K+ channel inhibition, calcium signaling, and vasomotor tone in canine pulmonary artery smooth muscle

Shouzaburoh Doi, Derek S. Damron, Koji Ogawa, Satoru Tanaka, Mayumi Horibe, and Paul A. Murray

Center for Anesthesiology Research, Division of Anesthesiology and Critical Care Medicine, The Cleveland Clinic Foundation, Cleveland, Ohio 44195


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We investigated the role of K+ channels in the regulation of baseline intracellular free Ca2+ concentration ([Ca2+]i), alpha -adrenoreceptor-mediated Ca2+ signaling, and capacitative Ca2+ entry in pulmonary artery smooth muscle cells (PASMCs). Inhibition of voltage-gated K+ channels with 4-aminopyridine (4-AP) increased the membrane potential and the resting [Ca2+]i but attenuated the amplitude and frequency of the [Ca2+]i oscillations induced by the alpha -agonist phenylephrine (PE). Inhibition of Ca2+-activated K+ channels (with charybdotoxin) and inhibition (with glibenclamide) or activation of ATP-sensitive K+ channels (with lemakalim) had no effect on resting [Ca2+]i or PE-induced [Ca2+]i oscillations. Thapsigargin was used to deplete sarcoplasmic reticulum Ca2+ stores in the absence of extracellular Ca2+. Under these conditions, 4-AP attenuated the peak and sustained components of capacitative Ca2+ entry, which was observed when extracellular Ca2+ was restored. Capacitative Ca2+ entry was unaffected by charybdotoxin, glibenclamide, or lemakalim. In isolated pulmonary arterial rings, 4-AP increased resting tension and caused a leftward shift in the KCl dose-response curve. In contrast, 4-AP decreased PE-induced contraction, causing a rightward shift in the PE dose-response curve. These results indicate that voltage-gated K+ channel inhibition increases resting [Ca2+]i and tone in PASMCs but attenuates the response to PE, likely via inhibition of capacitative Ca2+ entry.

alpha -adrenoreceptor activation; intracellular calcium; voltage-gated potassium channels; pulmonary circulation; vasoconstriction


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ACTIVATION OF SYMPATHETIC alpha -adrenoreceptors in the pulmonary circulation results in contraction of smooth muscle cells and vasoconstriction. A rapid rise in intracellular free Ca2+ concentration ([Ca2+]i) in pulmonary artery smooth muscle cells (PASMCs) is the trigger for contraction. Several factors contribute to the rise in [Ca2+]i, including influx of Ca2+ through sarcolemmal Ca2+ channels and release of Ca2+ from the sarcoplasmic reticulum (SR). A number of transarcolemmal Ca2+ influx pathways have been identified in vascular smooth muscle, including receptor-, voltage-, and store-operated Ca2+ channels (11).

Sarcolemmal K+ channels are key regulators of resting membrane potential (Em) in PASMCs and play an important role in regulating [Ca2+]i and vasomotor tone (14, 38, 40). Membrane depolarization via inhibition of voltage-gated K+ (KV) channels causes Ca2+ influx through L-type Ca2+ channels, which increases [Ca2+]i and vasomotor tone (40). However, changes in Em can also negatively affect other Ca2+ regulatory mechanisms such as agonist-induced inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] production (13, 22) and capacitative Ca2+ entry via store-operated Ca2+ channels (11). Membrane hyperpolarization induced by activation of ATP-sensitive (KATP) and/or Ca2+-activated (KCa) K+ channels can also modulate basal tone (6, 8, 33) as well as agonist-induced vasodilation (28) via a reduction in [Ca2+]i.

Hamada et al. (19) previously demonstrated that alpha -adrenoreceptor activation of PASMCs with phenylephrine (PE) causes oscillations in [Ca2+]i. These oscillations require the activation of phospholipase C and the presence of extracellular Ca2+ but do not involve activation of voltage-operated Ca2+ channels. In addition, the oscillations are dependent on the presence of thapsigargin-sensitive, but not ryanodine-sensitive, intracellular Ca2+ stores (19). Doi et al. (10) have also demonstrated that activation of tyrosine kinases and sarcolemmal Ca2+ influx via capacitative Ca2+ entry are required for the maintenance of PE-induced [Ca2+]i oscillations. In the present study, we investigated the extent to which KV, KCa and KATP channels modulate baseline [Ca2+]i, PE-induced oscillations in [Ca2+]i, and capacitative Ca2+ entry in canine PASMCs. We also investigated the effects of KV channel inhibition on isometric tension as well as on concentration-effect curves for KCl and PE in isolated pulmonary arterial rings. We tested two hypotheses. The first was that KV channel inhibition would cause membrane depolarization, an increase in [Ca2+]i, and an increase in vasomotor tone. The second was that KV channel inhibition would inhibit capacitative Ca2+ entry, decrease PE-induced [Ca2+]i oscillations, and decrease PE-induced contraction.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Pulmonary arteries were isolated from adult mongrel dogs. All procedures were performed aseptically, under general anesthesia with intravenous fentanyl citrate (20 µg/kg) and pentobarbital sodium (30 mg/kg), and with endotracheal intubation and positive-pressure ventilation. The dogs were exsanguinated and then killed by electrical fibrillation. (The method of euthanasia was approved by the Institutional Animal Care and Use Committee.) A thoracotomy was performed via the left fifth intercostal space. The heart and lungs were removed en bloc, and pulmonary arteries were isolated and dissected in a laminar flow hood under sterile conditions.

Cell culture of PASMCs. Primary cultures of PASMCs were obtained as previously described (19). Intralobar pulmonary arteries (2-4 mm ID) were carefully dissected and prepared for tissue culture. Explant cultures were prepared according to the method of Campbell and Campbell (7) with minor modifications. Briefly, the endothelium and adventitia were removed together with the most superficial part of the tunica media. The media was cut into 2-mm2 pieces, explanted in 25-cm2 culture dishes, nourished by DMEM-Ham's F-12 medium containing 10% fetal bovine serum and 1% antibiotic-antimycotic mixture solution (10,000 U/ml of penicillin, 10,000 µg/ml of streptomycin, and 25 µg/ml of amphotericin B), and kept in a humidified atmosphere of 5% CO2-95% air at 37°C. PASMCs began to proliferate from explants after 7 days in culture. Cells were allowed to grow for an additional 7-10 days before being subcultured nonenzymatically to 75-cm2 culture flasks and/or 35-mm glass dishes designed for fluorescence microscopy (Bioptechs). Cells were then used for experimentation within 72 h.

Fura 2-loading procedure. PASMCs were loaded with fura 2 as previously described (19). Twenty-four hours before experimentation, the culture medium containing 10% fetal bovine serum was replaced with a serum-free medium to arrest cell growth and allow for establishment of steady-state cellular events independent of cell division. PASMCs were washed twice in loading buffer (LB) that contained (in mM) 125 NaCl, 5 KCl, 1.2 MgSO4, 11 glucose, 1.8 CaCl2, and 25 HEPES in addition to 0.2% BSA at pH 7.4 adjusted with NaOH. PASMCs were then incubated in LB containing 2 µM fura 2-AM (Texas Fluorescence Laboratories) at ambient temperature for 30 min. After the 30-min loading period, the cells were washed twice in LB and incubated at ambient temperature for an additional 20 min before study.

Measurement of [Ca2+]i. [Ca2+]i was measured as previously described (19). Culture dishes containing fura 2-loaded PASMCs were placed in a temperature-regulated chamber (37°C; Bioptechs) and then mounted on the stage of an Olympus IX-70 inverted fluorescence microscope. Fluorescence measurements were performed on individual PASMCs in a culture monolayer with a dual-wavelength spectrofluorometer (Deltascan RFK6002, Photon Technology) at excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm. The volume of the chamber was 1.5 ml. The cells were superfused continuously at 1 ml/min with Krebs-Ringer buffer that contained (in mM) 125 NaCl, 5 KCl, 1.2 MgSO4, 11 glucose, 2.5 CaCl2, and 25 HEPES at pH 7.4 adjusted with NaOH. The temperature of all solutions was maintained at 37°C in a water bath. Solution changes were accomplished rapidly by aspirating the buffer in the dish and transiently increasing the flow rate to 10 ml/min. Just before data acquisition, the background fluorescence (i.e., fluorescence between cells) was measured and subtracted automatically from the subsequent experimental measurements. Fura 2 fluorescence signals (340 nm, 380 nm, and 340- to 380-nm ratio) originating from individual PASMCs were continuously monitored at a sampling frequency of 25 Hz and were collected with the use of the Felix software package from Photon Technology International.

Fura 2 titration. Estimations of [Ca2+]i were made by comparing the cellular fluorescence ratio with fluorescence ratios acquired with the use of fura 2 (free acid) in buffers containing known Ca2+ concentrations. [Ca2+]i was calculated as described by Grynkiewicz et al. (15).

Measurement of Em in PASMCs. The nystatin-perforated patch-clamp technique was used to measure Em in PASMCs as previously described (19). PASMCs were maintained at a temperature of 35°C (Delta T system, Bioptechs) on the stage of an inverted microscope. Cells were superfused (1-2 ml/min) with a control cell bath solution that contained (in mM) 1 CaCl2, 5 glucose, 5 HEPES, 5 KCl, 1 MgCl2, and 135 NaCl, pH 7.4. Sylgard-coated low-resistance electrodes (2-3 MOmega ) were front-filled with a pipette solution containing (in mM) 10 EGTA, 10 HEPES, 40 KCl, 100 potassium 2-(N-morpholino)ethanesulfonic acid, and 2 MgCl2 (pH 7.3) and then back-filled with a similar solution to which nystatin (100 µg/ml final concentration) had been added. Once nystatin was added to the buffer, the pipette solution was sonicated (30 s) and used within 3 h. After a stable seal was formed in voltage-clamp mode, the amplifier was switched to current-clamp mode and the superfusion solution was switched to one containing 4-aminopyridine (4-AP; 5 mM). Data acquisition was performed with pClamp 6.0 software controlling an Axopatch 200A amplifier (Axon Instruments). Em was monitored and recorded with Axotape software (Axon Instruments).

Organ chamber experiments. Intralobar pulmonary arteries (2-4 mm ID) were isolated and prepared as previously described (36). The arteries were cut into rings 0.5 cm wide. The endothelium was removed by inserting forceps tips into the lumen and rolling the rings over damp filter paper. Endothelial denudation was later confirmed by the absence of relaxation to acetylcholine (1 µM). The rings were suspended between two stainless steel stirrups in organ chambers filled with 25 ml of modified Krebs-Ringer bicarbonate solution (37°C) gassed with 95% O2 and 5% CO2. One of the stirrups was anchored, and the other was connected to a strain gauge (Grass FTO3) to measure isometric force. The rings were stretched at 10-min intervals in increments of 0.5 g to reach optimal resting tone (5 g). The contractile response to 60 mM KCl was assessed. After washout of KCl from the organ chamber and the return of isometric tension to prestimulation values, cumulative concentration-effect curves for 4-AP were performed. Concentration-effect curves for KCl and PE were performed in control rings and in paired rings pretreated with 4-AP (5 mM). Before PE administration, the rings were pretreated with 5 µM propranolol (incubated for 30 min) to inhibit the beta -agonist effects of PE.

Drug preparation. PE and propranolol (Sigma) were prepared as 10 and 5 mM stock solutions, respectively, in distilled water. Aliquots of the stock solutions were diluted in Krebs-Ringer buffer to achieve final concentrations of 10 and 5 µM, respectively. Acetylcholine (Sigma) was dissolved in distilled water to achieve a final organ bath concentration of 1 µM. 4-AP (Sigma) was dissolved directly into the Krebs-Ringer buffer on the day of use. The solutions containing 4-AP were buffered to pH 7.4 by adding HCl before each experiment. Glibenclamide, charybdotoxin, and nifedipine (Sigma) were dissolved in DMSO as stock solutions. Lemakalim (a gift from SmithKline Beecham Pharmaceuticals) was dissolved in distilled water to achieve a stock concentration of 1 mM. Aliquots of each stock solution were diluted in Krebs-Ringer buffer to achieve final concentrations. Similar dilutions of DMSO in Krebs-Ringer buffer had no effect on any of the end point measurements.

Data analysis. The amplitude and frequency of PE-induced increases in [Ca2+]i were measured in individual PASMCs. The amplitude was calculated by averaging the peak ratio value for 4-5 oscillations measured before (control) and after each intervention. Changes in the amplitude of the 340- to 380-nm fluorescence ratio in response to an intervention are expressed as a percent of control value. The frequency of oscillations was calculated by averaging the time interval between the oscillation peaks and is reported as the number of oscillations observed per minute. Changes in the frequency of oscillations are also expressed as a percent of control value. In some protocols, peak and sustained increases in [Ca2+]i were measured in individual PASMCs when the superfusion solution was switched from a Ca2+-free solution to a solution containing 2.2 mM Ca2+. Peak and sustained ratio values were averaged before and after each intervention and are expressed as a percent of control value. Contractile responses to KCl and PE in the presence and absence of 4-AP are expressed as 1) absolute increases in isometric tension, 2) normalized increases in tension after taking into account the fact that 4-AP induces increases in resting tension, and 3) a percent maximal response to KCl or PE. When comparing concentration-effect curves for KCl and PE, the concentrations of the agonists that caused 50% of the maximal response were interpolated from the concentration-effect curves by linear regression analysis and are presented as log ED50. All results are expressed as means ± SE. Statistical analysis was performed with repeated-measures ANOVA followed by Bonferroni or Dunn post hoc testing. Differences were considered significant at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of KV channel inhibition on Em in PASMCs. We tested the hypothesis that KV channel inhibition results in membrane depolarization. Current-clamp experiments revealed that the mean resting Em in PASMCs was -41 ± 4 mV. Addition of 4-AP (5 mM) to the superfusion buffer caused membrane depolarization, increasing the Em to -32 ± 3 mV (P < 0.05).

Effect of K+ channel modulators on baseline [Ca2+]i in PASMCs. We tested the hypothesis that KV channel inhibition would increase baseline [Ca2+]i in canine PASMCs. Baseline value of [Ca2+]i was 91 ± 6 nM. The effect of 4-AP (5 mM) on baseline [Ca2+]i consisted of a rapid transient increase in [Ca2+]i (175 ± 11% of control value) followed by a sustained elevation in [Ca2+]i above baseline (137 ± 6% of control value; Fig. 1). [Ca2+]i returned to baseline values after washout of 4-AP. The 4-AP-induced increase in baseline [Ca2+]i was markedly attenuated by the addition of nifedipine, a voltage-dependent Ca2+ channel blocker, or removal of extracellular Ca2+ (Fig. 2).


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Fig. 1.   A: representative trace depicting the effect of 4-aminopyridine (4-AP), a voltage-gated K+ (KV) channel blocker, on baseline intracellular free Ca2+ concentration ([Ca2+]i; 340- to 380-nm fluorescence ratio). B: summarized data demonstrating the peak and sustained increases in baseline [Ca2+]i induced by 4-AP (n = 8 cells). * P < 0.05.



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Fig. 2.   Summarized data depicting the inhibiting effects of nifedipine (NIF; 10 µM), a voltage-gated Ca2+ channel blocker, and removal of extracellular Ca2+ on 4-AP (5 mM)-induced peak (A) and sustained (B) increases in baseline [Ca2+]i (n = 8 cells). * P < 0.05.

We also examined the effects of KATP channel activation and KCa channel inhibition on baseline [Ca2+]i with the use of lemakalim and charybdotoxin, respectively. Neither lemakalim (10 µM; 98 ± 3% of control value) nor charybdotoxin (100 nM; 98 ± 2% of control value) had an effect on baseline [Ca2+]i (n = 6 cells). Similarly, inhibition of KATP channels with glibenclamide (10 µM, 101 ± 2% of control value) had no effect on baseline [Ca2+]i (n = 5 cells).

Effect of K+ channel modulators on PE-induced [Ca2+]i oscillations. We tested the hypothesis that PE-induced [Ca2+]i oscillations in canine PASMCs would be attenuated by KV channel inhibition. Continuous superfusion of PE (10 µM) stimulated repetitive [Ca2+]i oscillations at a frequency of 1.07 ± 0.16 transients/min. The [Ca2+]i reached an average peak value of 418 ± 23 nM. 4-AP (5 mM) attenuated the amplitude (14 ± 6% of control value) and frequency (28 ± 18% of control value) of PE-induced [Ca2+]i oscillations (P < 0.05; Fig. 3). Higher doses of 4-AP (10 mM) completely abolished PE-induced [Ca2+]i oscillations (Fig. 3). The oscillations returned when 4-AP was washed out.


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Fig. 3.   A: representative trace depicting the effect of 4-AP on phenylephrine (PE)-induced [Ca2+]i oscillations. 4-AP was added to the superfusion buffer as indicated. B and C: summarized data depicting the dose-dependent inhibitory effect of 4-AP on amplitude and frequency, respectively, of PE-induced [Ca2+]i oscillations (n = 9 cells). Nos. in parentheses, 4-AP concentration in mM. * P < 0.05.

We also examined the effects of KATP channel activation and KCa channel inhibition on oscillations in [Ca2+]i in response to PE (10 µM). Neither lemakalim (10 µM) nor charybdotoxin (100 nM) had an effect on the amplitude (lemakalim, 98 ± 4% of control value; charybdotoxin, 97 ± 4% of control value) or frequency (lemakalim, 96 ± 5% of control value; charybdotoxin, 99 ± 3% of control value) of PE-induced [Ca2+]i oscillations. Similarly, inhibition of KATP channels with glibenclamide (10 µM) had no effect on the amplitude (96 ± 4% of control value) or frequency (102 ± 4% of control value) of PE-induced oscillations in [Ca2+]i.

Effect of K+ channel modulators on capacitative Ca2+ entry. We tested the hypothesis that capacitative Ca2+ entry in canine PASMCs would be attenuated by KV channel inhibition. After depletion of SR Ca2+ stores with thapsigargin (1 µM) in the absence of extracellular Ca2+, restoration of extracellular Ca2+ resulted in a rapid peak increase (197 ± 11% of control value) and a sustained increase (140 ± 7% of control value) in [Ca2+]i (Fig. 4). The sustained increase in [Ca2+]i returned to baseline when extracellular Ca2+ was removed. We examined the effect of KV channel inhibition with 4-AP (5 mM) on capacitative Ca2+ entry (Fig. 5). Because 4-AP alone causes Ca2+ influx via voltage-operated Ca2+ channels (see Fig. 2), we added nifedipine (10 µM) to the bath just before restoring extracellular Ca2+ for the second time. Nifedipine had no effect on the peak or sustained increase in [Ca2+]i that was due to capacitative Ca2+ entry. In the continued presence of nifedipine, 4-AP was applied just before extracellular Ca2+ was restored the third time. 4-AP attenuated both the peak and sustained increases in [Ca2+]i that were due to capacitative Ca2+ entry (Fig. 5).


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Fig. 4.   A: representative trace depicting capacitative Ca2+ entry after depletion of sarcoplasmic reticulum (SR) Ca2+ stores with thapsigargin, a Ca2+-ATPase inhibitor. In the absence of extracellular Ca2+ (Ca2+-free buffer plus 2 mmol of EGTA), thapsigargin stimulated a transient increase in [Ca2+]i by releasing Ca2+ from SR Ca2+ stores. After [Ca2+]i had returned to baseline levels, Ca2+ was added back to the buffer, which caused sustained capacitative Ca2+ entry. [Ca2+]i returned to baseline levels after removal of extracellular Ca2+. B: summarized data depicting the peak and sustained increases in [Ca2+]i due to capacitative Ca2+ entry (n = 12 cells). * P < 0.05.



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Fig. 5.   A: representative trace depicting the effect of 4-AP on capacitative Ca2+ entry. After depletion of SR Ca2+ stores with thapsigargin, capacitative Ca2+ entry was compared in the presence and absence of nifedipine and 4-AP (5 mM), which were added to the superfusion buffer as indicated. B and C: summarized data showing that 4-AP, but not NIF, attenuated the peak and sustained increases, respectively, in [Ca2+]i because of capacitative Ca2+ entry (n = 6 cells). * P < 0.05.

We also examined the effects of KATP channel activation and KCa channel inhibition on capacitative Ca2+ entry. Neither lemakalim (10 µM) nor charybdotoxin (100 nM) had any effect on peak (lemakalim, 96 ± 4% of control value; charybdotoxin, 99 ± 4% of control value) or sustained (lemakalim, 102 ± 7% of control value; charybdotoxin, 98 ± 6% of control value) increases in [Ca2+]i that were due to capacitative Ca2+ entry (n = 6 cells). Similarly, inhibition of KATP channels with glibenclamide (10 µM) had no effect on peak (98 ± 5% of control value) or sustained (97 ± 5% of control value) increases in [Ca2+]i due to capacitative Ca2+ entry (n = 6 cells).

Effect of KV channel inhibition on tension in pulmonary arterial rings. We tested the hypothesis that KV channel inhibition would increase PASMC tone but would attenuate the contractile response to PE. The cumulative addition of 4-AP (1-10 mM) caused dose-dependent increases in tension in isolated pulmonary arterial rings (Fig. 6). To ascertain whether KV channel inhibition altered agonist-induced increases in tension, we examined the effect of 4-AP on concentration-effect curves for KCl and PE. When plotted as absolute changes in tension, 4-AP (5 mM) increased resting tension and increased the contractile responses to lower doses of KCl (Fig. 7A). However, the maximum tension achieved in response to KCl was the same in control and 4-AP-treated rings (Fig. 7A). Normalization of the 4-AP data to take into account the 4-AP-induced increase in resting tension revealed that the maximum response to KCl was attenuated in 4-AP-treated rings compared with control rings (Fig. 7A). 4-AP also caused a significant leftward shift (ED50, 13.9 ± 1.2 mM for 4-AP. 22.1 ± 0.5 mM for control; P < 0.05) in the concentration-effect curve for KCl (Fig. 7B). In contrast, 4-AP increased the maximum tension achieved in response to PE compared with that in control rings (Fig. 8A). However, this was due to the 4-AP-induced increase in baseline tension because the normalized data revealed no change in the maximum response to PE (Fig. 8A). 4-AP caused a rightward shift in the PE dose-response curve (Fig. 8B). Compared with control values, the PE ED50 (0.85 ± 0.17 vs. 1.63 ± 0.35 µM) and the PE 75% effective dose (2.61 ± 0.38 vs. 4.22 ± 0.62 µM) values were increased in 4-AP-treated rings (P < 0.05).


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Fig. 6.   Summarized data showing that 4-AP caused a dose-dependent increase in resting tension in isolated pulmonary arterial rings (n = 6 dogs).



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Fig. 7.   Effect of 4-AP on KCl-induced vasoconstriction in isolated pulmonary arterial rings. A: summarized data plotted as absolute increase in tension or normalized to same resting tension. B: summarized data plotted as percent maximal (MAX) response to KCl and normalized to same resting tension.



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Fig. 8.   Effect of 4-AP on PE-induced vasoconstriction in isolated pulmonary arterial rings. A: summarized data plotted as absolute increase in tension or normalized to same resting tension. B: summarized data plotted as percent maximal response to PE and normalized to same resting tension.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The overall goal of this study was to examine the extent to which K+ channels regulate baseline [Ca2+]i and alpha -adrenoreceptor-mediated Ca2+ signaling in PASMCs and to correlate our findings to functional responses in intact pulmonary arterial rings. Our results indicate that inhibition of KV channels in PASMCs causes inhibition of alpha -adrenoreceptor-mediated Ca2+ signaling and impairs contractile responses to alpha -adrenoreceptor activation in pulmonary arterial rings. The inhibitory action is mediated by an attenuation of capacitative Ca2+ entry through store-operated Ca2+ channels, which serves to refill SR Ca2+ stores after store depletion and to maintain oscillations in [Ca2+]i induced by PE (10).

Effect of K+ channel modulators on baseline Em and [Ca2+]i in PASMCs. Membrane depolarization and hyperpolarization through inhibition and activation of K+ channels, respectively, are important mechanisms regulating smooth muscle contraction and relaxation. KV channels sensitive to 4-AP are the primary K+ current at physiological Em values in rabbit pulmonary arteries (30). Inhibition of KV channels with 4-AP results in membrane depolarization of rabbit (30), canine (14), and rat (43) PASMCs. In our study, 4-AP increased Em and increased baseline [Ca2+]i, primarily via entry of Ca2+ through voltage-operated Ca2+ channels in the sarcolemma. These results confirm previous findings in rat PASMCs (39) and provide additional insight that KV channels are important regulators of baseline [Ca2+]i in canine PASMCs.

Although KATP and KCa channels are important mediators of agonist-induced vasodilation via membrane hyperpolarization (28), there is some evidence that they may also influence resting [Ca2+]i via regulation of resting Em (5, 8). In our studies, neither inhibition nor activation of KATP channels with glibenclamide or lemakalim had any effect on baseline [Ca2+]i, suggesting that KATP channels do not regulate resting Em in canine PASMCs. Similarly, inhibition of KCa channels with charybdotoxin had no effect on baseline [Ca2+]i. These results indicate that KATP and KCa channels do not play a role in the regulation of resting [Ca2+]i in canine PASMCs.

Effect of K+ channel modulators on PE-induced [Ca2+]i oscillations. Sympathetic alpha -adrenoreceptors on smooth muscle cells are G protein-coupled receptors that mediate contraction by increasing [Ca2+]i (24). The signaling pathway for the increase in [Ca2+]i involves activation of phospholipase C, production of Ins(1,4,5)P3, and Ca2+ release from the SR (4). Changes in Em can modify agonist-induced Ca2+ release from intracellular Ca2+ stores by altering Ins(1,4,5)P3 production. Membrane hyperpolarization inhibits norepinephrine-induced Ins(1,4,5)P3 production in rabbit mesenteric arteries (22), whereas membrane depolarization increases the rate of Ins(1,4,5)P3 accumulation in response to acetylcholine in guinea pig coronary vascular smooth muscle cells (13). Hamada et al. (19) previously demonstrated that PE-induced oscillations in [Ca2+]i were dependent on phospholipase C activation and Ins(1,4,5)P3-sensitive intracellular Ca2+ stores. Membrane depolarization induced by 4-AP should result in an increase in Ins(1,4,5)P3 production; thus the inhibitory effects of 4-AP on PE-induced [Ca2+]i oscillations likely are not due to decreases in Ins(1,4,5)P3 production and SR Ca2+ release.

KATP channels (8, 38) and KCa channels (1, 32) have also been identified in PASMCs and are important in regulating basal tone in coronary (33), mesenteric (26), cerebral (5), and saphenous (3) arteries. In pulmonary arteries, both KV (14, 39, 43) and KATP (8) channels are proposed to regulate resting Em. Moreover, inhibition of KATP (25) and KCa (35) channels mediates endothelin- and U-46619-induced vasoconstriction of coronary arteries via depolarization of vascular smooth muscle cells. In this study, neither glibenclamide, a KATP channel blocker (27), nor charybdotoxin, a KCa channel blocker (5), had any effect on the amplitude or frequency of PE-induced [Ca2+]i oscillations. Activation of KATP with lemakalim also had no effect on PE-induced [Ca2+]i oscillations. These results suggest that neither KATP nor KCa channels regulate or modulate PE-induced [Ca2+]i oscillations in PASMCs.

Effect of K+ channel modulators on capacitative Ca2+ entry. Hamada et al. (19) previously demonstrated that the amplitude and frequency of PE-induced [Ca2+]i oscillations were insensitive to voltage-operated Ca2+ channel blockers but were inhibited by SKF-96365, which blocks capacitative Ca2+ entry through store-operated Ca2+ channels in PASMCs (10). SKF-96365 is a nonselective Ca2+ channel blocker that has been utilized by a number of investigators (9, 34, 37) to inhibit capacitative Ca2+ entry. Inhibition of capacitative Ca2+ entry with SKF-96365 also attenuated alpha 1B-adrenoreceptor-mediated contraction in rat spleen (6). Capacitative Ca2+ entry is triggered by a depletion of intracellular Ca2+ stores and is believed to be important for refilling the SR Ca2+ pool after agonist stimulation. In this study, 4-AP attenuated capacitative Ca2+ entry in cells pretreated with nifedipine. This is consistent with the concept that capacitative Ca2+ entry is attenuated by membrane depolarization (11). Although we cannot rule out an effect on Na+-Ca2+ exchange, these data taken together with previous findings (10) suggest that KV channel inhibition attenuates PE-induced [Ca2+]i oscillations by inhibiting capacitative Ca2+ entry via store-operated Ca2+ channels. SR Ca2+ stores refill via capacitative Ca2+ entry after store depletion in canine PASMCs (10).

Effect of KV channel inhibition on tension in pulmonary arterial rings. The functional effects of KV channel inhibition were assessed in pulmonary arterial rings. Inhibition of KV channels with 4-AP caused dose-dependent contraction in canine pulmonary arterial rings, which confirms previous findings in rat pulmonary arterial rings and isolated perfused lung samples (20, 31). It has recently been demonstrated that KV channel inhibition in PASMCs is involved in hypoxic pulmonary vasoconstriction (31, 42) and primary pulmonary hypertension (40). Our data suggest that KV channel inhibition triggers Ca2+ influx via voltage-operated Ca2+ channels, resulting in pulmonary vasoconstriction.

We also investigated whether KV channel inhibition would alter contractile responses to agonist stimulation with KCl or PE. In absolute terms, pretreatment with 4-AP increased KCl-induced contraction, causing a leftward shift in the concentration-effect curve for KCl without altering maximal tension. However, the maximal response to KCl is attenuated in the presence of 4-AP when the data are normalized to take into account the 4-AP-induced increase in resting tension. Taken together, these results indicate that 4-AP and KCl increase tension in pulmonary arterial rings via a similar mechanism (e.g., membrane depolarization). The leftward shift in the KCl concentration-effect curve in 4-AP-treated rings is due to the depolarization effect of 4-AP (i.e., 4-AP-treated rings are already partially depolarized, which results in a leftward shift in response to a contractile stimulus).

The maximum contractile responses to PE were similar in control and 4-AP-treated rings after the 4-AP-induced increase in resting tension was taken into account. The PE concentration-effect curve was significantly shifted to the right by 4-AP. The rightward shift in the PE concentration-effect curve induced by 4-AP (Fig. 8) appears to be much smaller than the leftward shift in the KCl concentration-effect curve in 4-AP-treated rings. Actually, 4-AP decreased the KCl ED50 by about one-half, whereas it about doubled the PE ED50. The reason that the effects of 4-AP appear to be greater on the KCl vs. PE concentration-effect curves is that the PE concentrations are expressed in log units in Fig. 8. In addition, we believe that several factors masked an even larger inhibitory effect of 4-AP on PE contraction. First, 4-AP-treated rings are partially depolarized, which increases Ca2+ entry via voltage-gated Ca2+ channels. This effect would cause a leftward shift in the concentration-effect curve to a contractile stimulus as we observed with KCl. Second, membrane depolarization can also increase agonist-induced Ins(1,4,5)P3 production (13), which would stimulate the release of Ca2+ from the SR. This effect would also act to offset the inhibitory influence of 4-AP on PE-induced contraction. Third, PE-induced contraction is not only mediated by an increase in [Ca2+]i but also by an increase in myofilament Ca2+ sensitivity (23). There is no a priori reason to think that 4-AP would modify this effect. Thus regulation of PE-induced contraction is multifactorial. KV channel inhibition exerts opposing effects on PE-induced contraction. The net effect is an attenuation in PE contraction. Similar results have been observed in other tissues in which 4-AP has been shown to attenuate the positive inotropic action of PE, such as in rat ventricular (12) and atrial (29) muscle. We postulate that the 4-AP-induced membrane depolarization results in a reduction in capacitative Ca2+ entry after agonist stimulation with PE, causing a depression in the amplitude and frequency of [Ca2+]i oscillations in PASMCs and attenuation of PE-induced contraction in pulmonary arterial rings.

Ca2+ oscillations and signal transduction in pulmonary artery smooth muscle. A variety of vasoconstrictor stimuli, including angiotensin II (16), ATP (18), endothelin-1 (21), serotonin (41), and PE (19), have been shown to trigger oscillations in [Ca2+]i in PASMCs. It appears that the cyclic increase in [Ca2+]i is due to iterative Ca2+ release from Ins(1,4,5)P3-sensitive Ca2+ stores within the SR (17-19, 21). It has been hypothesized that Ca2+ release can then secondarily activate an oscillatory membrane Cl- current that depolarizes the cell membrane (17, 21). This results in an influx of Ca2+ via voltage-gated Ca2+ channels that contributes to the agonist-induced vasoconstrictor effect. Pretreatment with niflumic acid, an inhibitor of the membrane Cl- current, results in an attenuation of the contractile response to angiotensin II and endothelin-1 in isolated pulmonary arterial rings (17, 21). However, alternative signaling pathways may be activated by different agonists such as PE. It is difficult to definitively prove the physiological significance of [Ca2+]i oscillations. It should be noted that 4-AP abolished the PE-induced [Ca2+]i oscillations, whereas it caused a relatively modest attenuation in PE-induced contraction. At face value, these results would seem to suggest that [Ca2+]i oscillations are functionally important. However, these results could also be due to the multiple opposing effects of KV channel inhibition on PE-induced contraction. Doi et al. (10) recently demonstrated that capacitative Ca2+ entry and tyrosine kinase activation were required for PE-induced Ca2+ oscillations in PASMCs. In addition, the PE-induced contraction in isolated pulmonary arterial rings was attenuated by SKF-96365, an inhibitor of capacitative Ca2+ entry, as well as by tyrphostin 23, an inhibitor of tyrosine kinases (10). In intact rat tail artery preparations, oscillations in [Ca2+]i generated via Ins(1,4,5)P3-induced Ca2+ release from intracellular stores in response to locally produced angiotensin II contribute to the maintenance of vascular tone (2). Taken together, these results at least support the concept that [Ca2+]i oscillations may constitute an important signaling pathway in response to agonist activation in vascular smooth muscle.

In conclusion, our results indicate that KV channel inhibition in canine PASMCs causes membrane depolarization, an increase in [Ca2+]i, and an increase in isometric tension in pulmonary arterial rings. However, KV channel inhibition also attenuates capacitative Ca2+ entry, alpha -agonist-induced oscillations in [Ca2+]i, and alpha -agonist-induced increases in isometric tension.


    ACKNOWLEDGEMENTS

We gratefully acknowledge the technical support of Cindy Shumaker. We also thank Rhonda Schultz for outstanding work in preparing the manuscript.


    FOOTNOTES

This study was supported by National Heart, Lung, and Blood Institute Grants HL-38291 and HL-40361.

S. Doi was also supported by Dr. Jun-ichi Yata, Department of Pediatrics, Tokyo Medical and Dental University, Tokyo, Japan.

Address for reprint requests and other correspondence: P. A. Murray, Center for Anesthesiology Research, FF40, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195 (E-mail: murrayp{at}ccf.org).

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. §1734 solely to indicate this fact.

Received 18 August 1999; accepted in final form 2 March 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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