PKC activates BKCa channels in rat pulmonary arterial smooth muscle via cGMP-dependent protein kinase
Scott A. Barman,
Shu Zhu, and
Richard E. White
Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, Georgia 30912
Submitted 28 July 2003
; accepted in final form 9 February 2004
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ABSTRACT
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Normally, signaling mechanisms that activate large-conductance, calcium- and voltage-activated potassium (BKCa) channels in pulmonary vascular smooth muscle cause pulmonary vasodilatation. BKCa-channel modulation is important in the regulation of pulmonary arterial pressure, and inhibition (decrease in the opening probability) of the BKCa 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 BKCa-channel activity in pulmonary vascular smooth muscle. Accordingly, studies were done to determine the effect of PKC on BKCa-channel activity using patch-clamp studies in pulmonary arterial smooth muscle cells (PASMCs) of the Sprague-Dawley rat. The PKC activators phorbol myristate acetate (PMA) and thymeleatoxin opened BKCa channels in single Sprague-Dawley rat PASMC. The activator response to both PMA and thymeleatoxin on BKCa-channel activity was blocked by Gö-6983, which selectively blocks PKC-
, -
, -
, and -
, and by rottlerin, which selectively inhibits PKC-
. In addition, the specific cyclic GMP-dependent protein kinase antagonist KT-5823 blocked the responses to PMA and thymelatoxin, whereas the specific cyclic AMP-dependent protein kinase blocker KT-5720 had no effect. In isolated pulmonary arterial vessels, both PMA and forskolin caused vasodilatation, which was inhibited by KT-5823, Gö-6983, or the BKCa-channel blocker tetraethylammonium. The results of this study indicate that activation of specific PKC isozymes increases BKCa-channel activity in Sprague-Dawley rat PASMC via cyclic GMP-dependent protein kinase, which suggests a unique signaling mechanism for vasodilatation.
high-conductance calcium-and voltage-activated potassium channel; pulmonary arterial smooth muscle; protein kinase C isozymes; cyclic GMP-dependent protein kinase; protein kinase G
IN THE PULMONARY VASCULATURE, protein kinase C (PKC) is a key regulatory enzyme involved in the signal transduction of several cellular functions, including vascular smooth muscle growth and contractility (4, 7, 20). PKC consists of a family of serine/threonine kinases with at least 12 members. On the basis of their structures, the PKC family can be divided into three major subclasses: 1) the classical group, comprising the
,
I,
II, and
isozymes that are Ca2+ dependent and diacylglycerol (DAG) sensitive; 2) the novel group, comprising the
,
,
, and
isozymes, which are Ca2+ independent and DAG sensitive; and 3) the atypical group isozymes, consisting of the
,
,
, and µ isozymes, which are Ca2+ independent and DAG insensitive (27, 31, 37). Numerous PKC isozymes are expressed in vascular smooth muscle (
,
,
,
, and
), which may be dependent on species, type of vessel, and age of the vessel (17, 22, 24, 28).
Although multiple classes of K+ channels are expressed at varying densities in different vascular beds, the voltage- and Ca2+-activated K+ (BKCa) channel is the predominant K+-channel species in most arteries (26). Inhibition of BKCa channels produces membrane depolarization and subsequent vasoconstriction (30). The relationship between PKC and large conductance BKCa-channel modulation in pulmonary vascular smooth muscle is relatively unknown, although evidence suggests that PKC inhibits the activation of BKCa channels in other types of arterial smooth muscle. Minami et al. (25) observed that PKC blocks activation of the BKCa channel in coronary artery smooth muscle, Schubert et al. (34) reported that PKC reduces BKCa current in rat tail artery smooth muscle cells, and Barman (5) recently showed that K+ channels, including the BKCa 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 and mechanism(s) of PKC activation by phorbol myristate acetate (PMA) and thymeleatoxin on BKCa channels in pulmonary arterial smooth muscle cells (PASMCs) of the Sprague-Dawley rat (SDR). PMA, an ester derivative of croton oil, and thymeleatoxin, a phorbol derivative, have been used to study PKC-induced pulmonary vasoconstriction (2, 21). PMA is an activator of PKC (21), and thymeleatoxin has recently been shown to cause translocation and downregulation of multiple PKC isozymes (33). Specifically, the effect of PKC on BKCa-channel activity was investigated at the cellular and molecular level via the patch-clamp technique in SDR PASMC.
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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. Adult male SDR, weighing 250300 g, were purchased from a commercial vendor (Harlan), housed under temperature-controlled conditions (2123°C), and maintained on standard rat chow. All animals were allowed free access to food and water and exposed to a 12:12-h light-dark cycle.
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: papain (6.7 mg/5 ml), cysteine (3.5 mg/5 ml), and bovine serum albumin (5.0 mg/ml). After incubation, 5.0 ml of dissociation buffer, which contained 2.0 mg/ml bovine serum albumin, 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 12 h after cell dissociation.
Single-channel experiments.
Single K+ channels were measured in cell-attached patches by filling the patch pipette (25 M
) with normal Ringer 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 7.4). This manipulation allowed precise regulation of the patch-membrane voltage by the patch-clamp amplifier and yielded more accurate current measurements. In experiments measuring K+-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 N'-tetraacetic acid, 0.47 CaCl2 (pCa 7), 5.0 Mg-ATP, 0.1 GTP, and 20 HEPES (pH 7.4). The solution facing the external surface was the standard recording solution. Voltage-clamp and voltage-pulse generation were 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 2.0-ml recording chamber (Warner Instruments). Channel activity was quantified by calculating the single open-channel probability (NPo), as described previously (9, 12, 38, 39).
Isolated vessel preparation.
Male SDR were anesthetized with pentobarbital sodium (50 mg/kg ip) and intubated, and a midline incision was made to immediately excise the lung lobes. Extrapulmonary arteries (1- to 2-mm diameter) were dissected microscopically from the isolated lobes, cleaned of perivascular tissue, and cut into 2-mm rings. The endothelial cell layer was removed as previously described (9), and to confirm that the vessels were denuded, we measured the inability of acetylcholine to produce endothelium-dependent relaxation. Vessels not relaxing to acetylcholine after being preconstricted with 10 µM phenylephrine was pharmacological evidence that the endothelium had been removed. Isometric tension was measured and recorded with a Radnoti digital force transducer organ bath system (model 159920). The tissue was bathed in a Krebs solution of the following composition (in mM): 120 NaCl, 4.8 KCl, 18 NaHCO3, 1.2 MgCl, 2.5 CaCl, and 11 glucose. The solution was then oxygenated with 95% O2-5% CO2 and maintained at 37°C. The pulmonary vessels were equilibrated for 90 min under an optimal resting tension of 0.5 g, which was the tension where the maximum contractile response to 10 µM phenylephrine was achieved. After the initial equilibration period, the vessels were exposed to a maximally effective concentration of phenylephrine. After a stable, plateau tension developed, phenylephrine was removed by several washes, and a 30-min period was allowed for reequilibration. This procedure was repeated until the steady-state tension level remained constant. Isometric tension measurements were normalized by expressing force developed per cross-sectional area (g/mm2), which takes into account the variation in size of the vessels.
Experimental protocols.
Group 1 (n = 4) consisted of treating SDR PASMC for 30 min with PMA (100 nM). In group 2 (n = 4), PASMCs were treated with 100 nM thymeleatoxin for 30 min. For groups 3 (n = 3) and 4 (n = 6), PASMC were pretreated with 1 µM Gö-6983 for 15 min before being treated with PMA (group 3) or thymeleatoxin (group 4). In group 5 (n = 4), cells were pretreated with 1 µM rottlerin for 15 min before thymeleatoxin was added. In group 6 (n = 4), PASMCs were pretreated with 300 nM KT-5823 for 30 min before PMA addition, whereas group 7 (n = 4) cells were pretreated with 300 nM KT-5720 for 30 min before PMA was added. Groups 810 consisted of isolated pulmonary arterial vessels treated with either 100 nM PMA alone (group 8; n = 4), or after pretreatment with either 300 nM KT-5823 (group 9; n = 4) or 1 mM tetraethylammonium (TEA; group 10; n = 4) for 30 min. In groups 1114 (n = 4 for each), isolated vessels were treated with either 10 µM forskolin alone (group 11) or after pretreatment with 300 nM KT-5823 (group 12), 1 mM TEA (group 13), or 1 µM Gö-6983 (group 14) for 30 min.
Drugs.
Thymeleatoxin, Gö-6983, and rottlerin were purchased from Calbiochem. All other agents were purchased from Sigma Chemical.
Statistical analysis.
All data are expressed as means ± SE. Statistical significance between two groups was evaluated by Student's t-test for paired data. Comparison between multiple groups was made by using one-way ANOVA for multiple comparisons. A P value of <0.05 indicated a significant difference.
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RESULTS
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Identification of BKCa channels in PASMC from SDR.
Recordings from cell-attached patches on vascular smooth muscle cells isolated from SDR pulmonary arteries demonstrated the activity of a large conductance channel that carried outward current (Fig. 1). Single-channel activity in membrane patches of myocytes from pulmonary arteries was dominated by a high-amplitude channel conducting outward current at all test potentials. In cell-attached patches, channel activity was minimal; however, as seen in Fig. 1A, channel activity increased dramatically after the patch was excised into the inside-out configuration, with the cytoplasmic surface of the patch exposed to a Ca2+ concentration of 100 µM. In addition, single-channel, current-voltage relationships yield a calculated channel conductance of 142.4 ± 3 pS (n = 8; Fig. 1B), which agrees with previous studies on the identification of BKCa-channel conductance in pulmonary arterial smooth muscle (6) and systemic vascular smooth muscle (9, 12). In light of this pharmacological and biophysical characterization, we have identified this protein as the large-conductance BKCa channel that is expressed at high density in vascular smooth muscle cells.

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Fig. 1. Ca2+- and voltage-activated K+ (BKCa) channels are expressed in Sprague-Dawley rat (SDR) pulmonary arteries. A: recordings from the same membrane patch in the cell-attached configuration (100 nM Ca2+; +40 mV; top) and after excision into the inside-out configuration (100 µM Ca2+; +40mV; bottom). Upward deflections are channel openings from the channel closed state (dotted line). B: average current-voltage relationship (mean ± SE) for single-activity in inside-out patches in symmetrical (140 mM) K+ (n = 8).
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PKC activators stimulate BKCa-channel activity.
Intracellular activity of PKC was stimulated by treating cells with either PMA or thymeleatoxin (Figs. 2 and 3). As seen in Fig. 2, treating PASMC with 100 nM PMA (30 min) raised channel NPo from
0 to 0.1167 ± 0.0414 (n = 4, +40 mV; P < 0.05; Fig. 2B). Similarly, 100 nM thymeleatoxin (which, at this concentration, activates the
,
,
,
, and
isozymes of PKC) increased BKCa-channel NPo by over 1,000-fold (NPo: control, 0.0002 ± 0.0001; thymeleatoxin, 0.2530 ± 0.0400; n = 3; +40 mV; P < 0.05; Fig. 3B). In contrast, we did not observe increased activity of any other membrane channels after PKC stimulation.

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Fig. 2. Protein kinase C (PKC) activators stimulate BKCa channels in SDR pulmonary arterial smooth muscle cells (PASMCs). A: phorbol 12-myristate 13-acetate (PMA; 100 nM) induces BKCa-channel activity in the cell-attached patch configuration (100 nM Ca2+; +40 mV; n = 4). Channel openings are upward deflections from baseline (closed) state. B: treating cells with 100 nM PMA for 30 min activates BKCa channels as measured by open-channel probability (NPo) in the cell-attached patch configuration (100 nM Ca2+; +40 mV; n = 4).
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Fig. 3. PKC activators stimulate BKCa channels in SDR PASMC. A: thymeleatoxin (100 nM) induces BKCa-channel activity in cell-attached patch configuration (100 nM Ca2+; +40mV; n = 3). Channel openings are upward deflections from baseline (closed) state. B: treating cells with 100 nM thymeleatoxin for 30 min activates BKCa channels, as measured by NPo in the cell-attached patch configuration (100 nM Ca2+; +40 mV; n = 3).
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Inhibition of specific PKC isozymes prevents the stimulatory effect of PKC.
To identify and establish a role for specific PKC isozyme inhibition of PMA- or thymeleatoxin-stimulated gating of BKCa channels, we used 1 µM Gö-6983, a specific PKC isozyme inhibitor, which at this concentration inhibits the
,
,
,
and
isozymes of PKC. As observed in Figs. 4 and 5, the stimulatory effect of either PMA (Fig. 4) or thymeleatoxin (Fig. 5) was antagonized by pretreating cells with 1 µM Gö-6983. Specifically, in the presence of Gö-6983, 100 nM PMA did not stimulate BKCa-channel gating (NPo: Gö-6983 alone,
0; Gö-6983 with PMA, 0.0012 ± 0.0012; n = 3; P > 0.05; Fig. 4B). Gö-6983 also prevented the ability of 100 nM thymeleatoxin to enhance channel activity (NPo: Gö-6983 alone, 0.001 ± 0.0001; Gö-6983 with thymeleatoxin, 0.0015 ± 0.0011; n = 6; P > 0.05; Fig. 5B). In both cases, excision of the patch into the inside-out configuration (100 µM intracellular Ca2+ concentration) enhanced channel activity, indicating the viability of BKCa channels in these cells. Therefore, Gö-6983 had no direct channel-blocking activity but was most likely working via inhibition of PKC. In addition to antagonism by Gö-6983, the stimulatory effect of PKC activity in BKCa-channel gating was also inhibited by 1 µM rottlerin, a PKC antagonist, which at this concentration exhibits selectivity for the PKC-
isoform (16). Specifically, in the presence of rotterlin, thymeleatoxin was unable to increase BKCa activity significantly (NPo: rotterlin alone,
0; rotterlin with thymeleatoxin, 0.0046 ± 0.0029; n = 4; P > 0.05; Fig. 6B).

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Fig. 4. Inhibition of specific PKC isozymes blocks the effect of PMA. A: pretreating cells with 1 µM Gö-6983 for 15 min in combination with 100 nM PMA inhibits BKCa-channel activity. Channel openings are upward deflections from baseline (closed) state (100 nM Ca2+; +40 mV; n = 3). B: pretreating cells with 1 µM Gö-6983 for 15 min in combination with 100 nM PMA inhibits BKCa-channel activity, as measured by NPo in the cell-attached patch configuration (100 nM Ca2+; +40 mV; n = 3).
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Fig. 5. Inhibition of specific PKC isozymes blocks the effect of thymeleatoxin. A: pretreating cells with 1 µM Gö-6983 for 15 min in combination with 100 nM thymeleatoxin inhibits BKCa-channel activity. Channel openings are upward deflections from baseline (closed) state (100 nM Ca2+; +40 mV; n = 6). B: pretreating cells with 1 µM Gö-6983 for 15 min in combination with 100 nM thymeleatoxin inhibits BKCa-channel activity as measured by NPo in the cell-attached patch configuration (100 nM Ca2+; +40 mV; n = 6).
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Fig. 6. Inhibition of specific PKC isozymes blocks the effect of thymeleatoxin. A: pretreating cells with 1 µM rottlerin for 15 min has no effect on BKCa-channel activity in the cell-attached patch configuration (100 nM Ca2+; +40 mV; n = 4). Subsequent addition of 100 nM thymeleatoxin in the presence of rottlerin does not stimulate BKCa-channel activity in the cell-attached patch configuration (100 nM Ca2+; +40mV; n = 4). Channel openings are upward deflections from baseline (closed) state. B: pretreating cells with 1 µM rottlerin for 15 min in combination with 100 nM thymeleatoxin inhibits BKCa-channel activity, as measured by NPo in the cell-attached patch configuration (100 nM Ca2+; +40 mV; n = 4).
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Inhibition of cyclic GMP-dependent kinase activity blocks the effect of PKC.
To determine whether PKC-stimulated BKCa-channel activity involved cyclic nucleotide protein kinase activation, experiments were done with specific inhibitors of cyclic GMP (cGMP)-dependent protein kinase (PKG) and cyclic AMP-dependent protein kinase (PKA). As seen in Fig. 7, an inhibitor of PKG antagonized the effect of PKC stimulation on BKCa-channel activity. Specifically, pretreatment of PASMC with 300 nM KT-5823, a specific PKG antagonist, prevented PMA from opening BKCa channels in cell-attached patches (NPo: KT-5823 alone, 0.0007 ± 0.0007; KT-5823 with PMA, 0.0012 ± 0.0012; n = 4; P > 0.05; Fig. 7B). In contrast, inhibition of PKA with 300 nM KT-5720 had no effect on PMA-stimulated BKCa-channel activity in cell-attached patches. In the presence of 300 nM KT-5720, 100 nM PMA increased channel NPo from an average of 0.0006 ± 0.0003 to 0.3866 ± 0.0590 (n = 4; P < 0.05; Fig. 8B).

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Fig. 7. PKC opens BKCa channels via activation of cyclic GMP-dependent protein kinase (PKG) in SDR PASMC. A: effect of KT-5823 (300 nM), a specific inhibitor of PKG, on PMA. Pretreatment with KT-5823 for 30 min before PMA treatment prevents activation of BKCa channels by PMA in the cell-attached patch configuration (100 nM Ca2+; +40 mV; n = 4). Channel openings are upward deflections from baseline (closed) state. B: pretreating cells with 300 nM KT-5823 for 15 min in combination with 100 nM PMA inhibits BKCa-channel activity, as measured by NPo in the cell-attached patch configuration (100 nM Ca2+; +40 mV; n = 4).
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Fig. 8. PKC opens BKCa channels independent of PKA activation of in SDR PASMC. A: effect of KT-5720 (300 nM), a specific inhibitor of cyclic AMP-dependent protein kinase (PKA), on PMA. Pretreatment with 300 nM KT-5720 has no effect on PMA-induced stimulation of BKCa-channel activity in the cell-attached patch configuration (100 nM Ca2+; +40 mV; n = 4). Channel openings are upward deflections from baseline (closed) state. B: pretreating cells with 300 nM KT-5823 for 15 min in combination with 100 nM PMA has no effect on BKCa-channel activity, as measured by NPo in the cell-attached patch configuration (100 nM Ca2+; +40 mV; n = 4).
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Inhibition of cGMP-dependent kinase and BKCa activity blocks the effect of PKC in isolated vessels.
To determine whether the effect of PKC on isolated pulmonary arterial vessels involved PKG and BKCa-channel activation, experiments were done using specific inhibitors of PKG and BKCa channels. As seen in Fig. 9, 100 nM PMA decreased tension in phenylephrine-preconstricted vessels by
30%, which was blocked by either 300 nM KT-5823 (n = 4; P < 0.05) or 1 mM TEA ions (n = 4; P < 0.05), which at this concentration is a specific BKCa-channel inhibitor (34), indicating that both PKG and BKCa-channel activation are involved in the vasodilatory response to PMA. Figure 10 shows the effect of the adenylate cyclase-activator forskolin on isolated pulmonary arterial vessel tension. Forskolin decreased vessel tension over 60%, which was inhibited by KT-5823, TEA, and Gö-6983. These data indicate that an increase in cyclic AMP (via adenylate cyclase) causes pulmonary vasodilatation through BKCa-channel activation by a signaling mechanism involving either PKG or PKC.

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Fig. 9. PKC activation causes partial vasodilation of pulmonary arterial vessels via activation of PKG and BKCa channels in SDR as pretreatment with either KT-5823 (300 nM), a specific inhibitor of PKG (n = 4), or as the specific BKCa-channel-inhibitor TEA (1 mM; n = 4) for 30 min to block the response to PMA (100 nM). Vessels were preconstricted with 10 µM phenylephrine. Values are means ± SE. *Significantly different from PMA (P < 0.05).
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Fig. 10. Forskolin (FSK) causes vasodilation of pulmonary arterial vessels via activation of PKG, PKC, and BKCa channels in SDR as pretreatment with KT-5823 (300 nM; n = 4), Gö-6893 (1 µM; n = 4), or TEA (1 mM; n = 4) for 30 min and blocks the response to FSK (10 µM). Vessels were preconstricted with 10 µM phenylephrine. Values are means + SE. *Significantly different from FSK (P < 0.05).
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DISCUSSION
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The results of the present study indicate that specific PKC isozymes activate BKCa channels in SDR PASMC, which, to our knowledge, is the first report of PKC being an activator of BKCa channels in vascular smooth muscle. The relationship between PKC and BKCa-channel modulation in pulmonary arterial smooth muscle is relatively unknown, although evidence suggests PKC inhibition of the BKCa and other types of K+ channels in arterial smooth muscle. Minami et al. (25) observed that PKC blocks activation of the BKCa channel in coronary artery smooth muscle, and Schubert and colleagues (34) recently reported that PKC reduces BKCa current in rat tail artery smooth muscle cells. Barman (5) recently showed that K+ channels, including the BKCa channel, modulate the canine pulmonary vasoconstrictor response to PKC activation, and Taguchi et al. (36) observed that PKC modulates BKCa channels in cultured rat mesenteric artery smooth muscle cells. In addition, Boland and Jackson (8) reported that voltage-gated K+ 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 K+ current in rat intrapulmonary arterial myocytes by endothelin-1 occurs via activation of the PKC pathway (35).
PKC represents an important component of a signal transduction pathway that regulates vascular smooth muscle vasoreactivity. The role of PKC in vascular smooth muscle contraction has been investigated using phorbol esters, such as PMA, and the phorbol derivative thymeleatoxin (2, 21). Phorbol esters appear to exert their effect through the activation of the enzyme PKC by substituting for DAG (10, 31). 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 (27). In the present study, both PMA and thymeleatoxin activated BKCa channels in SDR PASMC. Thymeleatoxin is a diterpene derivative of mezerein that has been reported to selectively activate PKC-
, -
, -
, -
, and -
(33). The activator response by PMA and thymeleatoxin was blocked by Gö-6983, which selectively blocks PKC-
, -
, -
, -
, and -
at the concentration used in this study (15). In addition, the BKCa-channel activation response to thymeleatoxin was blocked 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 (16). Collectively, based on the pharmacological selectivity of the PKC isozyme inhibitors, these results indicate that specific PKC isozymes (
in particular) activate BKCa channels in rat pulmonary arterial smooth muscle.
Another important finding in this study is that PKC appeared to activate BKCa channels in SDR PASMC via PKG but not through PKA. In vascular smooth muscle, cGMP has been reported to elicit vasodilatation by activation of PKG (9, 23). In the pulmonary circulation, cGMP and cyclic AMP have been implicated as mediators of smooth muscle vasodilation (9, 14, 18, 19), and in ovine pulmonary vascular smooth muscle it has recently been reported that pulmonary arteries are more sensitive to relaxation induced by cGMP, which involves activation of PKG (11). In addition, Gao et al. (14) observed PKG-mediated relaxation of newborn ovine pulmonary veins.
At the present time, the physiological mechanism(s) of PKC-induced activation of BKCa channels in rat pulmonary arterial smooth muscle is unknown. However, this phenomenon may represent a novel mechanism of vasorelaxation in vascular smooth muscle, because results from this study showed that both PMA and forskolin appeared to partially vasodilate pulmonary arterial vessels, which was blocked by specific inhibitors of PKG and BKCa channels. In addition, Dimitropoulou et al. (13) recently reported a novel mechanism of angiotensin II-induced relaxation of rat mesenteric microvessels, whereby angiotensin II type 2 receptor simulation activated BKCa channels, which appeared to be mediated via PKC. In the pulmonary circulation, PKC activates voltage-dependent Ca2+ channels (2, 29), which can increase the influx of Ca2+. An increase in Ca2+ can activate nitric oxide synthase type I and type III to generate PKG via cGMP, which would activate BKCa channels in vascular smooth muscle. A second possible mechanism may involve colocalization of the voltage-dependent Ca2+ channels with BKCa channels, whereby an increase in Ca2+ flux through activated Ca2+ channels would subsequently open BKCa channels in vascular smooth muscle (26). Further studies are currently in progress to clarify the role of PKC signaling on BKCa-channel physiology.
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 BKCa channel is the predominant K+-channel species in most arteries (26). BKCa channels are activated by submicromolar intracellular Ca2+ concentrations and blocked by external charybdotoxin, iberiotoxin, and TEA ions (26). The biophysical profile of the BKCa channel is that it is a large conducting channel (100150 pS in physiological K+ gradients), which is both Ca2+ and voltage dependent for activation (9, 26, 38). Because of their large conductance and high density, these channels influence resting membrane potential and provide an important repolarizing negative feedback mechanism. Inhibition of BKCa channels produces membrane depolarization and subsequent vasoconstriction (30).
In summary, the results of this study indicate that PKC activation by PMA and thymeleatoxin induces BKCa-channel activity in SDR PASMC, which is blocked by the specific PKC isozyme inhibitors Gö-6983 and rottlerin. In addition, these results suggest that PKC activates BKCa channels via PKG but not through PKA. Thus this study reveals that specific PKC isozymes may be involved in BKCa-channel activation and reveals a unique signaling mechanism to activate BKCa channels in pulmonary arterial smooth muscle.
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GRANTS
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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).
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ACKNOWLEDGMENTS
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The authors thank Louise Meadows for excellent technical assistance.
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FOOTNOTES
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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.
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REFERENCES
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- Aiello EA, Clement-Chomienne O, Sontag DP, Walsh MP, and Cole WC. Protein kinase C inhibits delayed rectifier K+ current in rabbit vascular smooth muscle. Am J Physiol Heart Circ Physiol 271: H109H119, 1996.[Abstract/Free Full Text]
- Allison RC, Marble KT, Hernandez EM, Townsley MI, and Taylor AE. Attenuation of permeability lung injury after phorbol myristate acetate by verapamil and OKY-046. Am Rev Respir Dis 134: 93100, 1986.[ISI][Medline]
- Assender JW, Irenius E, and Fredholm BB. Endothelin-1 causes a prolonged protein kinase C activation and acts as a co-mitogen in vascular smooth muscle cells. Acta Physiol Scand 157: 451460, 1996.[ISI][Medline]
- Barman SA. Potassium channels modulate canine pulmonary vasoreactivity to protein kinase C activation. Am J Physiol Lung Cell Mol Physiol 277: L558L565, 1999.[Abstract/Free Full Text]
- Barman SA, Zhu S, Han G, and White RE. cAMP activates BKca channels in pulmonary arterial smooth muscle via cGMP-dependent protein kinase. Am J Physiol Lung Cell Mol Physiol 284: L1004L1011, 2003.[Abstract/Free Full Text]
- Bobik A, Grooms A, Millar JA, Mitchell A, and Grinpukel S. Growth factor activity of endothelin on vascular smooth muscle. Am J Physiol Cell Physiol 258: C408C415, 1990.[Abstract/Free Full Text]
- Boland LM and Jackson KA. Protein kinase C inhibits Kv1.1 potassium channel function. Am J Physiol Cell Physiol 277: C100C110, 1999.[Abstract/Free Full Text]
- Carrier GO, Fuchs LC, Winecoff AP, Giulumar AD, and White RE. Nitrovasodilators relax mesenteric microvessels by cGMP-induced stimulation of Ca-activated K channels. Am J Physiol Heart Circ Physiol 273: H76H84, 1997.[Abstract/Free Full Text]
- Castagna M, Takai Y, Kaibuchi K, Sano K, Kikkawa U, and Nishizuka Y. Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters. J Biol Chem 257: 78477851, 1982.[Abstract/Free Full Text]
- Dhanakoti SN, Gao Y, Nguyen MQ, and Raj JU. Involvement of cGMP-dependent protein kinase in the relaxation of ovine pulmonary arteries to cGMP and cAMP. J Appl Physiol 88: 16371642, 2000.[Abstract/Free Full Text]
- Dimitropoulou C, Han G, Miller AW, Molero M, Fuchs LC, White RE, and Carrier GO. Potassium (BkCa) currents are reduced in microvascular smooth muscle cells from insulin-resistant rats. Am J Physiol Heart Circ Physiol 282: H908H917, 2002.[Abstract/Free Full Text]
- Dimitropoulou C, White RE, Fuchs LC, Zhang HF, Catravas JD, and Carrier GO. Novel mechanism of angiotensin II induced relaxation of rat mesenteric microvessels: activation of Ang II type 2 receptor opens BKCa channels via protein kinase C (PKC). Circulation 100: 18, I-846, 1999.
- Gao Y, Dhanakoti S, Tolsa JF, and Raj JU. Role of protein kinase G in nitric oxide- and cGMP-induced relaxation of newborn ovine pulmonary veins. J Appl Physiol 87: 993998, 1999.[Abstract/Free Full Text]
- Gschwendt M, Dieterich S, Rennecke J, Kittstein W, Mueller HJ, and Johannes FJ. Inhibition of protein kinase C µ by various inhibitors. Differentiation from protein kinase c isozymes. FEBS Lett 392: 7780, 1996.[CrossRef][ISI][Medline]
- Gschwendt M, Muller HJ, Kielbassa K, Zang R, Kittstein W, Rincke G, and Marks F. Rottlerin, a novel protein kinase inhibitor. Biochem Biophys Res Commun 199: 9398, 1994.[CrossRef][ISI][Medline]
- Haller H, Lindschau C, Quass P, Distler A, and Luft FC. Differentiation of vascular smooth muscle cells and the regulation of PKC
. Circ Res 76: 2129, 1995.[Abstract/Free Full Text]
- Haynes J Jr, Kithas PA, Taylor AE, and Strada SJ. Selective inhibition of cGMP-inhibitable cAMP phosphodiesterase decreases pulmonary vasoreactivity. Am J Physiol Heart Circ Physiol 261: H487H492, 1991.[Abstract/Free Full Text]
- Haynes JH Jr, Robinson J, Saunders L, Taylor AE, and Strada SJ. Role of cAMP-dependent protein kinase in cAMP-mediated vasodilation. Am J Physiol Heart Circ Physiol 262: H511H516, 1992.[Abstract/Free Full Text]
- Hug H and Sarre TF. Protein kinase C isoenzymes: divergence in signal transduction? Biochem J 291: 329343, 1993.[ISI][Medline]
- Johnson A. PMA-induced pulmonary edema: mechanisms of the vasoactive response. J Appl Physiol 65: 23022312, 1988.[Abstract/Free Full Text]
- Khalil RA, Lajoie C, and Morgan KG. Ca2+ independent isoforms of PKC differentially translocate in smooth muscle. Am J Physiol Cell Physiol 263: C714C719, 1992.[Abstract/Free Full Text]
- Lincoln TM and Cornwell TL. Intracellular cyclic GMP receptor proteins. FASEB J 7: 328338, 1993.[Abstract/Free Full Text]
- Liou YM and Morgan KG. Redistribution of protein kinase C isoforms in association with vascular hypertrophy of rat aorta. Am J Physiol Cell Physiol 267: C980C989, 1994.[Abstract/Free Full Text]
- Minami K, Fukuzawa K, Nakaya Y, Xeng XR, and Inoue I. Mechanism of activation of the Ca-activated K+ channel by cyclic AMP in cultured porcine coronary artery smooth muscle cells. Life Sci 53: 11291135, 1993.[CrossRef][ISI][Medline]
- Nelson MT and Quayle JM. Physiological roles and properties of potassium channels in arterial smooth muscle. Am J Physiol Cell Physiol 268: C799C822, 1995.[Abstract/Free Full Text]
- Nishizuka Y. The role of protein kinase C in cell surface signal transduction and tumor production. Nature 308: 693698, 1984.[ISI][Medline]
- Ohanian V, Ohanian J, Shaw L, Scarth S, Parker PJ, and Heagerty AM. Identification of protein kinase C isoforms in rat mesenteric small arteries and their possible role in agonist induced contraction. Circ Res 78: 806812, 1996.[Abstract/Free Full Text]
- Orton EC, Raffestin B, and McMurtry IF. Protein kinase C influences rat pulmonary vascular reactivity. Am Rev Respir Dis 141: 654658, 1990.[ISI][Medline]
- Post JM, Hume JR, Archer SL, and Weir EK. Direct role for potassium channel inhibition in hypoxic pulmonary vasoconstriction. Am J Physiol Cell Physiol 262: C882C890, 1992.[Abstract/Free Full Text]
- Rasmussen H, Calle R, Ganesan S, Smallwood J, Throckmorton D, and Zawalich W. Protein Kinase C: Role in Sustained Cellular Responses. Chichester, UK: Ellis Horwood, 1991.
- Robertson TP, Aaronson PI, and Ward JPT. Hypoxic vasoconstriction and intracellular Ca2+ in pulmonary arteries: evidence for PKC-independent Ca2+ sensitization. Am J Physiol Heart Circ Physiol 268: H301H307, 1995.[Abstract/Free Full Text]
- Roivainen R and Messing RO. The phorbol derivatives thymeleatoxin and 12-deoxyphorbol-13-O-phenylacetate-10-acetate cause translocation and down-regulation of multiple protein kinase C isozymes. FEBS Lett 319: 3134, 1993.[CrossRef][ISI][Medline]
- Schubert R, Noack T, and Serebryakov VN. Protein kinase C reduces the KCa current of rat tail artery smooth muscle cells. Am J Physiol Cell Physiol 276: C648C658, 1999.[Abstract/Free Full Text]
- Shimoda LA, Sylvester JT, and Sham JS. Inhibition of voltage-gated K+ current in rat intrapulmonary arterial myocytes by endothelin-1. Am J Physiol Lung Cell Mol Physiol 274: L842L853, 1998.[Abstract/Free Full Text]
- Taguchi K, Kaneko K, and Kubo T. Protein kinase C modulates Ca2+-activated K+ channels in cultured rat mesenteric artery smooth muscle cells. Biol Pharm Bull 23: 14501454, 2000.[ISI][Medline]
- Walsh MP, Horowitz A, Clement-Chomienne O, Andrea JE, Allen BG, and Morgan KG. Protein kinase C mediation of Ca2+ independent contractions of vascular smooth muscle. Biochem Cell Biol 74: 5165, 1996.[ISI][Medline]
- White RE, Darkow DJ, and Lang DL. Estrogen relaxes coronary arteries by opening BKCa channels through a cGMP-dependent mechanism. Circ Res 77: 936942, 1995.[Abstract/Free Full Text]
- White RE, Kryman JP, El-Mowafy AM, Han G, and Carrier GO. cAMP-dependent vasodilators cross-activate the cGMP-dependent protein kinase to stimulate BKCa channel activity in coronary artery smooth muscle cells. Circ Res 86: 897905, 2000.[Abstract/Free Full Text]