP (100 µM), a membrane-permeable derivative of cAMP, opened BKCa channels in single FHR PASMC. Treatment of FHR PASMC with 300 nM KT5823, a selective inhibitor of cGMP-dependent protein kinase (PKG) activity inhibited the effect of both forskolin and CPT-cAMP. In contrast, blocking PKA activation with 300 nM KT5720 had no effect on forskolin or CPT-cAMP-stimulated BKCa channel activity. These results indicate that cAMP-dependent vasodilators activate BKCa channels in PASMC of FHR via PKG-dependent and PKA-independent signaling pathways, which suggests cross-activation between cyclic nucleotide-dependent protein kinases in pulmonary arterial smooth muscle and therefore, a unique signaling pathway for cAMP-induced pulmonary vasodilation.

high-conductance calcium-and voltage-activated potassium channel; cAMP-dependent protein kinase; cross-activation


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SIGNAL TRANSDUCTION MECHANISMS defining the role of cyclic nucleotides in the modulation of vascular tone is currently an area of great interest. Agents that relax vascular smooth muscle via activation of adenylate cyclase, such as forskolin, are thought to elicit their biological effects by increasing the intracellular concentration of cAMP (16, 17, 34). Although the exact mechanism by which cAMP causes vascular relaxation is not known, it is believed that activation of PKA is involved (7, 17, 18). In addition to cAMP-mediated vascular relaxation, guanosine-3',5'-cyclic monophosphate (cGMP) has been reported to elicit vasodilatation by activation of cGMP-dependent PKG (6, 23). In the pulmonary circulation, cGMP and cAMP have been implicated as mediators of smooth muscle vasodilatation (13, 15, 16, 24), and in ovine pulmonary vascular smooth muscle, it has recently been reported that pulmonary arteries are more sensitive to relaxation induced by cGMP and that activation of PKG is involved in both cGMP- and cAMP-induced relaxation (10).

It is believed that cAMP and cGMP stimulate their associated protein kinases, PKA and PKG, respectively, but recent evidence suggests that the vasodilatory effects of cAMP-producing agents may involve "cross-activation" of PKG by cAMP (41, 43) and stimulation of PKA by cGMP (8). Lincoln and colleagues (24) originally showed that cross-activation of PKG by cAMP was important in cAMP-elevating agent vasodilatation, and it is thought that cAMP can activate both PKA and PKG. However, relaxation correlates most closely with PKG activation (13), and vasodilators that increase cGMP and cAMP seem to act synergistically on vasorelaxation (41). Thus, whereas studies have reported the existence of cross-activation of PKG by cAMP, few studies have investigated this signaling mechanism at the cellular and molecular levels in pulmonary arterial smooth muscle cells (PASMC).

Although multiple classes of K+ channels are expressed at varying densities in different vascular beds, the large-conductance, calcium- and voltage-activated potassium (BKCa) channel is the predominant potassium channel species in most arteries (27). Inhibition of BKCa channels produces membrane depolarization and subsequent vasoconstriction (29). Specific to cyclic nucleotide activity, evidence suggests that BKCa channels in arterial smooth muscle can be opened through activation of PKG, which phosphorylates the channel (31). It has been shown that PKG stimulates BKCa channel activity in coronary (43), cerebral (31), and pulmonary arteries (1), and BKCa channels are also opened by PKA (25) in a variety of tissues including in vascular smooth muscle to influence myogenic tone (37). In PASMC, it has been reported that nitric oxide causes vasodilation through PKG-mediated BKCa channel activation by channel phosphorylation (28, 35). Furthermore, Haynes et al. (17) observed that tetraethylammonium (TEA), a selective BKCa channel blocker at low concentrations (38), inhibited pulmonary vasodilation induced by the PKA activator 8-bromoadenosine 3',5'-cyclic monophosphate or forskolin, suggesting a vasodilatory mechanism involving cAMP and BKCa channel activation.

In light of these previous investigations, the present study was done to determine mechanisms of cAMP activation of BKCa channels in PASMC of the fawn-hooded rat (FHR), an animal model of idiopathic pulmonary hypertension. Because genetic factors are likely to contribute to the pulmonary hypertension that develops in the FHR, the pulmonary vasculature of this animal model is an excellent model to use to further understand potential mechanisms of pulmonary hypertension. Specifically, the effect of cAMP on PKA and PKG modulation of BKCa channel activity was investigated at the cellular and molecular levels via the patch-clamp technique in FHR PASMC.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. All procedures and protocols were approved by the Animal Care and Use Committee at the Medical College of Georgia. FHRs were a gift from Dr. I. McMurtry (Cardiovascular Pulmonary Research Laboratory, Department of Medicine, University of Colorado Health Sciences Center, Denver, CO), and a breeding colony was established. Animals were maintained on standard rat chow, allowed free access to food and water, and exposed to a 12:12-h light-dark cycle. Male FHRs at 12-16 wk of age were used for this study.

Isolation of PASMC. After the rats were anesthetized with pentobarbital sodium (50 mg/kg ip), a midline incision was made, and the lungs were immediately excised and placed into ice-cold dissociation buffer consisting of (in mM): 110 NaCl, 5 KCl, 0.16 CaCl2, 2 MgCl2, 10 HEPES, 0.5 NaH2PO4, 10 NaHCO3, 0.5 KH2PO4, 10 glucose, 0.49 EDTA, and 10 taurine. Proximal conduit pulmonary arterial vessels were dissected from the lung lobes under a stereomicroscope. Endothelium was removed, and the adventitia was carefully teased away. The vascular tissue was then dissociated enzymatically at 35°C for 1 h in an incubation solution of dissociation buffer containing the following: papain (6.7 mg/5 ml), cysteine (3.5 mg/5 ml), and BSA (5.0 mg/ml). After incubation, 5.0 ml of dissociation buffer containing 2.0 mg/ml BSA was added to the cell solution, and the tissue was then triturated gently, which allowed individual smooth muscle cells to fall away from the larger pieces of undigested tissue with minimal damage to the cells. These cells exhibited morphology characteristics of vascular smooth muscle cells. The solution was then removed and centrifuged at 1,200 rpm for 10 min. The pellet was then resuspended in dissociation buffer. For patch-clamp experiments, several drops of cell suspension were placed in a microscope chamber containing recording solution, and all experiments were performed within 1-2 h after cell dissociation.

Ion channel recording. Whole cell currents were measured from metabolically intact PASMC using the amphotericin-perforated-patch technique (6, 11). In contrast to standard whole cell techniques, perforated-patch recordings provide accurate current measurement with only minimal current decay or loss of soluble cytoplasmic components due to cellular dialysis. Furthermore, endogenous calcium buffering is not inactivated by dialyzing cells with calcium chelators, as are required during whole cell recordings. In addition, identification and characterization of single ion channels were also assessed in these cells.

Perforated-patch experiments. Cells were placed in a recording solution of the following composition (in mM): 140 NaCl, 5 KCl, 2 MgCl2, 2 CaCl2, 20 HEPES, and 20 glucose (pH 7.2). Patch pipettes with a resistance of 3 MOmega or less were fabricated from capillaries of Corning Glass 7052 or other appropriate formulations. To measure potassium currents, the tip of the patch pipette was filled with a solution containing (in mM) 90 KCH3SO3, 40 KCl, 5 MgCl2, and 20 HEPES to approximate normal cellular potassium concentration ([K+]) and chloride concentration ([Cl-]) (pH 7.2 with KOH). The remainder of the pipette was back filled with a similar solution to which 200 mg/ml amphotericin B (diluted by dimethyl sulfoxide) was added. Cells were studied only if the voltage drop across the series resistance was reduced to <= 5 mV within 10-20 min after forming a gigaohm seal. Voltage-clamp and voltage-pulse generation was controlled with an Axopatch 200A patch-clamp amplifier (Axon Instruments), and data were analyzed with pCLAMP 6.0.3 (Axon Instruments), which is a comprehensive software package for acquisition and analysis of both whole cell and single-channel currents. Voltage-activated currents were filtered at 2 kHz and digitized at 10 kHz, and capacitative and leakage currents were subtracted digitally. All drugs were diluted into fresh bath solution and perfused into a 0.5-ml recording chamber (Warner Instruments).

Single-channel experiments. Single potassium channels were measured in cell-attached patches by filling the patch pipette (2-5 MOmega ) with the standard bath solution and making a gigaohm seal on an intact cell. The solution in the recording chamber contained (in mM) 140 KCl, 10 MgCl2, 0.1 CaCl2, 10 HEPES, and 30 glucose (pH 7.2). This manipulation allowed precise regulation of the patch membrane voltage by the patch-clamp amplifier and yielded more accurate current measurements. In experiments measuring potassium channel activity in cell-free inside-out patches, the solution facing the cytoplasmic surface of the membrane had the following composition (in mM): 115 KCH3SO3, 26 KCl, 2 MgCl2, 1 BAPTA, 0.47 CaCl2 (pCa 7), 5.0 Mg-ATP, 0.1 GTP, and 20 HEPES (pH 7.2). The solution facing the external surface was the standard recording solution as described above. Currents were filtered at 2 kHz and digitized at 10 kHz. Channel activity was quantified by calculating the single, open-channel probability (NPo) as described previously (11, 42).

Right ventricular hypertrophy measurements. At the time of death, hearts from adult FHR and control male Sprague-Dawley rats (SDR) were removed and dissected to isolate the free wall of the right ventricle from the left ventricle and septum. The ratio of right ventricle weight to left ventricle plus septum weight (RV/LV+S) was used as an index of right ventricular hypertrophy (14).

Drugs. TEA, iberiotoxin (IBTx), forskolin, 8-4-chlorophenylthio (CPT)-cAMP, CPT-cGMP, Rp-8-CPT-cGMPs, KT5823, and KT5720 were purchased from Calbiochem (San Diego, CA). All other agents were purchased from Sigma (St. Louis, MO).

Statistical analysis. All data were expressed as means ± SE. Statistical significance between two groups was evaluated by Student's t-test for paired data. Comparison among multiple groups was made by using one-way ANOVA for multiple comparisons. A P value <0.05 indicated a significant difference.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Identification of right ventricular hypertrophy. The RV/LV+S ratios in FHR (0.44 ± 0.05; n = 8) vs. control SDR (0.28 ± 0.03, P < 0.05; n = 8) were indicative of right ventricular hypertrophy in FHR as reported in other studies (22, 26, 36, 42).

Identification of BKCa channels in PASMC from FHR. To date, we are aware of no previous electrophysiological studies that have characterized potassium channels in PASMC from FHR. Therefore, our initial experiments characterized outward currents in PASMC from these animals, and these experiments were done in metabolically intact muscle cells. We performed current-clamp experiments on single PASMC using the perforated-patch method and measured an average resting membrane potential (RMP) of -50.8 ± 2.8 mV (n = 4). After treating cells with 100 nM IBTx for 15-20 min, RMP was depolarized to an average of -25.8 ± 5.9 mV (n = 4), which represented a highly significant (P = 0.006) depolarization of RMP due to selective blockade of BKCa channels indicating that these channels are a major regulator of RMP in these cells.

Macroscopic potassium currents were generated by incremental 10-mV depolarizing steps (from -60 to +60 mV), and a representative family of outward currents is illustrated in Fig. 1A. Whole cell, steady-state potassium outward currents obtained from perforated-patch experiments demonstrated noninactivating outward currents, which increased with membrane depolarization (Fig. 1A, left). These currents were significantly attenuated (P < 0.02, n = 4) starting at -10 mV by 100 nM IBTx, a selective inhibitor of BKCa channels as shown by the representative electrophysiological tracing (Fig. 1A, right). A complete current-voltage relationship for average potassium current densities in control cells and the effect of IBTx are illustrated in Fig. 1B. Outward current (initially apparent at approximately -40 mV from a holding potential of -60 mV under control conditions) was measured up to +60 mV, activated slowly, and did not inactivate during the 200-ms depolarization pulse. IBTx inhibited this outward current from -10- to +50- mV depolarization potential steps. This kinetic profile strongly suggests that the majority of outward current in PASMC from FHR is due to activity of BKCa channels, and a definitive identification of channel species was made by measuring single-channel currents pharmacologically at the molecular level.


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Fig. 1.   A: Outward currents in single myocytes from pulmonary arteries are carried mainly via large-conductance, calcium- and voltage-activated potassium (BKCa) channels. Left, outward currents were measured in response to a series of 200-ms voltage steps from a holding potential of -60 mV as illustrated by this representative electrophysiological tracing (n = 4). Right, treating cells with 100 nM iberiotoxin (IBTx), a highly specific blocker of BKCa channels, reduced outward currents (n = 4). B: IBTx inhibits outward currents. Complete current-voltage relationship for steady-state outward current recorded from a single pulmonary arterial smooth muscle cell (PASMC; holding potential -60 mV) is shown. The addition of 100 nM IBTx reduced outward currents from -10 mV to +50 mV voltage (n = 4).

Single BKCa channel properties. Unitary current amplitude was plotted as a function of membrane potentials (-60 to +60 mV) as shown in Fig. 2. Recordings from excised (inside-out) patches under conditions of symmetrical [K+] (140 mM) demonstrated activity of a large-amplitude channel that reversed at ~0 mV, which is the calculated equilibrium potential for K+ in these experiments (Fig. 2A). Single-channel current-voltage relationships yielded an average single-channel conductance of 142 ± 5 pS (n = 3; Fig. 2B), which agrees with other studies reporting BKCa channel conductance in vascular smooth muscle (6, 11). Furthermore, raising Ca2+ concentration at the cytoplasmic face of the patch membrane increased channel gating dramatically (Figs. 5B and 6A), and channel activity was inhibited by 1 mM TEA (Fig. 3B), a selective inhibitor of BKCa channels at this concentration (37). Thus the specific biophysical and pharmacological profile obtained from both intact cells and isolated patches in PASMC from FHR clearly identify this protein as the BKCa channel, which is highly expressed in vascular smooth muscle.


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Fig. 2.   A: BKCa channels are the prominent ion channels in membranes of PASMC. In symmetrical gradients of K+ (140 mM), channel activity recorded from the same inside-out patch varies as a function of membrane potential (n = 3). Channel openings are upward or downward deflections from baseline (closed) state (dashed line). B: complete single-channel current-voltage relationship for BKCa channel activity (n = 3).



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Fig. 3.   cAMP-dependent vasodilators open BKCa channels. A: electrophysiological tracing showing the effect of 10 µM forskolin on BKCa channel activity (n = 5). B: time course plot of BKCa channel activity recorded from cell-attached patches. Channel activity during a 100-ms pulse to +40 mV is plotted as a bar on the graph. Recording time was 10 s for each condition. Breaks in the time axis denote drug incubation times when channel activity was recorded. Lines above the graph indicate periods of drug exposure. Channel activity was before and 10 min after 10 µM forskolin. The patch was then excised into an inside-out configuration, and the addition of 1 mM tetraethylammonium (TEA) reversed the effect of forskolin (n = 5).

cAMP-dependent vasodilators stimulate BKCa channels by a PKA-independent pathway. Intracellular levels of cAMP were increased in PASMC with either forskolin (stimulator of adenylyl cyclase) or a membrane-permeable derivative of cAMP (CPT-cAMP). As shown in Fig. 3, forskolin (10 µM; 10 min) stimulated BKCa channel activity (NPo from ~0.0000 ± 0.0000 to 0.2868 ± 0.0857; n = 5; P < 0.02; Fig. 3A). In contrast, 1 mM TEA inhibited BKCa channel activity after excision into the inside-out configuration (Fig. 3B). In addition to forskolin, treating cells with membrane-permeable 100 µM CPT-cAMP (20 min; Fig. 4B) dramatically increased the activity of BKCa channels in cell-attached patches (NPo ~0.0000 ± 0.0000 to 0.9832 ± 0.005; n = 5; P < 0.0001). Treatment with the PKA inhibitor KT5720 (300 nM) did not attenuate the effect of forskolin (n = 3; Fig. 4A) or CPT-cAMP (n = 3; Fig. 4B) on BKCa channel activity. These data indicate that cAMP activates BKCa channels in FHR PASMC by a PKA-independent signaling pathway.


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Fig. 4.   Forskolin and 8-4-chlorophenylthio (CPT)-cAMP-stimulated BKCa channel activity in fawn-hooded rat (FHR) PASMC does not involve PKA. A: recordings from cell-attached patches (+40 mV) before (left) and 10 min after the addition of 10 µM forskolin (middle; n = 3), then after 300 nM KT5720 (30 min), a specific PKA inhibitor (right; n = 3). B: Recordings from cell-attached patches (+40 mV) before (left) and 20 min after the addition of 100 µM CPT-cAMP, a cell membrane cAMP analog (middle; n = 3), then after 300 nM KT5720 (30 min) (right; n = 3). Channel openings are upward deflections from baseline (dotted line; closed) state.

cAMP-dependent vasodilators stimulate BKCa channels by a PKG-dependent pathway. Fig. 5 shows the effect of KT5823 (300 nM), an inhibitor of PKG activity, on CPT-cAMP (Fig. 5A) and forskolin-induced BKCa channel activation (Fig. 5B). As previously observed, 100 µM CPT-cAMP increased BKCa channel NPo (0.000 ± 0.000 to 0.997 ± 0.0057; P < 0.0001; n = 3), but the subsequent addition of 300 nM KT5823 completely reversed this effect (0.000 ± 0.000; P < 0.0001; n = 3) (Fig. 5A), a phenomenon not observed in SDR PASMC (control NPo: 0.000 ± 0.000; forskolin NPo: 0.3400 + 0.295; forskolin and KT5823 NPo: 0.5379 ± 0.071; n = 2). KT5823 was also a potent inhibitor of 10 µM forskolin-stimulated NPo, because pretreatment with 300 nM KT5823 prevented the response to 10 µM forskolin (n = 2) as illustrated in the time-course histogram of BKCa channel activity (Fig. 5B). Subsequent incision of the patch into an inside-out configuration and raising cytoplasmic Ca2+ concentration produced an immediate increase in BKCa channel activity in the presence of both KT5823 and forskolin, indicating the continued BKCa channel viability of these cells. Further experiments done with another inhibitor of PKG activity, 100 µM Rp-8-CPT-cGMPS, also prevented the stimulatory effect of CPT-cAMP on BKCa channels (n = 4) (Fig. 6A). Once again, excision into the inside-out configuration revealed the continued BKCa channel viability of these cells in the presence of Rp-8-CPT-cGMPS and CPT-cAMP (Fig. 6A, right).


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Fig. 5.   cAMP-dependent vasodilators open BKCa channels via cross-activation of PKG in FHR PASMC. A: effect of KT5823 (300 nM), a specific inhibitor of cGMP-dependent protein kinase (PKG) activity, on CTP-cAMP. The 100 µM CPT-cAMP increased BKCa channel single, open-channel probability (NPo) (n = 3), and the subsequent addition of 300 nM KT5823 completely reversed this effect (n = 3). B: pretreating myocytes with 300 nM KT5823 prevents the stimulatory effect of forskolin on BKCa channel activity. Subsequent incision of the patch into an inside-out configuration and raising cytoplasmic calcium concentration ([Ca2+]c) produced an immediate increase in BKCa channel activity in the presence of both KT5823 and forskolin, indicating the continued BKCa channel viability of these cells.



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Fig. 6.   A: 100 µM Rp-8-CPT-cGMPS (inhibitor of PKG activity) blocks the effect of 100 µM CPT-cAMP on BKCa channels (middle) (n = 4). Excision into the inside-out configuration revealed the continued BKCa channel viability of these cells in the presence of both Rp-8-CPT-cGMPS and CPT-cAMP (right). B: cGMP stimulates openings in FHR PASMC. Recordings were made from cell-attached patch (+40 mV) before (left) and 20 min after 100 µM CPT-cGMP (right), a cell membrane-permeable analog of cGMP (n = 4). Channel openings are upward deflections from baseline (closed) state.

cGMP mimics the effect of cAMP and forskolin on BKCa channels. If cAMP-dependent vasodilators activate BKCa channels through a PKG-dependent signaling pathway, then treating cells with a non-cAMP- dependent agent that increases levels of PKG via cGMP should also stimulate BKCa channel activity. To this end, subsequent experiments revealed that treating cells with CPT-cGMP (a membrane-permeable derivative of cGMP) stimulated BKCa channel openings in PASMC. Specifically, BKCa channel NPo was increased by >100-fold after a 20-min exposure to 100 µM CPT-cGMP (0.0003 ± 0.0002 to 0.0335 ± 0.0106; P < 0.03; n = 4) (Fig. 6B).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Results of the present study indicate that the cAMP-dependent vasodilators forskolin (activator of adenylate cyclase) and CTP-cAMP (membrane-permeable derivative of cAMP) activate BKCa channels in PASMC of FHR via PKG-dependent and PKA-independent signaling pathways. These findings suggest cross-activation between cyclic nucleotide-dependent protein kinases in pulmonary arterial smooth muscle. Earlier studies have shown that agents such as forskolin can activate PKG by increasing cAMP levels in both vascular and nonvascular smooth muscle (13, 18, 24). Also, it has been observed that cAMP levels are up to fivefold higher in some vascular smooth muscle tissues (13), and autophosphorylation of PKG greatly improves its affinity for cAMP, making it more readily activated by cAMP than in the unphosphorylated state (12).

Specific to pulmonary vascular smooth muscle, few studies have shown that cAMP can activate PKG, although recent studies have shown that cGMP can elicit physiological effects by stimulating PKA activity in systemic vascular smooth muscle (8). In addition, cAMP and its analogs can activate both PKA and PKG, with vascular relaxation correlating more closely with PKG activation (13). In the pulmonary circulation, cGMP and cAMP have been implicated as mediators of smooth muscle vasodilation (10, 15-17), and in ovine pulmonary vascular smooth muscle, it has recently been reported (10) that pulmonary arteries are more sensitive to relaxation induced by cGMP and that both cGMP- and cAMP-induced relaxation involves activation of PKG. In addition, Gao and colleagues (15) observed PKG-mediated relaxation of newborn ovine pulmonary veins, and Haynes et al. (17) reported in rat pulmonary circulation that cAMP-mediated pulmonary vasodilatation was mediated via PKA.

Potassium channel activity is the main determinant of membrane potential, and associated K+ efflux causes hyperpolarization, which inhibits voltage-gated calcium channels and promotes vascular relaxation. Although multiple classes of potassium channels are expressed at varying densities in different vascular beds, the BKCa channel is the predominant potassium channel species in most arteries (27). BKCa channels are activated by submicromolar intracellular Ca2+ concentration and are blocked by external charybdotoxin, IBTx, and TEA ions (27, 32). The biophysical profile of the BKCa channel is that it is a large conducting channel (100-150 pS in physiological K+ gradients) that is both calcium and voltage dependent for activation (6, 27, 43). Because of their large conductance and high density, these channels influence RMP and provide an important repolarizing negative feedback mechanism. Inhibition of BKCa channels produces membrane depolarization and subsequent vasoconstriction (29). Specific to this study, the BKCa single-channel conductance was slightly lower (~142 pS) than is typically measured in vascular smooth muscle (>160 pS). The reason for this slightly lower value in this particular vascular smooth muscle type is not apparent. However, because our data are the first to report single BKCa channel conductance in PASMC from FHRs, the BKCa channels in pulmonary vascular smooth muscle in this species may indeed have a conductance of 140-150 pS, which is only ~10% lower than average pS values measured in other types of vascular smooth muscle. Regardless, the conductance values measured in this study are clearly indicative of average single-channel conductance of BKCa channels.

Evidence suggests that ion channels, including BKCa channels, may be modulated by cyclic nucleotide second messengers in various tissues including pulmonary vascular smooth muscle (4, 5, 9, 23, 30, 33). Barman (3) recently reported that dibutyryl cAMP, a cell membrane-permeable analog of cAMP, the cGMP/cAMP phosphodiesterase inhibitor IBMX, and the cAMP-dependent vasodilator isoproterenol blocked the pulmonary vasoconstrictor response to serotonin. In addition, it has been shown that the activity of BKCa channels is potentiated by the cGMP-dependent activation of G kinase (31), and Archer et al. (1) provided compelling evidence for the opening of BKCa channels by increased cGMP levels in rat pulmonary vascular smooth muscle cells. In their experimental model, Archer et al. (1) suggest that pulmonary vascular signal transduction is modulated by both cGMP and cAMP, which regulate potassium channel phosphorylation. Specifically, increased levels of these cyclic nucleotide second messengers promote opening of BKCa channels, which leads to membrane hyperpolarization, inhibition of voltage-gated calcium channels, and subsequent vasorelaxation. Torphy (40), in a recent review, documented that increases in cAMP and cGMP can simultaneously activate the PKA and PKG pathways to promote opening of BKCa channels and elicit membrane hyperpolarization.

Because vascular myocytes are a rich source of PKG, several studies suggest that cGMP-dependent phosphorylation is important in regulating vascular tone (6, 31). Evidence suggests that BKCa channels in arterial smooth muscle can be opened through activation of PKG, which phosphorylates the channel (31), and Carrier et al. (6) reported that BKCa channel activity via cGMP-dependent phosphorylation contributes to vasodilatation in mesenteric vascular smooth muscle. In PASMC, it has been reported that nitric oxide causes vasodilation through PKG-mediated BKCa channel activation by channel phosphorylation (28, 35). Finally, it has been shown that purified PKG stimulates BKCa channel activity in coronary (43), cerebral (31), and pulmonary arteries (1).

Cyclic nucleotide-elevating vasodilators have been used to treat a variety of cardiovascular diseases including heart failure relating to pulmonary hypertension, and vasodilator therapy plays an important role in the management of pulmonary hypertension. Agents that stimulate the cAMP or cGMP transduction pathway in the pulmonary vasculature are presently among the primary treatment measures for this pathophysiological state. For example, some of the most commonly used vasodilators, sodium nitroprusside and nitroglycerin, stimulate the cGMP signaling pathway, whereas isoproterenol, amrinone, and forskolin activate the cAMP transduction pathway. Most recently, the selective phosphodiesterase inhibitor zaprinast has been shown to induce pulmonary vasodilation through the cGMP pathway (19) and has been reported to be effective in the management of pulmonary hypertension. Although the vasodilatory effects of these agents are well documented, there is currently very little knowledge available about the cellular and molecular basis of cAMP- and cGMP-dependent vasodilator mechanisms of action in pulmonary vascular smooth muscle.

The study of pulmonary hypertension has also been problematic because of lack of relevant animal models. In addition, it is difficult in many experimental models to separate the vascular changes that may be pathogenic from the changes that occur secondary to the hypertension (2). Recently, the FHR has been found to be an excellent animal model to study pulmonary hypertension (22, 36). The FHR strain is characterized by platelet abnormalties and systemic hypertension and has been widely used to study genetic risk factors for the development of idiopathic pulmonary hypertension (20, 36). Studies show that the platelet storage disease is not involved in the pathogenesis of pulmonary hypertension (2, 20) and that the FHR strain develops significant pulmonary hypertension, which is not associated with differences in blood gas tension, polycythemia, or parenchymal lung disease (45). Unlike other rat strains, FHR develop severe pulmonary hypertension, which is age dependent (20), a phenomenon accelerated under mild hypoxic conditions (36). Evidence also suggests that the genetic locus for the hypertensive condition is PH1 on chromosome 1 (39).

In summary, the results of this study indicate that cAMP-dependent vasodilators activate BKCa channels in PASMC of FHR via PKG-dependent and PKA-independent signaling pathways. Thus this study suggests that cAMP increases levels of PKG in an animal model of pulmonary hypertension and demonstrates the phenomenon of cross-activation of cyclic-nucleotide-dependent protein kinases in pulmonary arterial smooth muscle under potential pathophysiologcial conditions.


    ACKNOWLEDGEMENTS

The authors thank Louise Meadows for excellent technical assistance.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants HL-68026 (to S. A. Barman) and HL-64779 (to R. E. White) and by the American Heart Association Grant 9950179N (to R. E. White).

Address for reprint requests and other correspondence: S. A. Barman, Dept. of Pharmacology and Toxicology, Medical College of Georgia, Augusta, GA 30912 (E-mail: sbarman{at}mail.mcg.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.

First published January 24, 2003;10.1152/ajplung.00295.2002

Received 27 August 2002; accepted in final form 13 January 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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