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Protein kinase C inhibits BK_Ca channel activity in pulmonary arterial smooth muscle

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

Submitted 27 June 2003 ; accepted in final form 23 September 2003

	ABSTRACT

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Signaling mechanisms that elevate cyclic AMP (cAMP) activate large-conductance, calcium- and voltage-activated potassium (BK_Ca) channels in pulmonary vascular smooth muscle and cause pulmonary vasodilatation. BK_Ca channel modulation is important in the regulation of pulmonary arterial pressure, and inhibition (closing) of the BK_Ca channel has been implicated in the development of pulmonary vasoconstriction. Protein kinase C (PKC) causes pulmonary vasoconstriction, but little is known about the effect of PKC on BK_Ca channel activity. Accordingly, studies were done to determine the effect of PKC activation on cAMP-induced BK_Ca 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, opened BK_Ca channels in single FHR PASMC, which were blocked by the PKC activators phorbol 12-myristate 13-acetate (100 nM) and thymeleatoxin (100 nM). The inhibitory response by thymeleatoxin on forskolin-induced BK_Ca channel activity was blocked by Gö-6983, which selectively blocks the , , , , and PKC isozymes, and Gö-6976, which selectively inhibits PKC-, PKC-, and PKC-µ, but not by rottlerin, which selectively inhibits PKC-. Collectively, these results indicate that activation of specific PKC isozymes inhibits cAMP-induced activation of the BK_Ca channel in pulmonary arterial smooth muscle, which suggests a unique signaling pathway to modulate BK_Ca channels and subsequently cAMP-induced pulmonary vasodilatation.

high-conductance calcium- and voltage-activated potassium channel; protein kinase C isozymes; forskolin; cyclic adenosine 5'-monophosphate

Although multiple classes of K⁺ channels are expressed at varying densities in different vascular beds, the large-conductance, calcium- and voltage-activated potassium (BK_Ca) channel is the predominant K⁺ channel species in most arteries (24). Inhibition of BK_Ca channels produces membrane depolarization and subsequent vasoconstriction (30). The relationship between PKC and BK_Ca channel modulation in pulmonary vascular smooth muscle is relatively unknown, although scant evidence suggests PKC inhibition of the BK_Ca channel in other types of arterial smooth muscle. Minami et al. (22) observed that PKC blocks activation of the BK_Ca channel in coronary artery smooth muscle, Schubert and colleagues (33) reported that PKC reduces BK_Ca current in rat tail artery smooth muscle cells, and Barman (5) recently showed that K⁺ channels, including the BK_Ca channel, modulate the canine pulmonary vasoconstrictor response to PKC activation.

In light of these previous investigations, the present study was done to determine the effect of PKC activation by phorbol 12-myristate 13-acetate (PMA) and thymeleatoxin on cAMP-induced activation of BK_Ca channels by forskolin in pulmonary arterial smooth muscle cells (PASMC) of the fawn-hooded rat (FHR), an animal model of "idiopathic" pulmonary hypertension. PMA, an ester derivative of croton oil, and thymeleatoxin, a phorbol derivative, have been used to study PKC-induced pulmonary vasoconstriction (2, 6, 17). PMA is an activator of PKC (17), and thymeleatoxin has recently been shown to cause translocation and downregulation of multiple PKC isozymes (31). Specifically, the effect of PKC on cAMP-induced BK_Ca channel activity was investigated at the cellular and molecular level via the patch-clamp technique in FHR PASMC.

	METHODS

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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, Dept. of Medicine, Univ. 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. The 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: 6.7 mg/5 ml papain, 3.5 mg/5 ml cysteine, and 5.0 mg/ml BSA. After incubation, 5 ml of dissociation buffer containing 2 mg/ml of 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 removed and centrifuged at 1,200 rpm for 10 min, and 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.

Single-channel experiments. Single potassium channels were measured in cell-attached patches by filling the patch pipette (2–5 M) with normal Ringer solution and making a gigaohm seal on an intact cell. The solution in the recording chamber contained 140 mM KCl, 10 mM MgCl₂, 0.1 mM CaCl₂, 10 mM HEPES, and 30 mM glucose (pH 7.4). 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: 115 mM KCH₃SO₃, 26 mM KCl, 2 mM MgCl₂, 1 mM BAPTA, 0.47 mM CaCl₂ (pCa 7), 5.0 mM Mg-ATP, 0.1 mM GTP, and 20 mM HEPES (pH 7.4). The solution facing the external surface was the standard recording solution. 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 (1 ml) and perfused into a 2-ml recording chamber (Warner Instruments). Channel activity was quantified by calculating the single channel open probability (NP_o) over the duration of the entire data file, as described previously (10, 12, 39, 40).

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 (13).

Drugs. Thymeleatoxin, Gö-6976, Gö-6983, and rottlerin were purchased from Calbiochem. All other agents were purchased from Sigma Chemical.

Statistical analysis. All data were expressed as means ± SE. Statistical significance between two groups was evaluated by the 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 was considered to indicate a significant difference.

	RESULTS

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Identification of right ventricular hypertrophy. The RV/LV+S in FHR (0.46 ± 0.06; n = 9) vs. control SDR (0.29 ± 0.04, P < 0.05; n = 9) was indicative of right ventricular hypertrophy in FHR as reported in other studies (20, 23, 32, 37).

Identification of BK_Ca channels in PASMC from FHR. Recordings from cell-attached patches on vascular smooth muscle cells isolated from FHR pulmonary arteries demonstrated that forskolin, an activator of adenylyl cyclase, stimulated the activity of a large-conductance channel that carries outward current (Figs. 1 and 2). As shown in Fig. 1, forskolin increased BK_Ca channel activity in a time-dependent manner, with representative tracings shown at 15 min (Fig. 1B) and 30 min (Fig. 1C). When NP_o was calculated, on average, 10 µM forskolin increased channel activity by 100-fold at ± 40 mV (NP_o: control, 0.0006 ± 0.0003; forskolin, 0.0599 ± 0.0174; n = 15; P = 0.002). Figure 2 also shows a representative tracing of forskolin-induced BK_Ca channel activity. BK_Ca channel activity was inhibited by 1 mM tetraethylammonium (TEA; Fig. 2C, n = 5), a selective inhibitor of BK_Ca channels at this concentration (33). When patches were excised into the inside-out configuration, raising [Ca²⁺] to 0.1 mM at the cytoplasmic face of the patch membrane increased channel gating dramatically, indicating the BK_Ca channel viability of these cells. Furthermore, previous studies show that this channel has a microscopic conductance of 142 ± 5 pS in symmetric [K⁺] (7), which agrees with other studies reporting BK_Ca channel conductance in vascular smooth muscle (10, 12). Collectively, given this specific pharmacological and biophysical profile, we have identified this ion channel as the BK_Ca channel, which is highly expressed in vascular smooth muscle.

View larger version (19K): [in this window] [in a new window]   Fig. 1. cAMP-dependent vasodilators open large-conductance, calcium- and voltage-activated potassium (BKCa) channels in a time-dependent manner. A: control electrophysiological tracing. B: electrophysiological tracing showing the effect of 10 µM forskolin on BKCa channel activity for 15 min (n = 15). C: electrophysiological tracing showing the effect of 10 µM forskolin on BKCa channel activity for 30 min (n = 15). On average, 10 µM forskolin increased BKCa channel activity by 100-fold. Channel openings are upward deflections from baseline (closed) state.

View larger version (15K): [in this window] [in a new window]   Fig. 2. Forskolin activation of BKCa channels is blocked by tetraethylammonium (TEA). A: control electrophysiological tracing. B: electrophysiological tracing showing the effect of 10 µM forskolin on BKCa channel activity (n = 15). C: electrophysiological tracing showing the effect of TEA ions on forskolin-induced BKCa channel activity. When patches were excised into the inside-out configuration, BKCa channel activity was inhibited by 1 mM TEA (n = 5), a specific blocker of BKCa channels at this concentration. Channel openings are upward deflections from baseline (closed) state.

PKC activators inhibit cAMP-dependent stimulation of BK_Ca channels. Intracellular levels of cAMP were increased in PASMC with forskolin (stimulator of adenylyl cyclase), which opened BK_Ca channels as previously shown (Figs. 1 and 2). The ability of forskolin to open BK_Ca channels was inhibited by compounds that stimulate the activity of PKC. As illustrated in Fig. 3, A and B, application of PMA (100 nM) in the presence of forskolin almost completely (98%) reversed the forskolin-stimulated activity of BK_Ca channels in cell-attached patches (NP_o: forskolin, 0.0903 ± 0.0198; forskolin + PMA, 0.0014 ± 0.0014; n = 3; P < 0.001). In addition, 100 nM thymeleatoxin (which activates the , , , , and isozymes of PKC) also abolished forskolin-induced BK_Ca channel activity (NP_o: forskolin, 0.0216 ± 0.0051; forskolin + thymeleatoxin, 0.0007 ± 0.0005; n = 4; P < 0.001; Fig. 4, A and B). As shown in Fig. 5, pretreating cells with either thymeleatoxin or PMA (data not shown) prevented forskolin from opening BK_Ca channels. In contrast, thymeleatoxin (100 nM) was unable to inhibit calcium-stimulated BK_Ca channel activity in inside-out patches (Fig. 5C), thus excluding a potential direct inhibitor effect of this toxin on BK_Ca channel proteins. Also, incision of the patch into an inside-out configuration and raising cytoplasmic [Ca²⁺] to produce an immediate increase in BK_Ca channel activity in the presence of thymeleatoxin indicated the continued BK_Ca channel viability in these cells (Fig. 5C).

View larger version (24K): [in this window] [in a new window]   Fig. 3. PKC activators inhibit cAMP-dependent stimulation of BKCa channels. A: intracellular levels of cAMP were increased in pulmonary arterial smooth muscle cells (PASMC) with treatment by forskolin for 15 min (stimulator of adenylyl cyclase), which opens BKCa channels (n = 15). A and B: phorbol 12-myristate 13-acetate (PMA; 100 nM, 10 min) in the presence of forskolin almost completely reverses forskolin-stimulated activity of BKCa channels in cell-attached patches (n = 3). Channel openings are upward deflections from baseline (closed) state.

View larger version (19K): [in this window] [in a new window]   Fig. 4. PKC activators inhibit cAMP-dependent stimulation of BKCa channels. A: intracellular levels of cAMP were increased in PASMC with treatment by forskolin for 15 min (stimulator of adenylyl cyclase), which opens BKCa channels (n = 15). A and B: 100 nM thymeleatoxin treatment for 15 min inhibits forskolin-induced BKCa channel activity (n = 4). Channel openings are upward deflections from baseline (closed) state.

View larger version (20K): [in this window] [in a new window]   Fig. 5. PKC activation inhibits forskolin-induced BKCa channel activation. A: electrophysiological recording of thymeleatoxin treatment alone. B: pretreatment of PASMC with thymeleatoxin (15 min) inhibits forskolin activation of BKCa channels. C: subsequent incision of the patch into an inside-out configuration and raising cytoplasmic [Ca2+] produces an immediate increase in BKCa channel activity in the presence of both thymeleatoxin and forskolin, indicating the continued BKCa channel viability of these cells.

Inhibition of specific PKC isozymes reverses the effect of thymeleatoxin. To identify and establish a role for specific PKC isozyme inhibition of forskolin-stimulated gating of BK_Ca channels, we used 1 µM Gö-6983, a specific PKC isozyme inhibitor, that at this concentration inhibits the , , , , and isozymes of PKC. When PKC activity was inhibited with Gö-6983, thymeleatoxin was unable to prevent the activation of BK_Ca channels by forskolin. Specifically, as illustrated in Fig. 6A, pretreating cells with 1 µM Gö-6983 for 15 min in combination with 100 nM thymeleatoxin had no effect on BK_Ca channel activity (NP_o 0.0005 ± 0.0003; n = 4). However, subsequent addition of 10 µM forskolin in the presence of both Gö-6983 and thymeleatoxin stimulated channel activity by nearly 50-fold, as shown in Fig. 6, B and C (NP_o 0.0232 ± 0.0057; n = 4; P < 0.001). These data indicate that Gö-6983 reverses the inhibition of BK_Ca channel activity produced by thymeleatoxin.

View larger version (24K): [in this window] [in a new window]   Fig. 6. Inhibition of specific PKC isozymes reverses the effect of thymeleatoxin. A: pretreating cells with 1 µM Gö-6983 for 15 min in combination with 100 nM thymeleatoxin (15 min) has no effect on BKCa channel activity (n = 4). B and C: subsequent addition of 10 µM forskolin (15 min) in the presence of both Gö-6983 and thymeleatoxin stimulates BKCa channel activity by nearly 50-fold (n = 4). Channel openings are upward deflections from baseline (closed) state.

Subsequent experiments to gain further insight into the nature of the PKC isozyme(s) that inhibits the effect of forskolin on BK_Ca channel activity were done using 1 µM Gö-6976 and 1 µM rottlerin. At these concentrations, Gö-6976 has a higher degree of selectivity compared with Gö-6983, as it preferentially inhibits the , , and µ isozymes of PKC, and rottlerin, at this concentration, is a specific inhibitor of PKC-. As observed with Gö-6983, pretreating cells with 1 µM Gö-6976 for 15 min in the presence of 100 nM thymeleatoxin negated the inhibitory effect of thymeleatoxin-induced PKC activity (Fig. 7). Specifically, as shown in Fig. 7A, the combination of Gö-6976 and thymeleatoxin did not affect BK_Ca NP_o (0.0010 ± 0.0006; n = 4). However, subsequent treatment with forskolin in the continued presence of Gö-6976 and thymeleatoxin significantly enhanced BK_Ca channel activity above control levels (NP_o: 0.1622 ± 0.0513; n = 4; P = 0.02; Fig. 7, B and C). In contrast, 1 µM rottlerin did not reverse the thymeleatoxin inhibitory effect of forskolin on BK_Ca channels (Fig. 8, B and C), since NP_o recorded from cell-attached patches was not significantly different before or after addition of 10 µM forskolin (NP_o: rottlerin + thymeleatoxin, 0.0007 ± 0.0007; rottlerin + thymeleatoxin + forskolin, 0.0042 ± 0.0025; n = 4).

View larger version (27K): [in this window] [in a new window]   Fig. 7. Inhibition of specific PKC isozymes reverses the effect of thymeleatoxin. A: pretreating cells with 1 µM Gö-6976 for 15 min in combination with 100 nM thymeleatoxin (15 min) has no effect on BKCa channel activity (n = 4). B and C: subsequent addition of 10 µM forskolin for 15 min in the presence of both Gö-6976 and thymeleatoxin stimulates BKCa channel activity (n = 4).

View larger version (23K): [in this window] [in a new window]   Fig. 8. Inhibition of specific PKC isozymes does not reverse the effect of thymeleatoxin. A: pretreating cells with 1 µM rottlerin for 15 min in combination with 100 nM thymeleatoxin (15 min) has no effect on BKCa channel activity (n = 4). B and C: subsequent addition of 10 µM forskolin for 15 min in the presence of both rottlerin and thymeleatoxin has little effect on BKCa channel activity (n = 4). Channel openings are upward deflections from baseline (closed) state.

	DISCUSSION

TOP ABSTRACT METHODS RESULTS DISCUSSION REFERENCES

 
The study of pulmonary hypertension has been problematic because of the 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 (3). Recently, the FHR has been found to be an excellent animal model to study pulmonary hypertension (20, 32). The FHR strain is characterized by platelet abnormalities and systemic hypertension and has been widely used to study genetic risk factors for the development of idiopathic pulmonary hypertension (18, 32). Studies show that the platelet storage disease is not involved in the pathogenesis of pulmonary hypertension (3, 18), and the FHR strain develops significant pulmonary hypertension, which is not associated with differences in blood gas tension, polycythemia, or parenchymal lung disease (41). Unlike other rat strains, the FHR develops severe pulmonary hypertension that is age dependent (18), a phenomenon accelerated under mild hypoxic conditions (32). Evidence also suggests that the genetic locus for the hypertensive condition is PH1 on chromosome 1 (35).

The results of the present study indicate that specific PKC isozyme activation inhibits BK_Ca channel activity in PASMC of pulmonary hypertensive FHR (as defined by right ventricular hypertrophy) by the cAMP-dependent vasodilator forskolin, which suggests a unique signaling pathway to modulate BK_Ca channels in pulmonary arterial smooth muscle. The relationship between PKC and BK_Ca channel modulation in pulmonary arterial smooth muscle is relatively unknown, although evidence suggests PKC inhibition of the BK_Ca channel in other types of arterial smooth muscle. Minami et al. (22) observed that PKC blocks activation of the BK_Ca channel in coronary artery smooth muscle, and Schubert and colleagues (33) recently reported that PKC reduces BK_Ca current in rat tail artery smooth muscle cells. Barman (5) recently showed that K⁺ channels, including the BK_Ca channel, modulate the canine pulmonary vasoconstrictor response to PKC activation, and Taguchi et al. (36) observed that PKC modulates BK_Ca channels in cultured rat mesenteric artery smooth muscle cells. In addition, Boland and Jackson (9) reported that voltage-gated Kv current blocked by PMA (an activator of PKC) was reversed by PKC inhibition. PKC inhibits the delayed rectifier current K⁺ channels in rabbit vascular smooth muscle cells (1), and inhibition of the voltage-gated Kv current in rat intrapulmonary arterial myocytes by endothelin-1 occurs via activation of the PKC pathway (34). PKC represents an important component of a signal transduction pathway that regulates vascular smooth muscle contraction. The role of PKC in vascular smooth muscle contraction has been investigated using phorbol esters such as PMA and the phorbol derivative thymeleatoxin (2, 16, 17). Phorbol esters appear to exert their effect through the activation of the enzyme PKC by substituting for DAG (11). DAG is thought to be one of the endogenous lipids that activates PKC by increasing the affinity of the enzyme for Ca²⁺ and phosphatidylserine at normal Ca²⁺ levels (25).

In the present study, both PMA and thymeleatoxin blocked activation of the BK_Ca channel by forskolin. Thymeleatoxin is a diterpene derivative of mezerein that has been reported to selectively activate the , , , , and PKC isozymes (31). The inhibitory response by thymeleatoxin on forskolin was blocked by Gö-6983, which selectively blocks the , , , , and PKC isozymes and Gö-6976, which selectively inhibits PKC-, PKC-, and PKC-µ at the concentrations used in this study. It is of interest that the BK_Ca channel activation (open probability) response to forskolin appeared to be somewhat greater in cells blocked with Gö-6976 than in cells treated with Gö-6983. The reason for this observation is unknown but may be related to the degree of response of individual PASMC to forskolin, the differential blockade of the PKC isozymes, or secondary nonspecific pharmacological effects of the PKC isozyme inhibitors at these concentrations. In contrast, the BK_Ca channel activation response to forskolin was still blocked by thymeleatoxin in the presence of the PKC isozyme inhibitor rottlerin, a compound isolated from Mallotus philippinensis, which selectively inhibits PKC- at the concentration used in this study (14). Although rottlerin also exhibits selectivity for calmodulin (CaM) kinase III at low concentrations (IC₅₀ = 5.3 µM), the concentration used in this study (1 µM) would suggest that CaM kinase III is minimally affected by rottlerin. Collectively, on the basis of the pharmacological selectivity of the PKC isozyme inhibitors, these results indicate that activation of specific PKC isozymes (, , and µ) may modulate cAMP-induced activation of the BK_Ca channel in pulmonary arterial smooth muscle.

K⁺ channel activity is the main determinant of membrane potential, and associated K⁺ efflux causes hyperpolarization, which inhibits voltage-gated Ca²⁺ channels and promotes vascular relaxation. Although multiple classes of K⁺ channels are expressed at varying densities in different vascular beds, the BK_Ca channel is the predominant K⁺ channel species in most arteries (24). BK_Ca channels are activated by submicromolar [Ca²⁺]_i and blocked by external charybdotoxin, iberiotoxin, and TEA ions (24, 25). The biophysical profile of the BK_Ca 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 (10, 24, 39). Because of their large conductance and high density, these channels influence resting membrane potential and provide an important repolarizing negative feedback mechanism. Inhibition of BK_Ca channels produces membrane depolarization and subsequent vasoconstriction (28). It should be noted that as the findings from this study are reported in an animal model of pulmonary hypertension, the nature of BK_Ca channel modulation in pulmonary vascular smooth muscle may be specific to the physiological conditions present.

In summary, the results of this study indicate that PKC activation by PMA and thymeleatoxin inhibits cAMP-induced BK_Ca channel activity in FHR PASMC, which is reversed by the specific PKC isozyme inhibitors Gö-6983 and Gö-6976 but not by rottlerin. Thus this study reveals that specific PKC isozymes may be involved in BK_Ca channel inhibition and suggests an important vasoconstrictor signaling mechanism in pulmonary arterial smooth muscle.

	ACKNOWLEDGMENTS

 
The authors thank Louise Meadows for excellent technical assistance.

GRANTS

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 American Heart Association Grant 9950179N (to R. E. White).

	FOOTNOTES

 

Address for reprint requests and other correspondence: Scott A. Barman, Dept. of Pharmacology and Toxicology, Medical College of Georgia, Augusta, Georgia 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.

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